# Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health

Laurent Dufossé, Mireille Fouillaud, Yanis Caro

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Laurent Dufossé, Mireille Fouillaud, Yanis Caro. Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health. 2021, 978-3-0365-1465-9. hal-04149858

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Journal of
***
*Fungi*

Fungi and Fungal
Metabolites for
the Improvement
of Human and
Animal Nutrition
and Health

Edited by

Laurent Dufossé, Mireille Fouillaud and Yanis Caro

Printed Edition of the Special Issue Published in *Journal of Fungi*
# Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health
<figure><img src="image_3.png" /></figure>
Fungi and Fungal Metabolites for the
Improvement of Human and Animal
Nutrition and Health

Editors

Laurent Dufossé

Mireille Fouillaud

Yanis Caro

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

<figure><img src="image_4.png" /></figure>
Editors

Laurent Dufossé
Laboratoire CHEMBIOPRO
Université de La Réunion
Sainte-Clotilde
Reunion

Mireille Fouillaud
Laboratoire CHEMBIOPRO
Université de La Réunion
Sainte-Clotilde
Reunion

Yanis Caro
IUT-Laboratoire CHEMBIOPRO
Université de La Réunion
Saint-Pierre
Reunion

Editorial Office

MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Journal of Fungi* (ISSN 2309-608X) (available at: www.mdpi.com/journal/jof/special_issues/fungal metazoans).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

Last Name, A.A.; Last Name, B.B.; Last Name, C.C. Article Title. *Journal Name Year, Volume Number, Page Range*.

ISBN 978-3-0365-1466-6 (Hbk)

ISBN 978-3-0365-1465-9 (PDF)

© 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.
# Contents

About the Editors vii

Preface to "Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health" ix

Laurent Dufossé, Mireille Fouillaud and Yanis Caro

Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health

Reprinted from: *Journal of Fungi* **2021**, *7*, 274, doi:10.3390/jof7040274. 1

Apinun Kanpiengjai, Chartchai Khanongnuch, Saisamorn Lumyong, Aksarakorn Kummasook and Suwapat Kittibunchakul

Characterization of *Sporidiobolus ruineniae* A45.2 Cultivated in Tannin Substrate for Use as a Potential Multifunctional Probiotic Yeast in Aquaculture

Reprinted from: *Journal of Fungi* **2020**, *6*, 378, doi:10.3390/jof6040378. 9

Birgit Keller, Henrike Kuder, Christian Visscher, Ute Siesenop and Josef Kamphues

Yeasts in Liquid Swine Diets: Identification Methods, Growth Temperatures and Gas-Formation Potential

Reprinted from: *Journal of Fungi* **2020**, *6*, 337, doi:10.3390/jof6040337. 23

Alexandros Mavrommatis, Christina Mitsiopoulou, Christos Christodoulou, Dimitris Karabinas, Valentin Nenov, George Zervas and Eleni Tsiplakou

Dietary Supplementation of a Live Yeast Product on Dairy Sheep Milk Performance, Oxidative and Immune Status in Peripartum Period

Reprinted from: *Journal of Fungi* **2020**, *6*, 334, doi:10.3390/jof6040334. 43

Theresa P. T. Nguyen, Margaret A. Garrahan, Sabrina A. Nance, Catherine E. Seeger and Christian Wong

Assimilation of Cholesterol by *Monascus purpureus*

Reprinted from: *Journal of Fungi* **2020**, *6*, 352, doi:10.3390/jof6040352. 61

Madelaine M. Aguilar-Pérez, Daniel Torres-Mendoza, Roger Vásquez, Nivia Rios and Luis Cubilla-Rios

Exploring the Antibacterial Activity of *Pestalotiopsis* spp. under Different Culture Conditions and Their Chemical Diversity Using LC-ESI-Q-TOF-MS

Reprinted from: *Journal of Fungi* **2020**, *6*, 140, doi:10.3390/jof6030140. 77

Talaat H. Habeeb, Mohamed Abdel-Mawgoud, Ramy S. Yehia, Ahmed Mohamed Ali Khalil, Ahmed M. Saleh and Hamada AbdElgawad

Interactive Impact of Arbuscular Mycorrhizal Fungi and Elevated CO₂ on Growth and Functional Food Value of *Thymus vulgare*

Reprinted from: *Journal of Fungi* **2020**, *6*, 168, doi:10.3390/jof6030168. 93

Yong-Woon Kim, Yuanzheng Wu, Moon-Hee Choi, Hyun-Jae Shin and Jishun Li

Alginate-Derived Elicitors Enhance -Glucan Content and Antioxidant Activities in Culinary and Medicinal Mushroom, *Sparassis latifolia*

Reprinted from: *Journal of Fungi* **2020**, *6*, 92, doi:10.3390/jof6020092. 107
Diego Ibarra-Cantún, María Elena Ramos-Cassellis, Marco Antonio Marín-Castro and Rosalía del Carmen Castelán-Vega

Secondary Metabolites and Antioxidant Activity of the Solid-State Fermentation in Apple (*Pirus malus* L.) and Agave Mezcalero (*Agave angustifolia* H.) Bagasse

Reprinted from: *Journal of Fungi* **2020**, *6*, 137, doi:10.3390/jof6030137. 123

Simona Dzurendova, Boris Zimmermann, Valeria Tafintseva, Achim Kohler, Svein Jarle Horn and Volha Shapaval

Metal and Phosphate Ions Show Remarkable Influence on the Biomass Production and Lipid Accumulation in Oleaginous *Mucor circinelloides*

Reprinted from: *Journal of Fungi* **2020**, *6*, 260, doi:10.3390/jof6040260. 137

Ondrej Slaný, Tatiana Klempová, Volha Shapaval, Boris Zimmermann, Achim Kohler and Milan Čertík

Biotransformation of Animal Fat-By Products into ARA-Enriched Fermented Bioproducts by Solid-State Fermentation of *Mortierella alpina*

Reprinted from: *Journal of Fungi* **2020**, *6*, 236, doi:10.3390/jof6040236. 161

Juliana Lebeau, Thomas Petit, Mireille Fouillaud, Laurent Dufossé and Yanis Caro

Aqueous Two-Phase System Extraction of Polyketide-Based Fungal Pigments Using Ammonium- or Imidazolium-Based Ionic Liquids for Detection Purpose: A Case Study

Reprinted from: *Journal of Fungi* **2020**, *6*, 375, doi:10.3390/jof6040375. 177

Fernanda de Oliveira, Caio de Azevedo Lima, André Moreni Lopes, Daniela de Araújo Viana Marques, Janice Izabel Druzian, Adalberto Pessoa Júnior and Valéria Carvalho Santos-Ebinuma

Microbial Colorants Production in Stirred-Tank Bioreactor and Their Incorporation in an Alternative Food Packaging Biomaterial

Reprinted from: *Journal of Fungi* **2020**, *6*, 264, doi:10.3390/jof6040264. 195

Mardonio E. Palomino Agurto, Sarath M. Vega Gutierrez, R. C. Van Court, Hsiou-Lien Chen and Seri C. Robinson

Oil-Based Fungal Pigment from *Scytalidium cuboideum* as a Textile Dye

Reprinted from: *Journal of Fungi* **2020**, *6*, 53, doi:10.3390/jof6020053. 209

Jacqueline A. Takahashi, Bianca V. R. Barbosa, Bruna de A. Martins, Christiano P. Guirlanda and Marília A. F. Moura

Use of the Versatility of Fungal Metabolism to Meet Modern Demands for Healthy Aging, Functional Foods, and Sustainability

Reprinted from: *Journal of Fungi* **2020**, *6*, 223, doi:10.3390/jof6040223. 225

Yuanming Ye, Jingwang Qu, Yao Pu, Shen Rao, Feng Xu and Chu Wu

Selenium Biofortification of Crop Food by Beneficial Microorganisms

Reprinted from: *Journal of Fungi* **2020**, *6*, 59, doi:10.3390/jof6020059. 253

Xiaokang Zhang, Boqiang Li, Zhanquan Zhang, Yong Chen and Shiping Tian

Antagonistic Yeasts: A Promising Alternative to Chemical Fungicides for Controlling Postharvest Decay of Fruit

Reprinted from: *Journal of Fungi* **2020**, *6*, 158, doi:10.3390/jof6030158. 269

Pedro Pais, Vanda Almeida, Melike Yılmaz and Miguel C. Teixeira

*Saccharomyces boulardii*: What Makes It Tick as Successful Probiotic?

Reprinted from: *Journal of Fungi* **2020**, *6*, 78, doi:10.3390/jof6020078. 285
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# About the Editors

## Laurent Dufossé

Laurent Dufossé, has held the position of Professor of Food Science and Biotechnology since 2006, at the Reunion Island University, which is located on a volcanic island in the Indian Ocean, near Madagascar and Mauritius. The island is one of France's overseas territories, with almost one million inhabitants, and the university has 15,000 students. Previously, Professor Dufossé was a researcher and senior lecturer at the Université de Bretagne Occidentale, Quimper, Brittany, France. He attended the University of Burgundy, D, where he received his PhD in Food Science in 1993 and has been involved in the field of biotechnology of food ingredients for more than 30 years. His main research over the last 20 years has focused on microbial production of pigments and studies are mainly devoted to aryl carotenoids, such as isorenieratene, C50 carotenoids, azaphilones, and anthraquinones.

## Mireille Fouillaud

Mireille Fouillaud is a senior lecturer at the University of Reunion and a researcher at the Laboratory of Chemistry and Biotechnology of Natural Products (ChemBioPro). She graduated in 1990 as an industrial microbiology engineer. Following a Ph.D. in Cell Biology and Microbiology from the University of Aix-Marseille I, defended in 1994, she was recruited at the Faculty of Sciences and Technology of the University of Reunion. Between 2009 and 2020, she joined the Ecole Supérieure d'Ingénieurs Réunion Océan Indien (ESIROI), in the food engineering department. She taught biology and microbiology applied to agribusiness there. In 2020, she came back at the Faculty of Sciences of University of La Réunion to teach microbiology and project management. Her main research interests range from the microbial diversity of ecosystems and organisms to the production of metabolites of interest for industries, through biotechnology.

## Yanis Caro

Dr Yanis Caro has held the position of Associate Professor of Chemistry and Biotechnology at University of La Reunion and senior researcher at Chemistry and Biotechnology of Natural Products Laboratory (CHEMBIOPRO) since 2010. He first joined the food engineering department of the engineering school ESIROI, and then the Department of Health, Safety and Environment (HSE) at the Institute of Technology (IUT) of La Reunion in 2017. Holder of a Master Degree in Agro-Resources Sciences (1997), Dr Caro obtained his Ph.D. (2001) in Lipid Chemistry and Biotechnology at the school INP of Toulouse (France) and his Habilitation at the University of La Reunion (2020). Dr Caro worked at CIRAD of Montpellier (1998 - 2001) and, from 2004 until 2010, he worked as graduate engineer and advisor technological development in charge of food safety innovation projects at the CRITT centre of La Reunion.
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Preface to "Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health"

Fungi comprise 1, 2, 3, ..., or maybe around 5.1 million species? Even scientists do not currently agree on how many fungi species may be found on planet Earth, with only around 120,000 having been described so far. Fungi have been classified as a separate kingdom of organisms, as complex and diverse as plants and animals, of which only a few percent have been named and described. Fungal biomasses and fungal metabolites share a long common history with human and animal nutrition and health. Macrofungi and filamentous fungi constitute a large portfolio of proteins, lipids, vitamins, minerals, oligo elements, pigments, colorants, bioactive compounds, antibiotics, pharmaceuticals, etc. For example, industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. Some start-ups convert byproducts and side streams rich in carbohydrates into a protein-rich fungal biomass. This biomass can then be processed into a vegan meat substitute for food applications. In recent years, there has also been a significant increase (in fact, a significant revival) in the number of publications in the international literature dealing with the production of lipids by microbial sources (the single-cell oils (SCO) that are produced by the so-called "oleaginous"microorganisms, including "oleaginous"fungi such as zygomycete species, e.g., *Cunninghamella echinulata* and *Mortierella isabellina*). Fungi are potential sources of polyunsaturated fatty acids (PUFA), as these microorganisms can accumulate large amounts of high-valued PUFAs, such as gamma-linolenic acid (GLA) and arachidonic acid (ARA).

The purpose of this Special Issue of Journal of *Fungi* (MDPI) was not to provide a comprehensive overview of the vast arena of how fungi and fungal metabolites are able to improve human and animal nutrition and health; rather, we, as Guest Editors, wished to encourage authors working in this field to publish their most recent work in this rapidly growing journal in order for the large readership to appreciate the full potential of wonderful and beneficial fungi. Thus, this Special Issue welcomed scientific contributions on applications of fungi and fungal metabolites, such as bioactive fatty acids, pigments, polysaccharides, alkaloids, terpenoids, etc., with great potential in human and animal nutrition and health.

Laurent Dufossé, Mireille Fouillaud, Yanis Caro
*Editors*
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Editorial

# Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health

Laurent Dufossé <fnref n="1" />,<fnref n="2" />,* D, Mireille Fouillaud <fnref n="1" />,<fnref n="2" /> D and Yanis Caro <fnref n="1" />,<fnref n="3" /> D

**Keywords:** micro-fungi; macro-fungi; filamentous fungi; antioxidant; *Ganoderma*; kombucha; anticancer; carotenoid; medicinal mushroom; mycobiome; antimicrobial; antifungal; bioconversion; feed additive; cheese; dairy; *Sclerotinia*; secondary metabolite

Fungi: 1, 2, 3, ... 5.1 million species? Even scientists do not currently agree on how many fungi there might be on the planet Earth but only about 120,000 have been described so far [1]. They have been grouped into a separate kingdom of organisms, as complex and diverse as plants and animals, of which only a small percentage have been named and described. Fungal biomasses and fungal metabolites have a long common history with human and animal nutrition and health. Macro fungi and filamentous fungi bring a large portfolio of proteins, lipids, vitamins, minerals, oligo elements, pigments, colorants, bioactive compounds, antibiotics, pharmaceuticals, etc. For example, industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. Some start-ups convert by-products and side-streams rich in carbohydrates into a protein-rich fungal biomass. The biomass is then processed into a vegan meat substitute for food applications. The last years there has also been a significant rise (in fact, a significant revival) in the number of publications in the international literature dealing with the production of lipids by microbial sources (the "single cell oils; SCOs" that are produced by the so-called "oleaginous" microorganisms, including "oleaginous" fungi, such as the zygomycete species, e.g., *Cunninghamella echinulata* and *Mortierella isabellina*). Fungi are potential sources of polyunsaturated fatty acids (PUFAs) as these microorganisms can accumulate large amounts of high-valued PUFAs, such as gamma-linolenic acid (GLA) and arachidonic acid (ARA).

The objective of the invitation to contribute to this MDPI Fungi Special Issue was not to give a complete coverage of how fungi and fungal metabolites are able to improve human and animal nutrition and health. We, as co-guest editors, simply wanted to encourage authors working in this field to publish their most recent work in a rapidly expanding journal, in order to make a large audience discover the full potential of wonderful and beneficial fungi. Thus, this Special Issue welcomes 22 scientific contributions (13 original research papers and 9 reviews) on applications of fungi and fungal metabolites, such as bioactive fatty acids, pigments, polysaccharides, alkaloids, terpenoids, etc., with great potential in human and animal nutrition and health.

Three original research papers investigate the advantages of using probiotics and living fungi in feed. Kanpiengjiai et al. [2] provide insights into a potential multifunctional probiotic yeast in aquaculture produced from *Sporidiobolus ruineniae* cultivated in tannin

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check for
updates

**Citation:** Dufossé, L.; Fouillaud, M.; Caro, Y. Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health. *J. Fungi* 2021, 7, 274. https://doi.org/10.3390/jof7040274

Received: 17 March 2021

Accepted: 1 April 2021

Published: 4 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

<footnote n="1">¹ Laboratoire de Chimie et Biotechnologie des Produits Naturels—CHEMBIOPRO, Université de la Réunion, 15 Avenue René Cassin, CEDEX 9, CS 92003, F-97744 Saint-Denis, Ile de la Réunion, France; mireille.fouillaud@univ-reunion.fr (M.F.); yanis.caro@univ-reunion.fr (Y.C.)</footnote>

<footnote n="2">² Ecole Supérieure d'Ingénieurs Réunion Océan Indien—ESIROI, 2 Rue Joseph Wetzell, F-97490 Sainte-Clotilde, Ile de la Réunion, France</footnote>

<footnote n="3">³ IUT de La Réunion, Département Hygiène, Sécurité, Environnement (HSE), 40 Avenue de Soweto, CEDEX 9, BP 373, F-97455 Saint-Pierre, Ile de la Réunion, France</footnote>

<footnote>* Correspondence: laurent.dufosse@univ-reunion.fr</footnote>
substrate. *Sporidiobolus ruineniae* A45.2, a carotenoid-producing yeast, was able to co-produce cell-associated tannase (CAT), gallic acid and viable cells with antioxidant activity when grown in a tannic acid substrate. Furthermore, viable cells were characterized by moderate hydrophobicity, high auto-aggregation and moderate co-aggregation with *Staphylococcus aureus*, *Salmonella ser. thyphimurium* and *Streptococcus agalactiae*. In another aspect of yeasts in animal feed, Keller et al. [3] investigate the gas-formation potential of yeasts present in liquid swine diets. Yeasts are said to cause sudden death in swine due to intestinal gas formation. As not all animals given high yeast content feed fall ill, growth and gas formation potential at body temperature were investigated as possible causally required properties. Most *Candida krusei* isolates formed high gas amounts within 24-h, whereas none of the *Candida lambica*, *Candida holmii* or most of the other isolates did. The gas pressure formed by yeast isolates varied more than tenfold. Only a minority of the yeasts were able to produce gas at temperatures common in the pig gut. Moving to dairy sheep milk performance, Mavrommatis et al. [4] evaluate the dietary administration of *Saccharomyces cerevisiae* live yeast on milk performance and composition, oxidative status of both blood plasma and milk, and gene expression related to the immune system of lactating ewes during the peripartum period. In conclusion, the dietary supplementation of ewes with *S. cerevisiae* improved the energy utilization and tended to enhance milk performance with simultaneous suppression of mRNA levels of pro-inflammatory genes during the peripartum period. Of course, fungi may also have an impact on human nutrition and health. Nguyen et al. [5] focus on *Monascus purpureus*, a filamentous fungus known for its fermentation of red yeast rice, that produces the metabolite monacolin K used in statin drugs to inhibit cholesterol biosynthesis. In their study, they show that active cultures of *M. purpureus* CBS 109.07, independent of secondary metabolites, use the mechanism of cholesterol assimilation to lower cholesterol in vitro. The findings demonstrate that active growing of *M. purpureus* CBS 109.07 can assimilate cholesterol, removing 36.38% of cholesterol after 48-h of incubation at 37 °C. The removal of cholesterol by resting or dead *M. purpureus* CBS 109.07 was not significant, with cholesterol reduction ranging from 2.75%–9.27% throughout a 72-h incubation. Cholesterol was also not shown to be catabolized as a carbon source.

The World Health Organization (WHO; Geneva, Switzerland) has established an urgent pathogen list of antibiotic-resistant bacteria to guide the research, discovery, and development of new antibiotics. This list includes carbapenem-resistant *Pseudomonas aeruginosa*, Enterobacteriaceae, and third generation cephalosporin-resistant bacteria as critical priorities as a result of the continuous and indiscriminate use of antibiotics, not only in the treatment of human diseases, but also in animals. This list includes antifungal compounds. Aguilar-Pérez et al. [6] explore and compare the antibacterial activity and chemical diversity of two endophytic fungi isolated from the plant *Hyptis dilatata* and cultured under different conditions by the addition of chemical elicitors, changes in the pH, and different incubation temperatures. A total of 34 extracts were obtained from both *Pestalotiopsis mangiferae* and *Pestalotiopsis microspora* and were tested against a panel of pathogenic bacteria. Three active extracts obtained from *P. mangiferae* were analyzed by Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry (LC-ESI-Q-TOF-MS) to screen the chemical diversity and the variations in composition. This allows the proposal of structures for some of the determined molecular formulas, including the previously reported mangiferaelactone, an antibacterial compound.

Arbuscular mycorrhizal fungi interact with plants, beneficial to humans or not. Habeeb et al. [7] highlight the integration of Arbuscular mycorrhizal fungi (AMF) and elevated CO₂ (eCO₂) into agricultural procedures as an ecofriendly approach to support the production and quality of plants. This study was conducted to investigate the effects of AMF and eCO₂, individually or in combination, on growth, photosynthesis, metabolism and functional food value of *Thymus vulgare*. Results revealed that both AMF and eCO₂ treatments improved photosynthesis and biomass production, however, much more positive impact was obtained by their synchronous application. Moreover, the levels of the
majority of the detected sugars, organic acids, amino acids, unsaturated fatty acids, volatile compounds, phenolic acids and flavonoids were further improved as a result of the synergistic action of AMF and eCO₂, as compared to the individual treatments.

Elicitation effects of fungal products on plants and mushrooms are screened all over the world. Kim et al. [8] investigate the elicitation effects of alginate oligosaccharides extracted from brown algae (*Sargassum* species) on β-glucan production in cauliflower mushroom (*Sparassis latifolia*). Sodium alginate was refined from *Sargassum fulvellum*, *S. fusiforme*, and *S. horneri*, and characterized by proton nuclear magnetic resonance spectroscopy. The Solid Fraction (SF) of *S. fusiforme* and Liquid Fraction (LF) of *S. horneri* were chosen for elicitation on *S. latifolia*, yielding the highest β-glucan contents of 56.01 ± 3.45% and 59.74 ± 4.49% in the stalk, respectively. Total polyphenol content (TPC), antioxidant activities (2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging and Superoxide dismutase (SOD)-like activity) of aqueous extracts of *S. latifolia* were also greatly stimulated by alginate elicitation.

Vegetal biomass, available in large amounts on the planet, could be a source of fungal substrate to produce useful compounds for humans, animals or plants. The project conducted by Ibarra-Cantún et al. [9] determines the quantity of secondary metabolites and the antioxidant activity of the extracts obtained by the solid-state fermentation of apple and agave mezcalero bagasse over 28 days, inoculated with the *Pleurotus ostreatus* strain. Solid-state fermentation (SSF) is used in enzyme and antibiotic production, bioethanol and biodiesel as an alternative energy source, biosurfactants with environmental goals, and the production of organic acids and bioactive compounds. The results showed a higher presence of phenolic compounds, flavonoids, total triterpenes and antioxidant activity in the apple bagasse from the SSF on day 21 in the extract of acetone and water (80:20 v/v), 100% methanol and aqueous; while the agave bagasse showed a significant presence of phenolic compounds and flavonoids only in the aqueous extract. In conclusion, the presence of secondary metabolites exhibiting antioxidant activities from the solid-state fermentation in the residues of the cider and mezcal industry is an alternative use for wasted raw material, and reduces the pollution generated from agro-industrial residues.

Fungal biotechnology brings much progress to humans, animals, and plants. Five research papers illustrate this aspect by the production of lipids and pigments or colorants. The biomass of *Mucor circinelloides*, a dimorphic oleaginous filamentous fungus, has a significant nutritional value and can be used for single cell oil production. Metal ions are micronutrients supporting fungal growth and metabolic activity of cellular processes. Dzurendova et al. [10] study the effect of 140 different substrates, with varying amounts of metal and phosphate ions concentration, on the growth, cell chemistry, lipid accumulation, and lipid profile of *M. circinelloides*. It was observed that Mg and Zn ions were essential for the growth and metabolic activity of *M. circinelloides*. An increase in Fe ion concentration inhibited fungal growth, while higher concentrations of Cu, Co, and Zn ions enhanced the growth and lipid accumulation. Lack of Ca and Cu ions, as well as higher amounts of Zn and Mn ions, enhanced lipid accumulation in *M. circinelloides*. Generally, the fatty acid profile of *M. circinelloides* lipids was quite constant, irrespective of media composition. In a second study dedicated to lipids, *Mortierella alpina* was used by Slaný et al. [11] in SSF for the bioconversion of animal fat by-products into high value fermented bioproducts enriched with arachidonic acid (ARA). Although in general the addition of an animal fat by-product caused a gradual cessation of ARA yield in the obtained fermented bioproduct, the content of ARA in fungal biomass was higher. Thus, *M. alpina* CCF2861 effectively transformed exogenous fatty acids from animal fat substrate to ARA. Maximum yield of 32.1 mg of ARA/g of bioproduct was reached when using cornmeal mixed with 5% (w/w) of an animal fat by-product as substrate.

Fungal pigments and colorants tend to replace synthetic, chemical ones as consumers request green and sustainable processing. Demand for microbial colorants is now becoming a competitive research topic for food, cosmetics, textile, and pharmaceutics industries. Textile dyeing is one of the most polluting aspects of the global fashion industry, devastating
the environment and posing health hazards to humans. Many fungal genera/species are
currently in use for large-scale, industrial-scale production of pigments and colorants.
*Talaromyces albobiverticillius* (deep red pigment producer), *Emericella purpurea* (red pigment
producer), *Paecilomyces marquandii* (yellow pigment producer) and *Trichoderma harzianum*
(yellow-brown pigment producer) are discussed by Lebeau et al. [12]. Polyketide-based
pigments from fungal submerged cultures are usually extracted by conventional liquid-
liquid extraction methods requiring large volumes of various organic solvents and time.
To address this question from a different angle, the authors proposed an investigation
the use of three different aqueous two-phase extraction systems using either ammonium-
or imidazolium-based ionic liquids. Their findings led them to conclude that (i) these
alternative extraction systems using ionic liquids as the means for greener extractant
worked well for this extraction of colored molecules from the fermentation broths of the
filamentous fungi investigated; (ii) tetrabutylammonium bromide, [N4444]Br-, showed the
best pigment extraction ability, with a higher putative affinity for azaphilone red pigments;
(iii) the back extraction and recovery of fungal pigments from ionic liquid phases remained
the limiting point of the method under their selected conditions for potential industrial
applications. In another study, de Oliveira et al. [13] produce yellow-orange-red colorants
with *Talaromyces amestolkiae* in a stirred-tank bioreactor. The effect of the pH-shift control
strategy from 4.5 to 8.0 after 96-h of cultivation is evaluated at 500 rpm, resulting in an
improvement of natural colorant production, with this increase being more significant
for the orange and red examples, both close to four-fold. Next, the fermented broth
containing the colorants is applied to the preparation of cassava starch-based films in order
to incorporate functional activity in biodegradable films for food packaging. The presence
of fermented broth did not affect the water activity and total solids of biodegradable films
as compared with the standard. In the end, the films are used to pack butter samples
(for 45 days) showing excellent results regarding antioxidant activity. It is demonstrated
that the presence of natural colorants is obtained by a biotechnology process, which can
provide protection against oxidative action, as well as be a functional food additive in
food packing biomaterials. The last original paper and the third on fungal pigments and
colorants deals with pigments derived from spalting fungi that have previously shown
promise as textile dyes; however, their use has required numerous organic solvents with
human health implications. Palomino Agurto et al. [14] explore the possibility of using
linseed oil as a carrier for the pigment from *Scytalidium cuboideum* as a textile dye. Colored
linseed oil effectively dyed a range of fabrics, with natural fibers showing better coloration.
Scanning electron microscopy (SEM) revealed a pigment film over the fabric surface. While
mechanical testing showed no strength loss in treated fabric, colorfastness tests showed
significant changes in color in response to laundering and bleach exposure with variable
effects across fabric varieties. SEM investigation confirmed differences in pigmented oil
layer loss and showed variation in pigment crystal formation between fabric varieties.
Heating of the pigmented oil layer was found to result in a bright, shiny fabric surface,
which may have potential for naturally weatherproof garments.

A special issue is also an opportunity to go deeper into some scientific subjects via
reviews. The first of six published in this issue describes how fungal metabolism meets mod-
ern demands for healthy aging, functional foods, and sustainability. Takahashi et al. [15]
remind us that aging-associated, non-transmissible chronic diseases (NTCD) such as cancer,
dyslipidemia, and neurodegenerative disorders have been challenged through several
strategies including the consumption of healthy foods and the development of new drugs
for existing diseases. Consumer health consciousness is guiding market trends toward
the development of additives and nutraceutical products of natural origin. Fungi produce
several metabolites with bioactivity against NTCD as well as pigments, dyes, antioxi-
dants, polysaccharides, and enzymes that can be explored as substitutes for synthetic food
additives. Research in this area has increased the yields of metabolites for industrial appli-
cations through improving fermentation conditions, application of metabolic engineering
techniques, and fungal genetic manipulation. Several modern hyphenated techniques have
impressively increased the rate of research in this area, enabling the analysis of a large number of species and fermentative conditions. Their review thus focuses on summarizing the nutritional, pharmacological, and economic importance of fungi and their metabolites resulting from applications in the aforementioned areas, examples of modern techniques for optimizing the production of fungi and their metabolites, and methodologies for the identification and analysis of these compounds.

The second review by Ye al. [16] focuses on selenium. Selenium (Se) is essential for human health, however, Se is deficient in soil in many places all around the world, resulting in human diseases, such as the notorious Keshan disease and Kashin-Beck disease. Therefore, Se biofortification is a popular approach to improve Se uptake and maintain human health. Beneficial microorganisms, including mycorrhizal and root endophytic fungi, dark septate fungi, and plant growth-promoting rhizobacteria (PGPRs), show multiple functions, especially increased plant nutrition uptake, growth and yield, and resistance to abiotic stresses. Such functions can be used for Se biofortification and increased growth and yield under drought and salt stress. The work summarizes the use of mycorrhizal fungi and PGPRs in Se biofortification, aiming to improving their practical use.

Another way to protect humans from chemical hazards is to find biological and sustainable alternatives to chemical fungicides. Zhang et al. [17] highlight this specific point for controlling postharvest decay of fruit. Fruit plays an important role in human diet. Whereas fungal pathogens cause huge losses of fruit during storage and transportation, abuse of chemical fungicides leads to serious environmental pollution and endangers human health. Antagonistic yeasts (also known as biocontrol yeasts) are promising substitutes for chemical fungicides in the control of postharvest decay owing to their widespread distribution, antagonistic ability, environmentally friendly nature, and safety for humans. Over the past few decades, the biocontrol mechanisms of antagonistic yeasts have been extensively studied, such as nutrition and space competition, mycoparasitism, and induction of host resistance. Moreover, combination of antagonistic yeasts with other agents or treatments was developed to improve biocontrol efficacy. Several antagonistic yeasts are used commercially. In the review of Zhang et al. [17], the application of antagonistic yeasts for postharvest decay control is summarized, including the antagonistic yeast species and sources, antagonistic mechanisms, commercial applications, and efficacy improvement. Issues requiring further study are also discussed.

In addition to the research papers dedicated to probiotics, two reviews illustrate the importance of this scientific field. Pais et al. [18] discuss how *Saccharomyces boulardii* became so successful. *Saccharomyces boulardii* is a probiotic yeast often used for the treatment of gastro-intestinal (GI) tract disorders such as diarrhea symptoms. It is genetically close to the model yeast *Saccharomyces cerevisiae* and its classification as a distinct species or a *S. cerevisiae* variant has long been discussed. Here, the authors review the main genetic divergencies between *S. boulardii* and *S. cerevisiae* as a strategy to uncover the ability to adapt to host physiological conditions by the probiotic. *S. boulardii*, which possesses discernible phenotypic traits and physiological properties that underlie its success as probiotic, such as optimal growth temperature, resistance to the gastric environment and viability at low pH. Its probiotic activity has been elucidated as a conjunction of multiple pathways, ranging from improvement of gut barrier function, pathogen competitive exclusion, production of antimicrobial peptides, immune modulation, and trophic effects. This work summarizes the participation of *S. boulardii* in these mechanisms and the multifactorial nature by which this yeast modulates the host microbiome and intestinal function. The second paper assigned to probiotics, written by Kunyeit et al. [19], summarize how probiotic yeasts can be applied against *Candida* species associated infections. Superficial and life-threatening invasive *Candida* infections are a major clinical challenge in hospitalized and immuno-compromised patients. Emerging drug-resistance among *Candida* species is exacerbated by the limited availability of antifungals and their associated side-effects. In the current review, authors discuss the application of probiotic yeasts as a potential alternative/combination therapy against *Candida* infections. Preclinical studies have identified several probiotic yeasts
that effectively inhibit virulence of *Candida* species, including *Candida albicans*, *Candida tropicalis*, *Candida glabrata*, *Candida parapsilosis*, *Candida krusei* and *Candida auris*. However, *Saccharomyces cerevisiae* var. *boulardii* is the only probiotic yeast commercially available. In addition, clinical studies have further confirmed the in vitro and in vivo activity of the probiotic yeasts against *Candida* species. Probiotics use a variety of protective mechanisms, including posing a physical barrier, the ability to aggregate pathogens and to render them avirulent. Secreted metabolites such as short-chain fatty acids effectively inhibit the adhesion and morphological transition of Candida species. Overall, the probiotic yeasts could be a promising effective alternative or combination therapy for *Candida* infections.

Mushrooms have been valued as food and health supplements by humans for thousands of years. They are rich in dietary fiber, essential amino acids, minerals, and many bioactive compounds, especially those related to human immune system functions. Mushrooms contain diverse immunoregulatory compounds such as terpenes and terpenoids, lectins, fungal immunomodulatory proteins (FIPs) and polysaccharides. The distributions of these compounds differ among mushroom species and their potent immune modulation activities vary depending on their core structures and fraction composition chemical modifications. Zhao et al. [20] provide insight into the current status of clinical studies on immunomodulatory activities of mushrooms and mushroom products.

Mycotoxin is an important issue when dealing with human and animal health or nutrition. A specific outline by Chen et al. [21] discuss sphinganine-analog mycotoxins (SAMs). Sphinganine-analog mycotoxins (SAMs) include fumonisins from the *Fusarium* genus and *Alternaria alternata* f. sp. *lycopersici* (AAL) toxins. SAMs have shown diverse cytotoxicity and phytotoxicity, causing adverse impacts on plants, animals, and humans, and are a destructive force to crop production worldwide. The review summarizes the structural diversity of SAMs and encapsulates the relationships between their structures and biological activities. The toxicity of SAMs on plants and animals is mainly attributed to their inhibitory activity against the ceramide biosynthesis enzyme, influencing the sphingolipid metabolism and causing programmed cell death.

With the last articles, number 21 and 22 of this Journal of Fungi special issue, we return to fungal pigments. Fungi can produce a myriad of secondary metabolites, including pigments. Some of these pigments play a positive role in human welfare while others are detrimental. The review by Lin and Xu [22] summarizes the types and biosynthesis of fungal pigments, their relevance to human health, including their interactions with host immunity, and recent progress in studying their structure-activity relationships. Fungal pigments are grouped into carotenoids, melanin, polyketides, and azaphilones, etc. These pigments are phylogenetically broadly distributed. While the biosynthetic pathways for some fungal pigments are known, the majority remain to be elucidated.

Synthetic pigments/non-renewable coloring sources used normally in the textile industry release toxic substances into the environment, causing perilous ecological challenges. To be safer from such challenges, both academia and industry have explored the use of natural colorants such as microbial pigments. As explained by Venil et al. [23] such explorations have created a fervent interest among textile stakeholders in undertaking the dyeing of textile fabrics, especially with fungal pigments. The biodegradable and sustainable production of natural colorants from fungal sources is comparatively advantageous to synthetic dyes. The prospective scope of fungal pigments has emerged in the opening of many new avenues in textile colorants for wide ranging applications.

As a concluding remark, we, the Guest Editors, wish to thank all the authors and the reviewers for their significant contributions to this Special Issue and for making it a highly successful and timely collection of papers. Our acknowledgements also go to the whole MDPI team, i.e., assistant editors, editors, Editor-in-Chief, production office, website management, etc.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
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Article

# Characterization of *Sporidiobolus ruineniae* A45.2 Cultivated in Tannin Substrate for Use as a Potential Multifunctional Probiotic Yeast in Aquaculture

Apinun Kanpiengjai ¹,²,*, Chartchai Khanongnuch ³, Saisamorn Lumyong ²,⁴,⁵, Aksarakorn Kummasook ⁶ and Suwapat Kittibunchakul ⁷

1 Division of Biochemistry and Biochemical Technology, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand

2 Research Center of Microbial Diversity and Sustainable Utilization, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand

3 Division of Biotechnology, Faculty of Agro-Industry, Chiang Mai University, Mae-Hia, Chiang Mai 50100, Thailand; chartchai.k@cmu.ac.th

4 Division of Microbiology, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand; scboi009@gmail.com

5 Academy of Science, The Royal Society of Thailand, Sanam Suea Pa, Dusit, Bangkok 10300, Thailand

6 Unit of Excellence in Infectious Disease, Department of Medical Technology, School of Allied Health Sciences, University of Phayao, Muang, Phayao 56000, Thailand; aksarakorn.ku@up.ac.th

7 Institute of Nutrition, Mahidol University, 999 Phutthamonthon 4 Rd., Nakhon Pathom 73170, Thailand; suwapatkt@gmail.com

* Correspondence: ak.apinun@gmail.com or apinun.k@cmu.ac.th

Received: 4 November 2020; Accepted: 14 December 2020; Published: 18 December 2020

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**Abstract:** At present, few yeast species have been evaluated for their beneficial capabilities as probiotics. *Sporidiobolus ruineniae* A45.2, a carotenoid-producing yeast, was able to co-produce cell-associated tannase (CAT), gallic acid and viable cells with antioxidant activity when grown in a tannic acid substrate. The aim of this research study was to identify the potential uses of *S. ruineniae* A45.2 obtained from a co-production system as a potential feed additive for aquaculture. *S. ruineniae* A45.2 and its CAT displayed high tolerance in pH 2.0, pepsin, bile salts and pancreatin. Furthermore, its viable cells were characterized by moderate hydrophobicity, high auto-aggregation and moderate co-aggregation with *Staphylococcus aureus*, *Salmonella* ser. Thyphimurium and *Streptococcus agalactiae*. These attributes promoted *S. ruineniae* A45.2 as a multifunctional probiotic yeast. In addition, the intact cells possessed antioxidant activities in a 100–150 µg gallic acid equivalent (GAE)/mL culture. Remarkably, the fermentation broth demonstrated higher antioxidant activity of 9.2 ± 1.8, 9.0 ± 0.9, and 9.8 ± 0.7 mg GAE/mL culture after FRAP, DPPH and ABTS assays, respectively. Furthermore, higher antimicrobial activity was observed against *Bacillus cereus*, *Staphylococcus aureus* and *Strep. agalactiae*. Therefore, cultivation of *S. ruineniae* A45.2 with a tannic acid substrate displayed significant potential as an effective multifunctional feed additive.

**Keywords:** feed additive; probiotic; yeast; *Sporidiobolus ruineniae*; tannase

1. Introduction

Aquaculture is the most rapidly growing sector of food production throughout the world. Its global demand continues to rise as it is applied to farming practices in ever-increasing proportions [1]. Industrial-scale aquaculture production is recognized as a challenge within the farming sector as it requires sustainable and efficient technologies [2] that address concerns of potential human exposure
to microbial diseases and the possibility of severe economic losses across the industry [3]. The use of antibiotics is a simple approach in the control of diseases, however, excess antibiotics that are discharged into the environment are known to be responsible for the spread of antibiotic-resistant genes of pathogenic and commensal bacteria, all of which can lead to increases in drug resistance among animal and human populations. The addition of additives to antibiotics, vaccines, immunostimulants, prebiotics and probiotics, in particular, is an environmentally friendly alternative and recognized as a sustainable strategy [2]. The Food and Agriculture Organization (FAO)/World Health Organization (WHO) defined probiotics as live microorganisms that, when administered in adequate amounts, confer a health benefit upon the host [4]. In terms of their potential applications in aquaculture, yeasts are considered a second leading source of probiotics after bacteria. However, the use of probiotic yeasts is not as popular as bacteria. Indicative of a greater potential for profit than bacterial probiotics, yeasts are not affected by antibacterial compounds and are known to contain various immunostimulant compounds [3,5]. However, their applicable use among a wide variety of animals is limited due to the fact that the normal body temperature of animals is higher than the temperature for the optimal growth of yeast. To date, two yeast species, namely *Saccharomyces cerevisiae* and *Debaryomyces hansenii*, are widely recognized as potential probiotic yeasts [5]. Additionally, yeasts isolated from fish microbiota exhibit certain probiotic properties. These yeast species include *Candida deformans*, *Rhodotorula mucilaginosa*, *Yarrowia lipolytica*, *Metschnikowia viticola* and *Cryptococcus laurentii* [3].

*Sporidiobolus ruineniae* A45.2, isolated from fermented tea-leaves of northern Thailand, namely Miang, is a pigment-producing and tannin-tolerant type of yeast [6]. It is among the range of yeasts commonly found in the intestines of humans [7]. Based on evidence established by previous studies [6,8], *S. ruineniae* A45.2 is assumed to have a unique cell wall structure that serves its tannin-tolerance and may promote the organism as a potential probiotic yeast. On the other hand, its carotenoid pigment is considered highly valuable in terms of the enhancement of some aquaculture pigmentations [9]. Moreover, *S. ruineniae* A45.2 is capable of producing thermostable and pH-stable cell-associated tannase (CAT) and can degrade tannic acid to gallic acid [10]. Tannase is a feed additive enzyme that plays an important role in the reduction of tannins, an antinutritional factor in animal feed. The enzymatic degradation of tannins releases gallic acid that can be used as both an antimicrobial and an antioxidant agent. In aquaculture feed ingredients, tannins come from plant-derived, alternate fish feed ingredients that are used as protein sources, such as soybean meal, rapeseed meal, pea seed meal and mustard oil cake [11]. This drawback leads to significantly decreased levels of cumulative feed intake and digestibility [12]. However, this can be overcome by the addition of tannase.

In previous studies involving the co-production of gallic acid and CAT derived from tannins, both cells and the culture broth rich in gallic acid may be used as a potential source of tannase and gallic acid in the feed industry, respectively. The aims of this research study were to evaluate *S. ruineniae* A45.2 for its potential to be used as a probiotic in aquaculture. Our objectives were to also investigate the potential for fermented broth cultivated in a tannic acid substrate to be further applied as a multifunctional feed additive. Further carotenoids produced by this yeast were also characterized.

## 2. Materials and Methods

### 2.1. *Chemicals*

Bile salts, 40× pancreatin, pepsin, methyl gallate, gallic acid, rhodanine, 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothi-azoline-6-ylsulfonic acid (ABTS) and potassium persulfate were all of analytic grade and of the highest quality available from Sigma and Sigma-Aldrich (St. Louis, MO. USA). All media used in this research study, including yeast-malt extract broth (YMB), nutrient broth (NB), trypticase soy broth (TSB) and agar, were purchased from HiMedia (Nashik, India).
## 2.2. Microorganisms and Culture Conditions

S. ruineniae A45.2 was maintained on yeast-malt extract agar (YMA) at 4 °C for further use. To prepare the seed inoculum, a single colony of yeast was inoculated in YMB and incubated at 30 °C on a 150 rpm rotary shaker for 15–18 h or until the optical density at 600 nm reached 2.0–3.0. *Escherichia coli* TISTR 527, *Salmonella* ser. Thyphimurium TISTR 1472, *Staphylococcus aureus* TISTR 746 and *Bacillus cereus* TISTR 747 were maintained on nutrient agar (NA) and were grown in a nutrient broth (NB) at 37 °C on a 100 rpm rotary shaker when necessary. Furthermore, *Listeria monocytogenes* DMST 17303 and *Streptococcus agalactiae* DMST 11366 were maintained on trypticase soy agar (TSA). These pathogenic bacteria were grown in TSB at 37 °C on a 100 rpm rotary shaker when necessary.

## 2.3. Co-Production of Gallic Acid, CAT and Viable Cells of S. ruineniae A45.2

A total of 10% (v/v) of the prepared seed inoculum was transferred into a 1 L stirred tank fermenter (B.E. Marubishi Co. Ltd., Tokyo, Japan) with a 60% working volume of the optimized medium [10] that contained 12.3 g/L tannic acid, 6.91 g/L glucose, 10 g/L yeast extract, 2 g/L (NH₄)₂SO₄, 0.5 g/L tween 80 and 1 g/L glutamate (pH 6.0). Culture conditions were administered at 30 °C with an aeration rate of 1 vvm and an agitation speed of 250 rpm. After 48 h of cultivation, the cells were harvested by centrifugation at 8000 rpm, 4 °C for 10 min and washed twice with phosphate buffer saline (PBS) supplemented with 0.1% (v/v) triton X-100. The cell pellets were then resuspended in 0.85% (w/v) NaCl to obtain a concentration of 10⁸ cells/mL for further experimentation.

## 2.4. Tolerance of S. ruineniae A45.2 and Stability of Tannase at Low pH Values

A total of 0.5 mL of the prepared cell suspension (10⁸ cells/mL) was transferred to a 125 mL Erlenmeyer flask containing 49.5 mL of 0.85% (w/v) NaCl adjusted to pH 2.0 and 3.0 by 0.1 N HCl. The cell suspension in PBS was then used as a control. All mixtures were incubated at 30 °C for 4 h. Samples were periodically taken for measurement of viable cells by plate count technique and residual tannase activity. Initial cell concentration and tannase activity without incubation were set to 100%.

## 2.5. Tolerance of S. ruineniae A45.2 and Stability of Tannase in Simulated Gastric Juice

A total of 0.5 mL of the prepared cell suspension (10⁸ cells/mL) was transferred to a 125 mL Erlenmeyer flask containing 49.5 mL of simulated gastric juice (0.3% (w/v) pepsin, 0.85% (w/v) NaCl, pH 2.0). Cell suspension in PBS was used as a control. All mixtures were incubated at 30 °C for 4 h. Samples were periodically taken for measurement of viable cells by plate count technique and to determine residual tannase activity. Initial cell concentration and tannase activity without incubation was set to 100%.

## 2.6. Bile Salt Tolerance of S. ruineniae A45.2 and Stability of Tannase

A total of 0.5 mL of the prepared cell suspension (10⁸ cells/mL) was transferred to a 125 mL Erlenmeyer flask containing 49.5 mL of solution that consisted of 0.85% (w/v) NaCl and 0.3% (w/v) bile salts. Cells suspended with PBS were used as a control. All mixtures were incubated at 30 °C for 6 h. Samples were periodically taken for measurement of viable cells by the plate count technique to determine residual tannase activity. Initial cell concentration and tannase activity without incubation were set to 100%.

## 2.7. Tolerance of S. ruineniae A45.2 and Stability of Tannase in Simulated Intestinal Fluid

A total of 0.5 mL of the prepared cell suspension (10⁸ cells/mL) was transferred to a 125 mL Erlenmeyer flask containing 49.5 mL of simulated intestinal fluid (0.3% (w/v) bile salts, 0.3% (w/v) pancreatin and 0.85% (w/v) NaCl). Cells suspended with PBS were used as a control. All mixtures were incubated at 30 °C for 8 h. Samples were periodically taken for measurement of viable cells by plate
count technique to determine residual tannase activity. Initial cell concentration and tannase activity without incubation were set to 100%.

## 2.8. Assay of Tannase

Tannase activity was assayed using the previously described method [13]. Briefly, 50 µL of enzyme solution was mixed with 50 µL of 12.5 mM methyl gallate in 100 mM citrate-phosphate buffer pH 6.5 and incubated at 30 °C. After the incubation procedure, the reaction was stopped by adding 60 µL of 0.667% (w/v) methanolic rhodanine and the mixture was left at room temperature (25 °C) for 5 min. Subsequently, 40 µL of 0.5 M KOH was added to the mixture, which was then left at room temperature for 5 min prior to adding 800 µL of distilled water. Absorbance of the mixture was measured at 520 nm. One unit of tannase was defined as the amount of enzyme that liberated 1 µmol of gallic acid per minute under assay conditions.

## 2.9. Cell Surface Hydrophobicity

Yeast adherence was determined by cell surface hydrophobicity. The cell suspension (3 mL) (Ainitial) was transferred to a glass tube (12 × 100 mm) containing 1 mL of chloroform, agitated using a vortex mixer for 2 min and allowed to stand at room temperature for 30 min. The optical density of the aqueous phase (Afinal) was measured at a wavelength of 600 nm. The hydrophobicity index (HPBI) was calculated using the following equation:

$$ \text{HPBI (\%)} = \left(1 - \frac{A_{\text{final}}}{A_{\text{initial}}}\right) \times 100 $$

## 2.10. Auto-Aggregation Assay

A total of 3 mL of the yeast suspension in PBS (Ainitial) was transferred to a glass tube (12 × 100 mm), vortexed for 10 s and incubated at 30 °C for 2 h. Absorbance of the upper part of the mixture (approximately 1 mL) was measured at 600 nm (Afinal). Auto-aggregation was calculated using the following equation:

$$ \text{Auto - aggregation (\%)} = \left(1 - \frac{A_{\text{final}}}{A_{\text{initial}}}\right) \times 100 $$

## 2.11. Co-Aggregation Assay

Equal volumes (1.5 mL) of the yeast suspension (Ayeast) and pathogenic bacterium (Apathogen) were transferred into a glass tube (12 × 100 mm), vortexed for 30 s and incubated at 30 °C for 2 h. Absorbance of the upper part of the mixture (Amix) was measured at 600 nm. Co-aggregation was calculated using the following equation:

$$ \text{Co - aggregation (\%)} = \left(1 - \frac{A_{\text{mix}}}{(A_{\text{yeast}} + A_{\text{pathogen}})/2}\right) \times 100 $$

## 2.12. Adherence of Bacteria onto Yeast Cells

Adhesion of bacteria onto yeast cells was accomplished by mixing 1 mL of the yeast suspension (10⁸ cells/mL) in PBS and 1 mL of each pathogenic bacteria (10⁸ cells/mL). The specimens were then incubated at 30 °C. After 2 h of incubation, 10 µL of the mixture was smeared onto a microscopic slide for Gram-staining [14]. The Gram-stained slide was then used to visualize the adherence of the bacteria onto the yeast cells under a phase-contrast light microscope. The pathogenic bacteria used in the adherence test were B. cereus, E. coli, Sal. Thyphimurium, Staph. aureus, L. monocytogenes and Strep. agalactiae.
## 2.13. Determination of Antimicrobial Activity

The agar well diffusion method was used to determine antagonistic activity of culture broth obtained from the co-fermentation and fermentation in YMB. Briefly, an overnight culture (approximately $10^6-10^8$ CFU/mL) of the pathogenic bacteria was swabbed onto an NA plate for *B. cereus*, *E. coli*, *Sal. Typhimurium* and *Staph. aureus* and a TSA plate for *L. monocytogenes* and *Strep. agalactiae*. The wells were prepared by being punched with a 6 mm diameter sterile cork-borer and were filled with 50 µL of sterile culture broth or 50 µg/mL of chloramphenicol as the positive control. The plates were incubated at 30 °C for 18 h.

## 2.14. Analysis of Carotenoids

The previously obtained cell pellet of *S. ruineniae* A45.2 was lyophilized into a dry cell for carotenoid extraction. The freeze-dried cell (0.25 g) was then placed into a screw-cap glass tube (25 × 150 mm) containing 10 mL of acetone and 30 g of glass beads. Cell disruption was carried out through vigorous mixing for 10 min at room temperature. The mixture was filtered through filter paper to collect the cell extract and then centrifuged at 10,000 rpm for 10 min for the purposes of clarification. To quantify total carotenoids, absorbance of the clear extract was measured at 485 nm. The total carotenoid content of the yeast cells was calculated based on the extinction coefficient ($E_{1cm}^{1%}$) of 2680 and expressed in terms of total carotenoids (µg)/g dry cell weight. To determine the carotenoid composition, individual carotenoids were separated by Mightysil RP-18 GP prepacked column (150 × 2.0 mm; Kanto Chemical Co., Inc., Tokyo, Japan) equilibrated with a solution of methanol/acetonitrile (90:10 *v/v*). The conditions were carried out at 30 °C with a flow rate of 1.0 mL/min. The separated carotenoids were detected using a UV detector at 485 nm. Meanwhile, the evaporated cell extract was resuspended in acetone. This was then spotted on a thin layer chromatography (TLC) Silica gel plate (Silica gel 60 $F_{254}$, Merck Millipore, Germany) and developed in a chamber containing hexane/acetone (70:30 *v/v*). The pigments separated by TLC were recovered and dissolved in acetone for measurement of the wavelength of maximum absorbance ($\lambda_{max}$) using a UV-visible spectrophotometer.

## 2.15. Assay of Antioxidants

A culture of *S. ruineniae* A45.2 obtained from the co-production system was harvested by centrifugation at 8000 rpm for 10 min. The cell pellet was washed twice with PBS solution, suspended in the same solution and the fermented broth was then collected. Both intact cells and cell-free extract were determined for antioxidant activity using three different methods, including ferric-reducing antioxidant power (FRAP), DPPH free-radical-scavenging activity and ABTS free-radical-scavenging activity.

For the FRAP assay, the FRAP reagent consisted of 300 mM acetate buffer pH 3.6, a solution of 10 mM TPTZ in 40 mM HCl and 20 mM $FeCl_3$ at a ratio of 10:1:1 (*v/v/v*). The sample solution (0.10 mL) was mixed thoroughly with 3.40 mL of the FRAP reagent for 30 min prior to measuring the absorbance at 593 nm. A standard curve was prepared using different concentrations of gallic acid. The results were expressed in terms of milligram gallic acid equivalent (GAE)/mL culture.

For the DPPH assay, a sample (0.25 mL) was mixed with freshly prepared 40 ppm methanolic DPPH (2.25 mL) and allowed to stand in the dark at room temperature. A decrease in absorbance at 517 nm was determined after 10 min of the incubation process. The concentration of the sample that produced between 20% and 80% inhibition of the blank absorbance was determined and adapted. Radical scavenging activity was expressed as the concentration of the extract required for reduction of the initial concentration of DPPH by 50% ($EC_{50}$) under specified experimental conditions. DPPH radical scavenging activity was expressed in terms of mg GAE/mL culture.
For the ABTS assay, 0.0384 g of ABTS was prepared in 10 mL of water. Subsequently, 5 mL of the solution was mixed with 88 µL of 140 mM potassium persulfate and adjusted to 25 mL with deionized water in a volumetric flask for further experimentation. A total of 1.75 mL of the ABTS solution was mixed thoroughly with 0.25 mL of the sample and allowed to stand in the dark at room temperature for 10 min. A decrease in absorbance at 734 nm was measured. Radical scavenging activity was expressed as the concentration of the extract required for reduction of the initial concentration of ABTS by 50% (EC₅₀) under specified experimental conditions. ABTS radical scavenging activity was expressed in terms of mg GAE/mL culture.

**3. Results**

**3.1. Survival of Yeast and CAT Stability under Simulated Gastrointestinal Tract (GIT) Conditions**

Gastric and intestinal conditions were simulated for evaluation of the probiotic properties of *S. ruineniae* A45.2. Under simulated gastric conditions, cell viability of *S. ruineniae* A45.2 and its residual CAT activity were evaluated at pH 2.0 and pH 3.0 (Figure 1a,b) and carried out with and without (Figure 1c,d) the supplementation of pepsin. The results revealed that the values of both pH and pepsin did not significantly affect cell viability of the yeast or its CAT. At a pH value of 2.0, both in the presence and in the absence of pepsin, approximately 90% of the initial viable cells and residual CAT activity were retained. Furthermore, at a pH value of 3.0, the supplementation of pepsin slightly decreased tannase activity but had no effect on cell viability.

<figure><img src="image_13.png" /><figcaption>**Figure 1.** Effect of pH 2.0 and pH 3.0 on (a) survival of *S. ruineniae* A45.2 and (b) residual CAT activity and effect of pepsin on (c) survival of *S. ruineniae* A45.2 and (d) residual CAT activity.</figcaption></figure>

The same results were also obtained when *S. ruineniae* A45.2 and CAT were incubated under simulated intestinal conditions, in which bile salts and pancreatin acted as key factors. It was revealed that *S. ruineniae* A45.2 and CAT were resistant to bile salts (Figure 2a,b) and pancreatin (Figure 2c,d), as they retained 100% residual viable cells and tannase activity after incubation.
<figure><img src="image_14.png" /><figcaption>**Figure 2.** Effect of bile salts on (a) survival of *S. ruineniae* A45.2 and (b) residual CAT activity and effect of pancreatin in combination with bile salts on (c) survival of *S. ruineniae* A45.2 and (d) residual CAT activity.</figcaption></figure>

## 3.2. Cell Surface Hydrophobicity, Auto-Aggregation and Co-Aggregation

Cell surface hydrophobicity, auto-aggregation and co-aggregation of *S. ruineniae* A45.2 are presented in Table 1. *S. ruineniae* A45.2 displayed 58.4 ± 2.7% cell surface hydrophobicity. Significantly high values of auto-aggregation were observed at up to 88.2 ± 1.2% along with the ability to be co-aggregated with pathogenic bacteria, including *B. cereus*, *E. coli*, *Staph. aureus*, *Sal. Thyphimurium*, *L. monocytogenes* and *Strep. agalactiae* at different percentages of co-aggregation. *S. ruineniae* A45.2 displayed a stronger co-aggregation ability with *Strep. agalactiae*, *Sal. Thyphimurium* and *Staph. aureus* than other tested pathogenic bacteria. This evidence was in agreement with their adherence ability (Figure 3), wherein pathogenic bacteria obviously adhered to the yeast cells.

<table><caption>**Table 1.** Cell surface hydrophobicity, auto-aggregation and co-aggregation against pathogenic bacteria.</caption><thead><tr><th>Properties</th><th>%</th></tr></thead><tbody><tr><td>Cell surface hydrophobicity</td><td>58.4 ± 2.7</td></tr><tr><td>Auto-aggregation</td><td>88.2 ± 1.2</td></tr><tr><td>Co-aggregation</td><td></td></tr><tr><td><i>B. cereus</i></td><td>36.2 ± 2.8</td></tr><tr><td><i>E. coli</i></td><td>33.8 ± 0.7</td></tr><tr><td><i>Staph. aureus</i></td><td>44.0 ± 0.8</td></tr><tr><td><i>Sal. Thyphimurium</i></td><td>45.8 ± 0.1</td></tr><tr><td><i>L. monocytogenes</i></td><td>37.8 ± 2.1</td></tr><tr><td><i>Strep. agalactiae</i></td><td>51.5 ± 2.6</td></tr></tbody></table>

## 3.3. Antimicrobial Activity

The antagonistic effects of *S. ruineniae* A45.2 were observed after being exposed to various indicator microorganisms, including *B. cereus*, *E. coli*, *Sal. Thyphimurium*, *Staph. aureus*, *L. monocytogenes* and *Strep. agalactiae*. Culturing periods of 24 and 48 h of the co-production system were tested in comparison with specimens cultured in YMB. Only the supernatant obtained from co-production exhibited antimicrobial activity against some pathogenic bacteria (Figure 4), i.e., *B. cereus*, *Staph. aureus* and *Strep. agalactiae*.
<figure><img src="image_15.png" /><figcaption>**Figure 3.** Adherence of (a) *B. cereus*, (b) *E. coli*, (c) *Staph. aureus*, (d) *Sal. Thyphimurium*, (e) *L. monocytogenes* and (f) *Strep. agalactiae* on yeast cell walls observed under a phase-contrast light microscope at 100× magnification.</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>**Figure 4.** Antimicrobial activity of cell-free extract of *S. ruineniae* A45.2 cultivated in tannic acid substrate at 30 °C for 24 (left) and 48 h (right) of cultivation against (a) *B. cereus*, (b) *E. coli*, (c) *Staph. aureus*, (d) *Sal. Thyphimurium*, (e) *L. monocytogenes* and (f) *Strep. agalactiae* compared to control (top) (50 µg/mL chloramphenicol)</figcaption></figure>

### 3.4. Carotenoids Produced by *S. ruineniae* A45.2

*S. ruineniae* A45.2 grown in tannic acid were harvested after 48 h of cultivation, lyophilized and used for carotenoid extraction. Identification and characterization of the carotenoid pigment was performed by HPLC. Three main peaks were separated from the carotenoid extracts (Figure 5a). These peaks corresponded to the three spots that were isolated and visualized by TLC (Figure 5b). The first spot from the bottom was rosy-red in color and migrated with slower mobility than the second spot, which appeared orange to red in color, while the third spot was yellow in color and
migrated with the same degree of mobility as β-carotene. These pigments were identified based on their Visible absorbance maxima (Figure 5c). The least degree of polar pigment was identified as β-carotene due to similar absorbance maxima values recorded at 429 nm and 485 nm, with the maximal degree of absorbance recorded at 456 nm. The most notable polar pigment revealed a spectrum with three absorption maxima at wavelengths of 474, 527 with the absorption optimum at 499 nm, thereby identified as torularhodin. The second most polar pigment was torulene with three absorbance maxima values recorded at 462 nm and 516 nm, with an absorption maximum value recorded at 488 nm. Total carotenoids produced by *S. ruineniae* A45.2 after cultivation at 30 °C for 48 h were recorded at 88.0 ± 0.2 µg/g dry cell weight, equivalent to 500 ± 130 µg/L culture.

<figure><img src="image_17.png" /><figcaption>**Figure 5.** Characterization of pigments produced by *S. ruineniae* A45.2 (a) Separation of pigments by HPLC and (b) TLC and (c) Visible absorption spectra of the major pigments.</figcaption></figure>

### 3.5. Antioxidant Activity

Antioxidant activities of intact cells of *S. ruineniae* A45.2 and cell-free extract obtained from the co-production system were measured using FRAP, DPPH and ABTS assays. Overall, there were no significant differences in the antioxidant activities among the different methods. Antioxidant activities ranging from 100–120 µg GAE/mL culture and 110–150 µg GAE/mL culture were detected from the intact cells obtained from 24-h and 48-h periods of cultivation, respectively (Table 2). The cell-free extract revealed significantly higher antioxidant activities than the intact cells. The antioxidant activities of 5.6–6.8 mg GAE/mL culture and 9.0–9.8 mg GAE/mL culture were obtained from the cell-free extract fraction after 24-h and 48-h periods of cultivation, respectively. Activities of both intact cells and cell-free extract increased relative to the incubation time of the co-production system.

<table><caption>**Table 2.** FRAP, DPPH and ABTS antioxidant activity of cell-free extract and intact cells obtained from co-production of gallic acid and viable cells of *S. ruineniae* A45.2.</caption><thead><tr><th rowspan="2">Time (h)</th><th colspan="3">Cell-Free Extract (mg GAE/mL)</th><th colspan="3">Intact Cells (µg GAE/mL)</th></tr><tr><th>FRAP</th><th>DPPH</th><th>ABTS</th><th>FRAP</th><th>DPPH</th><th>ABTS</th></tr></thead><tbody><tr><td>24</td><td>5.6 ± 0.8</td><td>6.8 ± 0.6</td><td>6.4 ± 0.4</td><td>122.1 ± 9.3</td><td>108.5 ± 2.4</td><td>104.7 ± 0.8</td></tr><tr><td>48</td><td>9.2 ± 1.8 *</td><td>9.0 ± 0.9 *</td><td>9.8 ± 0.7 *</td><td>143.6 ± 3.4 *</td><td>114.6 ± 1.4 *</td><td>111.1 ± 0.5 *</td></tr></tbody></table>

<footnote>* significant difference within a column (p < 0.05).</footnote>
**4. Discussion**

In this study, *S. ruineniae* A45.2 and its culture broth obtained from the co-production of cells, gallic acid and tannase were characterized for their potential use in animal feed, specifically in feed prepared for fish and other aquatic organisms. *S. ruineniae* A45.2 was isolated from Miang, which is rich in tannins and considered a microbial inhibitor [6,8]. The cell wall structure and composition of *S. ruineniae* A45.2 are believed to promote its growth along with high concentrations of tannic acid, yet this yeast was found to be a promising probiotic. When used as a functional probiotic yeast, growth temperature is a crucial limitation for the application of probiotics in animals, since the yeast must be able to survive and grow at the animal's normal body temperature in order to enhance the animal's growth performance and promote the health of the animal [15]. Typically, the growth temperatures of yeasts range from 0 to 47 °C with an optimal temperature between 25 and 30 °C [16], yet probiotic yeasts might actively function when they are used in aquaculture. Probiotic yeasts are less popular than bacteria but can offer some major physiological contributions over bacteria. These include their cell volume and the production of a wide spectrum of simple and more complex compounds that may be beneficial to the health of aquatic organisms. However, only a few varieties of probiotic yeasts have been isolated for aquaculture applications. It was reported that marine and other aquatic environments, along with the gut microbiota of aquatic organisms, are potential sources of probiotic yeasts. In addition to *S. cerevisiae*, *D. hansenii* is a ubiquitous yeast that is frequently associated with fish and marine environments [7]. As of yet, no reports of using *S. ruineniae* as a probiotic yeast have been identified.

To be a good probiotic yeast, it must be able to successfully survive under gastrointestinal tract (GIT) conditions and provide beneficial conditions for the enhancement of the health of the host. In this study, both *S. ruineniae* A45.2 and its CAT were exposed to GIT conditions in order to assess the degree of residual cell viability and CAT. The temperature used in this research study was 30 °C, as it was identified as an optimal temperature of *S. ruineniae* A45.2 (data not shown). Considering cell viability, *S. ruineniae* A45.2 resisted low pH values ranging from 2.0 to 3.0, which were within the range found in the stomachs of fish. The degree of acidity in the stomach of a fish can vary depending on the fullness of the stomach and the species of the fish [17,18]. Moreover, *S. ruineniae* A45.2 was not found to be affected by the digestive enzymes we tested, namely pepsin and pancreatin (a mixture of amylase, protease and lipase). These attributes are considered important selection criteria for a good probiotic yeast [19]. On the other hand, the CAT of *S. ruineniae* A45.2 exhibited a good degree of thermostability and pH stability. Surprisingly, positive stability values were observed under simulated GIT conditions by retaining more than 90% of initial activity after treatment. This indicates that the yeast species could be applicable in the aquafeed industry. Plant-based products in fish diets contain valuable proteins used to replace fishmeal. These plant feed ingredients contain considerable amounts of tannins that can have an adverse effect on animals by reducing the nutritional value of the feed [11]. This circumstance can also decrease the palatability of the feed due to an unpleasant taste caused by a high concentration of tannins [12]. The results of this study indicate that both cells of *S. ruineniae* A45.2 might be able to survive in transit through the stomach and small intestines and function effectively in the large intestines. However, its CAT might be stable in stomach environments and could be active in the intestines, as the environments are similar to the known optimal values for pH and temperature.

Cell surface hydrophobicity is defined as a nonspecific interaction in adhesion between probiotic microorganisms onto GIT epithelial cells, where they may provide prophylactic and therapeutic benefits [20]. Colonization in the intestinal epithelial cell wall and mucosal surfaces can prevent pathogenic bacteria adhesion and inflammatory reactions [21]. Yet, hydrophobicity is an important attribute for selecting potential probiotics. *S. ruineniae* A45.2 showed high cell surface hydrophobicity toward chloroform and was comparable with those reported in *Bacillus subtilis* [19,21], various strains of *Lactobacillus* sp. [22] and *Sac. unisporus* [20].

Auto-aggregation is defined as aggregation among yeast cells to form flocs and colonize the intestinal environment of the host when the cells approach harmful conditions [20,23]. Probiotic
microorganisms should be associated with higher auto-aggregation than pathogenic microorganisms [22], specifically *Strep. agalactiae*, a representative fish pathogen. Under the same experimental conditions as this study, the percentage auto-aggregation of pathogenic bacteria ranged between 15–35% for *L. monocytogenes*, *Sal. Typhimurium* and *Staph. aureus* [22]. Within 2 h of the auto-aggregation test, *S. ruineniae* reported 88.2 ± 1.2%, which was higher than previously reported probiotic yeasts, namely *P. kluyveri*, *Issatchenkia orientalis*, *P. kudriavzevii* [24], *Yarrowia lipolytica*, *Wickerhamomyces anomalus* and *Sac. cerevisiae* [23]. Auto-aggregation capacity is strain-specific, while a capacity greater than 50% displayed the potential to prevent the invasion of various other pathogenic microorganisms through film formation.

Co-aggregation is defined as the close interaction between probiotics and different pathogenic bacteria [23]. It was reported that adherence of enteric bacteria onto yeast cells is irreversible, thus transient passage of the bacteria occurs through GIT and subsequent flushing out in the feces [20]. The co-aggregation ability of *S. ruineniae* A45.2 agreed with its adherence ability. This could be explained by the specific fimbriae present on bacteria with mannan on yeast cells and the electrostatic and hydrophobic nonspecific bindings [20].

No antimicrobial activity of *S. ruineniae* A45.2 against the tested pathogenic bacteria was detected when it was cultivated in YMB. It is therefore implied that no antimicrobial metabolite was produced by the organisms typically identified in various yeasts [20,24–26]. Most yeasts scavenge pathogenic infection by indirect mechanisms such as auto-aggregation, co-aggregation and adherence ability [27]. On the contrary, the growing of *S. ruineniae* A45.2 in tannic acid containing medium led to the release of gallic acid, which enhanced the antimicrobial activity of the culture broth against *B. cereus*, *E. coli*, *Staph. aureus* and *Strep. agalactiae*. The results suggest that production of the yeast should be performed in the presence of tannic acid to promote gallic acid production and CAT, thereby gaining antimicrobial activity. Supplementation of gallic acid in animal feed, especially aquatic feed, was scarcely reported. Current research found that the supplementation of gallic acid in broiler diets at levels ranging from 75 to 100 mg/kg improved the performance of broiler chicks in terms of feed utilization, breast muscle yield and oxidative stability, while positively modulating jejunum intestinal morphology [28]. Hence, our results provide supplemental, supportive evidence for the use of gallic acid as an alternative to antibiotics in animal feed or for the determination of synergistic interactions of gallic acid that could enhance the effects of antibiotics.

*S. ruineniae* A45.2 is a basidiomycetous yeast that forms a natural pink-red pigment made up of carotenoids. The pigments extracted from the yeast were separated into three types of carotenoids based on the separation by HPLC and TLC. These pigments displayed distinctively different visible spectra. The most polar pigment showed a rosy-red color and had a similar visible spectrum to torularhodin, while the others displayed a similar spectrum to torulene and β-carotene as the second most polar and the least polar pigments, respectively [29–31]. However, structural elucidation of these compounds must be confirmed. Currently, carotenoid-producing yeasts are mainly represented by the genera *Rhodosporidium*, *Xanthophylomyces Rodotorula* and *Sporobomyces*. The latter genus has a close relationship to the genus *Sporidiobolus* and represents the main source of torulene and torularhodin [32]. The quantity of total carotenoids produced by *S. ruineniae* A45.2 was in the range of those produced by the yeast studied in previously published reports [31,33].

After cell wall components, some probiotic yeasts exhibit multifunctional potential in the production of bioactive compounds with certain antioxidant properties, such as carotenoids, organic acids and glutathione [24]. As *S. ruineniae* A45.2 is a carotenoid-producing yeast, it is likely that it possesses antioxidant capacity. The intact cells and cell-free extract obtained from YMB were evaluated for their antioxidant activity (data not shown). No antioxidant activity was detected in the cell-free extract, while the intact cells possessed approximately 10 times lower the degree of antioxidant activity than that obtained from cultivation in tannic acid. This may have resulted from the presence of β-glucan as a component of yeast cell wall composition [34,35]. Cultivation of *S. ruineniae* A45.2 in tannic acid could potentiate the antioxidant activity of not only intact cells but also cell-free extracts. It was
determined that the fermentation of *S. ruineniae* A45.2 induced the production of CAT which strongly affected the degradation of tannic acid, resulting in gallic acid production. During the degradation of tannic acid, large amounts of gallic acid were released into the fermentation broth and attached to the yeast cell surface, reported in previous studies [10,36]. The antioxidant activity in terms of gallic acid equivalent is likely a consequence of gallic acid content, as reported in previously published studies [10]. This result agrees with previously reported evidence published on the fermentation of plant-based foods [37], including grape seed flour and extracts [38], as well as Miang [39], as sources of *S. ruineniae* A45.2.

Overall, *S. ruineniae* A45.2 may be capable of exhibiting the beneficial characteristics attributed to a probiotic yeast that can be used for aquaculture. Cultivation of the yeast in tannic acid substrate might provide a number of benefits. These benefits include the assertion that yeast cells can be a source of antioxidant agents, tannase and carotenoids for aquatic organisms. Furthermore, it is believed that the resulting culture broth can display strong antioxidant activity as well as the potential to display antimicrobial activity against some pathogenic bacteria, especially fish pathogens. Therefore, this research study described and verified an alternative integrative strategy for the production of feed additives. To our knowledge, this is the first report to suggest that *S. ruineniae* exhibits probiotic properties.

## 5. Conclusions

*S. ruineniae* A45.2 was tolerant to simulated GIT conditions, displaying tolerance to pH 2.0, pepsin, bile salts and pancreatin. A high percentage of auto-aggregation was observed, and this species co-aggregated various pathogenic bacteria, specifically *Strep. agalactiae*, and adhered to some specific strains of pathogenic bacteria. These are considered beneficial attributes that support the use of *S. ruineniae* as a probiotic yeast. The fermentation of tannic acid to gallic acid has resulted in the co-production of CAT, gallic acid and viable yeast cells. Moreover, CAT was found to be stable and may be able to function under simulated GIT conditions, while the cells possessed antioxidant activity. Thus, *S. ruineniae* A45.2 as a carotenoid- and CAT-producing yeast could be labeled as a multifunctional probiotic yeast suitable for the feed of animals, particularly aquatic animals. In addition, its cell-free extract derived from the co-production system could be a potential alternative source of natural antioxidants and antimicrobial agents.

**Author Contributions:** Conceptualization, A.K. (Apinun Kanpiengjai); funding acquisition, A.K. (Apinun Kanpiengjai); investigation, A.K. (Apinun Kanpiengjai), C.K., S.L., A.K. (Aksarakorn Kummasook) and S.K.; methodology, A.K. (Apinun Kanpiengjai), A.K. (Aksarakorn Kummasook) and S.K.; project administration, A.K. (Apinun Kanpiengjai); supervision, A.K. (Apinun Kanpiengjai), C.K. and S.L.; writing—original draft, A.K. (Apinun Kanpiengjai); writing—review and editing, A.K. (Apinun Kanpiengjai). All authors read and approved of the final version of this manuscript.

**Funding:** This research study was partially funded by the Thailand Research Fund through the Research Grant for New Scholars (MRG6280057), and Chiang Mai University.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

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Article

# Yeasts in Liquid Swine Diets: Identification Methods, Growth Temperatures and Gas-Formation Potential

Birgit Keller<fnref n="1" />*, Henrike Kuder<fnref n="1" />, Christian Visscher<fnref n="1" /> ID, Ute Siesenop<fnref n="2" /> and Josef Kamphues<fnref n="1" />

Received: 30 September 2020; Accepted: 2 December 2020; Published: 4 December 2020

<figure><img src="image_20.png" /></figure>

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updates

**Abstract:** Liquid feed is susceptible to microbiological growth. Yeasts are said to cause sudden death in swine due to intestinal gas formation. As not all animals given high yeast content feed fall ill, growth and gas formation potential at body temperature were investigated as possible causally required properties. The best identification method for these environmental yeasts should be tested beforehand. Yeasts derived from liquid diets without (LD – S) and liquid diets with maize silage (LD + S) were examined biochemically (ID32C-test) and with MALDI-TOF with direct smear (DS) and an extraction method (EX). Growth temperature and gas-forming potential were measured. With MALDI-EX, most yeast isolates were identified: *Candida krusei* most often in LD – S, and *C. lambica* most often in LD + S, significantly more than in LD – S. Larger colonies, 58.75% of all yeast isolates, were formed at 25 °C rather than at 37 °C; 17.5% of all isolates did not grow at 37 °C at all. Most *C. krusei* isolates formed high gas amounts within 24 h, whereas none of the *C. lambica*, *C. holmii* and most other isolates did. The gas pressure formed by yeast isolates varied more than tenfold. Only a minority of the yeasts were able to produce gas at temperatures common in the pig gut.

**Keywords:** yeasts; liquid swine diets; MALDI-TOF; biochemical identification; growth temperature
Ancom Gas Production System; *Candida krusei*; *Candida lambica*

## 1. Introduction

Yeasts, about 600 species of which are known [1], are ubiquitous in nature and can also be found on feedstuffs [2]. They pose a risk factor regarding hygiene in liquid diets associated with off-flavor and loss of nutrients [3–5]. Depending on the species or strain, as well as on the growth conditions like temperature, substrate and its aw-value (activity of water), yeasts are able to metabolize numerous sugars, starch, protein, amino acids or even fats, and therefore lead to a loss of nutrients and energy in the feed [3–5]. In pig fattening, these energy losses in the feed are particularly undesirable [4,5]. In addition, the flavor and smell of the feed can be negatively affected [6,7]. High cell counts of yeasts in liquid swine diets due to pronounced metabolic activity are often seen in the presence of easily fermentable, low molecular weight sugars [5]. Choosing maize silage for pig feed was used with the aim of feeding the pigs to increase the feeling of satiety without making them fat [8]. The relatively high initial yeast flora of the feed has to be taken into account [8]. Therefore, feed hygiene related to yeast content was of special concern.

In liquid feeds, mostly microflora develops, which is dominated by lactic acid-producing bacteria [7]. A pH-value lower than 5.0, which significantly reduces several bacteria, is often achieved

<footnote n="1">¹ Institute for Animal Nutrition, University of Veterinary Medicine Hannover, Foundation, 30173 Hannover, Germany; henrike.kuder@uzh.ch (H.K.); christian.visscher@tiho-hannover.de (C.V.); josef.kamphues@tiho-hannover.de (J.K.)</footnote>

<footnote n="2">² Institute for Microbiology, University of Veterinary Medicine Hannover, Foundation, 30173 Hannover, Germany; ute.siesenop@tiho-hannover.de</footnote>

<footnote>* Correspondence: birgit.keller@tiho-hannover.de</footnote>
in a shorter time with the use of starter cultures for fermented liquid feeds [3,9,10]. Yeasts are not only
able to stay alive but also continue growing in fermented feeds [11], even if the pH-value is 4.5 [12].

Besides these complications concerning feed composition and quality, animal health may be affected
due to the yeast content in the feed [11,13–15]. Hemorrhagic bowel syndrome (HBS), mainly caused by
yeasts [13], is supposed to be causally responsible for gastric torsion and gastrointestinal tympani [16],
being sometimes associated with liquid feeding [15]. HBS preferentially affects fattened pigs in the
second half of the fattening period [16]. Those animals most affected are, as a rule, the better developed
pigs in the group [14]. The fact that the affected animals are in excellent health makes this disease of
particular economic importance [15].

In feed analyses, yeasts, irrespective of the species, are classified as spoilage indicators in animal feed [17]. A liquid diet with more than 10⁶ cfu yeasts/g original substance (OS) is considered as significantly increased, while less than 10⁵ cfu/g feed OS in liquid feed is considered as normal [18]. On the other hand, selected yeasts are authorized feed additives in human nutrition and animal feedstuffs as they synthesize vitamin B1, B2, B6, B12, folic acid, niacin, pantothenic acid and biotin, as well as containing some minerals (potassium, sodium, calcium, zinc and iron) [1]. For swine diets, viable *S. cerevisiae* is authorized as a feed additive as intestinal flora stabilizers, digestibility enhancers and microorganisms with a minimum concentration of 1 × 10⁹ cfu/kg complete feed (88% DM) [19].

Pathogenicity factors of yeasts have been analyzed to identify high-risk yeasts and their effects on humans and animals. In their study about potential virulence of food-borne yeasts, Rajkowska et al. [20] stated that the ability to grow at 37 °C was crucial; hence, they referred to this as preliminary criterion for pathogenicity. Adaptation to pH-value was also suggested to be a key to pathogenicity, especially important for yeasts entering the digestive tract where the pH-value changes from pH 2 to pH 8 [21,22]. The ability to form biofilms also on abiotic surfaces [21] or even to colonize them is a prerequisite for colonizing the liquid feeding system, which allows the yeasts to stay alive even if the hygiene of the liquid feed was improved [23]. Stalljohann et al. [3] distinguished yeasts according to their ability to produce high or low amounts of CO₂ with regard to their pathogenicity for swine, but did not mention which species produced the high gas amounts. Such detailed information on these possible indicators of pathogenicity is provided in the present paper.

The hypothesis of this study was that different yeast species could be found in different feedstuffs.
For this reason, a comparison of biochemical differentiation and identification with MALDI-TOF was
performed to determine the method with the most reliable identification. Presumably, only distinct
species would be able to grow and to produce high amounts of gas at 37 °C.

A comparison between gas amounts produced from yeasts measured with the Ancom Gas Production System under defined conditions in a standardized Sabouraud glucose bouillon, had, to the best of the authors' knowledge, never been carried out previously. This permits a comparison of yeast isolates not only to see whether but also how much gas can be produced by yeasts within a certain time period regardless of feedstuffs. Further studies must clarify whether and to what extent these properties have an influence on the development of diseases such as HBS. These new aspects could then allow to make better predictions concerning the ability of high yeast cell counts in liquid diets to cause clinical problems.

**2. Materials and Methods**

2.1. *Sample Origin*

For our project, samples from farms with liquid feeding common to all samples were collected.
We obtained these samples either by contacting farms in the area, or from our own studies, which were
also carried out on similar farms. In total, 42 liquid feed samples were analyzed. Of these samples,
33 were submitted for diagnostic purposes to the Institute for Animal Nutrition, University of Veterinary
Medicine Hannover, Foundation, Germany. These included common liquid feeds to which no silage
had been added, referred to as liquid diets without silage (LD – S). The remaining nine samples
obtained from field trials carried out by the Institute for Animal Nutrition, additionally contained whole plant maize-silage (up to 66% DM; liquid diets with silage, LD + S). For collecting the liquid feed samples, a standard laboratory protocol was used for both the submissions and the samples from the studies. The protocol required that the samples were taken fresh, packed directly into a sterile, unbreakable vessel, filled to 2/3 at most, immediately cooled and not sent before the weekend. All samples were processed directly, or in case they arrived late in the afternoon, refrigerated and processed the following morning.

## 2.2. *Detection Techniques*

Yeasts were isolated and morphologically characterized on Sabouraud glucose agar (SAB-Agar, PO 5096A, Thermo Fisher Scientific GmbH, Bremen, Germany) and then incubated at 30 °C. Only yeasts that grew at the highest decimal dilution levels of the agar plates were considered.

### 2.2.1. Biochemical Differentiation

Biochemical differentiation of the yeast isolates was performed by ID 32 C strip (bioMérieux SA, Marcy-l'Étoile, France). This was performed in accordance with the manufacturer's instructions. The strip consisted of 32 cavities, each containing a dehydrated carbohydrate substrate, testing the assimilation by the yeast. Pure culture yeast material of 44–48 h-grown subcultures was suspended in 3 mL aqua destillatum. Turbidity was set in accordance with a McFarland standard of 2.0 using a Densitometer DEN-1B (BioSan, Riga, Lettland). From this solution, 250 µL were added to the API C medium included in the test kit. After careful vortexing, 135 µL were transferred from this liquid medium to each well of the test strip. The strip was incubated at 30 °C for 44–48 h. Yeast growth resulted in turbidity of the liquid medium in the cupules, which was visually evaluated. The obtained results were noted on a result sheet. The values corresponding to the positive reactions were then added up within groups. Three results each were added up for a group. Group values were coded into a numerical profile. This was analyzed by means of identification software (APIWEBTM, bioMérieux). The results of two of these carbohydrates, N-acetylglucosamine (NAG) and lactic acid (LAT), are examined in more detail below. Only results that received good, very good or excellent (classified as "Very good identification") ratings were evaluated. Rice agar (Thermo Fisher Scientific GmbH), incubated at 25 °C for 44–48 h, was selected for some isolates if the identification software required this deficiency medium, with a cover glass placed over the inoculum for an oxygen-reduced atmosphere.

### 2.2.2. MALDI-TOF

In the MALDI-TOF analysis, the sample (e.g., bacteria or yeasts) was ionized by a laser beam. These ions were then accelerated differently depending on their mass and charge. The time required to pass through the length of the flight tube was determined [24]. In this way, a characteristic spectrum can be generated for bacteria or fungi, which usually allows a species diagnosis [24,25]. As an advantage, less time is required for this method compared to biochemical methods [24].

MALDI-TOF analyses were performed on a Microflex LT/SH MALDI-MS Biotyper (Bruker Daltonik GmbH, Bremen, Germany) with the direct smear method (MALDI-DS) and with a formic acid-ethanol extraction (MALDI-EX). The latter is used for hardly soluble bacteria or yeasts.

For MALDI-DS, direct on-plate smearing was performed with yeasts incubated 44–48 h on an SAB-plate at 30 °C. Small amounts of colony material of every isolate were evenly applied with a toothpick to two circles of the target plate (8280800 MSP 96 Target polished steel BC, Bruker Daltonik). After air drying the sample material at room temperature for five to ten minutes, 1 µL of an α-cyano-4-hydroxycinnamic acid (HCCA, 19182, Sigma Aldrich Inc., St. Louis, MO, USA) matrix solution was applied to each circle of the target plate and dried again at room temperature.

For MALDI-EX, a 1 µL loop of 20–24 h-grown yeast material on an SAB-plate was vortexed in 300 µL deionized water at 30 °C; 900 µL of ethanol absolute, HPLC-grade, was added and it was vortexed again. The samples were pelleted by centrifuging for two minutes (13,000 U/min;
approximately 3000× g); the supernatant was discarded, centrifuged and then the supernatant was discarded again. The pellet in the Eppendorf tube was air dried for three to five minutes and resuspended in 40 µL 70% formic acid. After adding 40 µL acetonitrile (ACN, Acetonitrile HPLC Gradient Grade, 20060.320 VWR International Inc., Radnor, PA, USA), this was followed by the same centrifugation step as above. Eppendorf tubes were taken carefully out of the centrifuge and 1 µL of the supernatant was applied to a circle of the target and air dried for 5 min at room temperature. Immediately afterwards, 1 µL of the same matrix solution that had been previously used for the MALDI-DS method was applied to a circle of the target and air dried for 5 min at room temperature.

For the control of both methods, 1 µL BTS (Bacterial Test-Standard, 8290190 Bruker IVD Bacterial Test-Standard) was placed on every target plate and, additionally, a control strain, *E. coli* DH5 α, was tested on every target plate in two circles.

Each sample was analyzed by a Microflex LT/SH MALDI-TOF MS in the linear mode across a mass-to-charge ratio range between 2000 and 20,000. The obtained data were analyzed automatically by using the MBT Compass Library BDAL and MBT Flex Control software, BTyp2.0-Sec.library, 1.0. Every strain was tested in two circles, with a decreased concentration in the second circle. The result from the two circles that achieved the higher score value was used for analysis.

The identification cut-off scores were interpreted as per Bruker's recommendation scores as follows: obtaining scoring thresholds between 2.30 and 3.0 suggested highly probable species identification; 2.00-2.29, probable species identification; 1.70-1.99, identification at the genus level was postulated; whereas cut-off scores <1.70 indicated no reliable identification.

### 2.2.3. Method Comparison

The results "excellent identification" and "very good identification" adopted in the software APIWEBTM were equated to "highly probable species identification" from the MALDI-TOF analysis. "Good identification" was equated to "probable species identification"; "probable genus identification" was equated to "good identification at genus level"; and "doubtful profile" and "no identification" were equated to "unacceptable profile".

### 2.3. Temperature Comparison

After cultivating the yeasts from liquid feeds on SAB-agar and subcultivating a single colony, a subculture on two SAB-agar plates was produced for the temperature comparison. One agar plate each was cultivated in an incubator at either 25 °C or 37 °C for 48 h. The colony growth (diameter) was compared visually.

### 2.4. PH-Value

Using a calibrated glass electrode (HI 2211 pH/ORP Meter, Hanna Instruments Inc., Woonsocket, RI, USA), pH-values were measured. Results of 25/33 LD - S and 16/17 LD + S were obtained immediately after dividing the samples for further microbiological testing.

### 2.5. Gas Pressure Measurement

To measure yeast activity in gas production, 40 selected yeast isolates were examined for 24 h at 37 °C with the ANKOM RF Gas Production System (ANCOM Technology, Macedon, NY, USA), which remotes pressure under controlled pressure measurements and records these on a standard Excel spreadsheet. Gas production curves were generated. In 100 mL glass bottles containing 100 mL Sabouraud glucose broth (SAB-B; CM 0147 B, Thermo Fisher Scientific GmbH), a 10 µL yeast suspension, McFarland standard 0.3 (Densitometer DEN-1B, Biosan, Riga, Lettland), in a physiologic salt solution, was added. SAB-B conforms to the parameters from the harmonized EP/USP/JP Microbial Limit Testing for the microbial enumeration tests and tests for specified microorganisms. The bottles were placed on magnetic stirring panels (MIXdrive magnetic e motion with Mixcontrol 20, 2mag AG, Munich, Germany) for permanent mixing at 210 rpm. Gas pressure was measured over a 24-h period, taking into
account that feed normally does not normally stay longer in animals' gastrointestinal tract. Line charts of the cumulated gas production were generated with the Ancom Gas Pressure Monitor. Each isolate was tested at least twice.

The results were divided into two groups. A very small pressure increase (<100 mbar) at the beginning was also observed if no further gas was produced thereafter. If less than 100 mbar of cumulated pressure within 24 h was observed, the result was determined as negative. If a yeast was able to produce more than 800 mbar within 24 h, the result was determined as gas production. This value was defined based on the results found, because no yeast produced gas amounts between 100 and 800 mbar.

## 2.6. Statistics

Data were statistically analyzed using the SAS® Enterprise Guide® (version 7.1, Fa. SAS Institute Inc. Cary, NC, USA). Pearson's chi-square homogeneity test and Fisher's exact test, used to analyze qualitative analytical characteristics, were applied to check if a yeast was found significantly more with one feed; if one of the identification tests found significantly more reliable results; whether yeasts grew better at a certain temperature; or if yeasts built up a distinct pressure at 37 °C within 24 h. Fisher's exact test was used especially for low absolute frequencies.

# 3. Results

In total, 95 morphologically different yeast colonies (color, size and surface structure) were isolated from a total of 42 feed samples. In each feed sample, one to four different yeast species were found.

## 3.1. Identification

In spite of a different morphology, yeast identification led to the same result for 15 yeast isolates. Yeasts diagnosed twice in the same sample were not considered in the evaluation of the number of yeast species or species of yeast-like organisms that were found in the respective feed sample.

The isolates originated from six genera (*Candida* (76.25%; *n* = 61), *Geotrichum* (7.5%, *n* = 6), *Trichosporon* (3.75%, *n* = 3), *Saprochaete* (2.5%, *n* = 2), *Rhodotorula* (1.25%, *n* = 1), *Pichia* (1.25%, *n* = 1) and non-identified yeasts (7.5%, *n* = 6)). A total of 19 different yeast species were identified.

In Table 1, the most often isolated species are listed. Less frequently isolated (one to three times) were *C. pelliculosa* (*n* = 3, 2× LD + S, 1× LD - S), *C. valida* (*n* = 3; 2× LD - S, 1× LD + S), *Sap. suaveolens* (*n* = 2, both from LD - S), *C. rugosa* (*n* = 2, both from LD - S), *C. kefyr* (*n* = 1, LD - S), *C. variabilis* (*n* = 1, LD - S), *C. spherica* (*n* = 1, LD - S), *T. asahii* (*n* = 1, LD + S), *T. coremiiforme* (*n* = 1, LD - S), *T. laibachii* (*n* = 1, LD - S), *P. manshurica* (*n* = 1, LD - S), *Rhodotorula mucilaginosa* (*n* = 1, LD - S) and *Candida* spp. (*n* = 1, LD - S). Six isolates (5× in LD - S; 1× in LD + S) were not reliably identified.

<table><caption>**Table 1.** Often isolated yeast species.</caption><thead><tr><th>Liquid Diet</th><th>Often Isolated Yeast Species in %</th></tr></thead><tbody><tr><td>LD - S<br>(n = 63)</td><td>C. krusei (23.8), C. holmii (12.7), C. humilis (7.9), isolates not identified (7.9), C. lambica (6.3),<br>S. cerevisiae (6.3), G. silvicola (6.3)</td></tr><tr><td>LD + S<br>(n = 17)</td><td>C. lambica (29.4), C. krusei (23.5), C. holmii (17.6), C. pelliculosa (11.8), S. cerevisiae (5.9),<br>C. valida (5.9), isolates not identified (5.9)</td></tr><tr><td>Total<br>(n = 80)</td><td>C. krusei (23.75), C. holmii (13.75), C. lambica (11.25), isolates not identified (7.5),<br>C. humilis (6.25), S. cerevisiae (6.25), G. silvicola (6.25)</td></tr></tbody></table>

C. = *Candida*; S. = *Saccharomyces*; G. = *Geotrichum*.

Only *C. lambica* was determined to have a significantly higher incidence in LD + S (*p* < 0.0126). The occurrence in LD - S or LD + S did not significantly change (Figure 1).
<figure><img src="image_21.png" /><figcaption>**Figure 1.** Incidence of yeasts according to their feed origin. An asterisk (*) indicates a significant difference (* *p* < 0.05). C. = *Candida*; S. = *Saccharomyces*.</figcaption></figure>

If, despite different colony morphology, two identical yeast species were diagnosed in one feed sample, only one yeast species was further examined. In 33 LD – S, 7/70 yeast colonies with different morphologies were selected, but identified as the same yeast species within the same diet. In LD + S, 8/25 morphologically different yeasts were identified as the same yeast within the same diet. Therefore, 63 yeasts from LD – S and 17 yeast from LD + S were further examined. As a result of this, significantly more different yeast morphologies were observed from the LD + S samples (*p* < 0.0211).

On the other hand, no differences between the two feeding groups were found regarding the actual (not morphologically) different yeasts. In both diet groups, an average of 1.9 different yeasts per feed sample were diagnosed: 63 different yeasts of 33 LD – S diets, and 17 different yeasts of nine LD + S diets.

### 3.1.1. Method Comparison

The comparison of methods revealed the most reliable results with MALDI-EX (78.75% reliable species identification), closely followed by ID32C with 75.0% reliable results. These test results did not differ statistically significantly (*p* < 0.6762). Among the reliable results, ID32C provided differentiation of ten isolates (8%), seven isolates with species identification and three isolates were at least identified up to genus level, which MALDI-EX was not able to differentiate. Hence, taking the results of both methods together, 71 (88.75%) of all isolates were identified up to species level. A probable identification or rather identification only at genus level was possible in three (3.75%) isolates. No identification with any of the three diagnostic methods was made for 6/80 (7.5%) yeast isolates. Only few results (37.5%) were provided by MALDI-DS (Table 2). When evaluating the reliable results, both MALDI-EX and ID32C differed significantly (both *p* < 0.001) from the MALDI-DS. No identification with any of the three diagnostic methods was made for 6/80 yeast isolates (7.5%). For better comparison, the results of all three methods were presented together in one table. However, the yeast names (according to their teleomorphic or anamorphic growth) given in the results varied sometimes according to the evaluation software.

Some yeasts were diagnosed more accurately with one or the other method. Five isolates differentiated with MALDI-EX as *C. humilis* were diagnosed as *C. holmii* in ID 32 C. *C. humilis* was not included in the database used to evaluate the ID 32 C. Both of these species had very similar biochemical reactions. Therefore, identification with MALDI-EX was chosen to be more accurate.

The two yeasts identified as *C. pararugosa* in MALDI-EX were identified as *C. rugosa* in ID 32 C. Results in MALDI-EX were only 1.88 and 1.79, respectively. Therefore, they had to be named according to ID 32 C, where the results for both isolates revealed very good identification scores, namely, 99.8% and 99.5% for *C. rugosa*. On the other hand, *C. pararugosa* was not included in the database used to evaluate the ID 32 C.
<table><caption>**Table 2.** Comparison of performance of methods with 80 yeast isolates in %.</caption><thead><tr><th>Evaluation Score of<br>MALDI-TOF</th><th>MALDI-EX</th><th>MALDI-DS</th><th>ID-32C</th></tr></thead><tbody><tr><td>Highly probable species<br>identification (&gt;2.3)</td><td>38.75 (n = 31)</td><td>78.75 *<sup>a</sup><br>(n = 63)</td><td>5.0 (n = 4)</td><td>47.5 (n = 38)</td></tr><tr><td>Probable species<br>identification (2.0–2.29)</td><td>40.0 (n = 32)</td><td></td><td>32.5 (n = 26)</td><td>27.5 (n = 22)</td></tr><tr><td>Probable genus<br>identification (1.7–1.99)</td><td>7.5 (n = 6)</td><td>21.25 ***<br>(n = 17)</td><td>33.75 (n = 27)</td><td>3.75 (n = 3)</td></tr><tr><td>No identification (&lt;1.7)</td><td>13.75 (n = 11)</td><td></td><td>28.75 (n = 23)</td><td>21.25 (n = 17)</td></tr></tbody></table>

<sup>a,b</sup> Different superscripts differ significantly in a row. * Reliable identification: Highly probable species identification and probable species identification; ** Reliable identification: Good and very good identification together; ***: No species identification.

Even with the formic acid extraction method, the slimy and red growing *Rhodotorula* (R.) mucilaginosa could not be detected with MALDI-TOF; there was no reliable identification, although it was registered in the database.

In the ID 32 C, two isolates were diagnosed as *Cryptococcus* (C. curvatus and C. laurentii), which showed no mucus capsule in the Indian ink preparation, but showed hyphal growth, arthro- and blastosporogenesis on rice agar under the cover glass (Figure 2), and were identified in MALDI-EX as *T. coremiiforme* and *T. laibachii* (Table 3). On the other hand, two *T. asahii* isolates could be recognized well or very well by both methods.

<figure><img src="image_22.png" /><figcaption>**Figure 2.** *Trichophyton coremiiforme* on rice agar: arthrospores (a) and blastospores (b). 400× magnification.</figcaption></figure>

<table><caption>**Table 3.** Isolates differentially diagnosed with MALDI-EX and ID 32C.</caption><thead><tr><th>Number of Isolates</th><th>MALDI-EX</th><th>ID 32C</th></tr></thead><tbody><tr><td>5</td><td><i>Candida humilis</i></td><td><i>Candida holmii</i></td></tr><tr><td>2</td><td><i>Candida pararugosa</i></td><td><i>Candida rugosa</i></td></tr><tr><td>1</td><td>No identification</td><td><i>Rhodotorula mucilaginosa</i></td></tr><tr><td>1</td><td><i>Trichosporon coremiiforme</i></td><td><i>Cryptococcus curvatus</i></td></tr><tr><td>1</td><td><i>Trichosporon laibachii</i></td><td><i>Cryptococcus laurentii</i></td></tr></tbody></table>

Yeasts highlighted in bold represent the selected diagnoses.

Six isolates had a score between 1.79 and 1.98 in the MALDI-EX. Of these isolates, *S. cerevisiae* (score 1.98) and *C. holmii* (score 1.97) had the same result in ID32C (see Table 4), with very good identification. Furthermore, two isolates were identified as *C. pararugosa* in the MALDI-EX (with scores
1.88 and 1.63). *C. pararugosa* was not included in the identification software (APIWEBTM, bioMérieux). The remaining two isolates consisted of *Saprochaete* (*Sap.*) *suaveolens* (score 1.88), which was diagnosed as a *Geotrichum* spp. in ID32C, and *Pichia occidentalis* (score 1.83), diagnosed with 99.7% as *C. krusei* in ID32C. Bearing in mind that *Sap. suaveolens* was formerly diagnosed as *Geotrichum fragrans*, the diagnosis made by MALDI-EX was most likely the one with the currently correct name. The name of the yeast in ID32C was probably out of date; the yeast was still correctly identified. Only one isolate was differently identified by the two methods as *P. occidentalis* (MALDI-TOF) and *C. krusei* (ID32C; Table 4).

<table><caption>**Table 4.** Isolates in MALDI-EX rated as probable genus identification (1.70 and 1.99).</caption><thead><tr><th colspan="2">MALDI-EX</th><th colspan="2">ID32C</th></tr><tr><th>Diagnosis</th><th>Score</th><th>Diagnosis</th><th>Identification</th></tr></thead><tbody><tr><td><i>C. pararugosa</i></td><td>1.88</td><td><i>C. rugosa</i></td><td>very good</td></tr><tr><td><i>Sap. suaveolens</i></td><td>1.88</td><td><i>Geotrichum spp.</i></td><td>very good</td></tr><tr><td><i>P. occidentalis</i></td><td>1.83</td><td><i>C. krusei</i></td><td>very good</td></tr><tr><td><i>C. holmii</i></td><td>1.97</td><td><i>C. holmii</i></td><td>very good</td></tr><tr><td><i>C. pararugosa</i></td><td>1.79</td><td><i>C. rugosa</i></td><td>very good</td></tr><tr><td><i>S. cerevisiae</i></td><td>1.98</td><td><i>S. cerevisiae</i></td><td>very good</td></tr></tbody></table>

Yeasts in bold represent the selected diagnoses *C. = Candida*; *Sap. = Saprochaete*; *P. = Pichia*; *S. = Saccharomyces*.

### 3.1.2. Biochemical Reactions

In total, all the investigated yeasts were able to assimilate glucose and no yeast grew in the cupule where no substrate was present (cupule F). These reactions were considered as the positive growth control or negative control (no contamination). From the large number of biochemical reactions, two of them will be examined in more detail in the following section, since the ability of the yeasts to metabolize them could be an advantage, especially in maize silage.

#### Metabolization of N-Acetylglucosamine (NAG)

In the ID 32 C-Test, 27 yeasts from a total of 63 yeasts in the LD – S samples were able to metabolize N-acetylglucosamine and 36 yeasts were not. In LD + S, 11 yeasts were able to build N-acetylglucosamine and six yeasts were not. Despite the fact that this is insignificant (*p* = 0.0788), the ability to build NAG was more often seen in yeasts from LD + S.

#### Metabolization of Lactic Acid (LAT)

In the ID 32 C-Test, 36 yeasts of a total of 63 yeasts in the LD – S samples were able to metabolize lactic acid and 27 yeasts were not. In LD + S, 12 yeasts were able to metabolize lactic acid and five yeasts were not. These results were insignificant (*p* = 0.4075). Nevertheless, the ability to metabolize lactic acid could be found more often with yeasts that had to stay alive or even grow in maize silage than for yeasts in LD – S.

### 3.2. Temperature Comparison

Most yeast isolates (n = 47; 58.75%) formed larger colonies at 25 °C than at 37 °C, among them, 14 isolates (17.5%) did not grow at 37 °C at all. These included ten isolates from LD – S (3× C. holmii, 1× each for C. humilis, C. lambica, T. laibachii, C. pelliculosa, Geotrichum spp., S. cerevisiae and an isolate not identified) and four from LD + S (3× C. holmii and 1× isolate not identified). Among all yeasts, which grew better or only at 25 °C, many isolates of C. holmii, C. humilis and C. lambica were found. Only 23.75% of isolates grew better at 3 °C compared to 25 °C; this was often the case for C. krusei (12/19 isolates) and S. cerevisiae (4/5 isolates). Additionally, C. kefyr (1/1), C. holmii (1/11 isolates) and one isolate that could not be identified (1/6) showed better growth at 37 °C. All S. cerevisiae isolates were harvested from LD – S samples. Larger colonies at 37 °C were formed from nine C. krusei isolates
gained from LD – S and three from LD + S, while three isolates from LD – S and one from LS + S formed larger colonies at 25 °C; one isolate showed equal colony growth at 25 °C or 37 °C. Hence, for *C. krusei*, no difference was observed, regardless of which feed it was isolated from. When comparing both feeds, it was noticeable that especially yeasts isolated from LD + S grew poorly at 37 °C (see Table 5). Better growth at 37 °C than at 25 °C for yeasts harvested from LD + S was only seen for three isolates, all of which were *C. krusei*. However, there was no statistically significant difference (*p* < 0.3862) between the two feed sources concerning growth performance (colony size) of the yeasts at either of the temperatures.

<table><caption>**Table 5.** Growth performance of the yeasts at different temperatures depending on feed.</caption><thead><tr><th>Yeast Isolates</th><th>No Growth at 37 °C</th><th>Better Growth at 25 °C</th><th>25 °C = 37 °C</th><th>Better Growth at 37 °C</th></tr></thead><tbody><tr><td>LD – S (n = 63)</td><td>10 (15.9%)</td><td>24 (38.1%)</td><td>13 (20.6%)</td><td>16 (25.4%)</td></tr><tr><td>LD + S (n = 17)</td><td>4 (23.5%)</td><td>9 (52.9%)</td><td>1 (5.9%)</td><td>3 (17.6%)</td></tr><tr><td>Total (n = 80)</td><td>14 (17.5%)</td><td>33 (41.25%)</td><td>14 (17.5%)</td><td>19 (23.75%)</td></tr></tbody></table>

### 3.3. pH-Value in Liquid Swine Diets

The pH-values of LD – S (n 25/33) ranged from 3.87–5.78, while the pH-values of LD + S (n = 8/9) achieved higher pH-values ranging from pH 4.79 to pH 5.61. Related to the feed origin, 44 yeasts isolated from LD – S were harvested from liquid feed, with an average pH-value of 4.59. Yeasts gained from LD + S were harvested from liquid feed, with an average pH-value of 5.51. *C. krusei* was isolated from liquid swine diets with the lowest (pH 3.87) and highest pH-values (pH 5.78) as well. *C. humilis* and *C. holmii* were found in diets with lower pH-values (pH 3.9 to pH 5.11), whereas *C. lambica* was isolated once from a diet with a pH-value of 4.45. However, other isolates were harvested from diets with higher pH-values (ranging from pH 4.97 to pH 5.61).

### 3.4. Gas Production

The results of the duplicate testing of each isolate showed small deviations, possibly caused by small differences in cell counts at the beginning as well as differences in replication time and counts of spores formed by each yeast cell during the 24-h incubation period.

Only two groups were formed: yeasts that produced virtually no gas within 24 h at 37 °C and yeasts that produced more than 800 mbar. A further subdivision of the yeasts into groups producing little or a lot of gas was omitted, because too little information was available from the literature as to which quantities could be classified as a lot or little.

No yeast produced gas amounts between 100 mbar and 800 mbar. More yeasts harvested from LD – S produced gas than yeasts that were found in LD + S, but the quantity was not significant (*p* < 0.2216).

Gas production with more than 800 mbar was observed for a total of 13 (40.6%) isolates (Table 6): 10/11 *C. krusei*-isolates, 2/3 *S. cerevisiae*-isolates, 1/1 *C. kefyr* and 1/1 *C. humilis*. *C. kefyr* formed the highest gas pressure, with 10,419 mbar, followed by the *C. krusei* isolates (7134.5, 7073, 6659, 6487, 6383, 6164.5, 4839, 4147.5, 3954.5 and 3940.5 mbar), both isolates of *S. cerevisiae* (1160.5 and 1466.5 mbar) and *C. humilis* (888 mbar). Eleven of these isolates grew better at 37 °C than at 25 °C within 48 h (see Section 3.2). Nonetheless, two isolates, which also grew better at 37 °C, were not able to produce more than 100 mbar gas in 24 h. These two yeasts were one *C. holmii* and one *C. krusei* isolate harvested from LD + S. The latter one produced these high gas quantities only after a 40 h incubation time. On the other hand, one isolate of *C. krusei*, harvested from LD – S, which grew better at 25 °C than at 37 °C, nevertheless produced 3954.5 mbar gas within 24 h. Only one yeast isolate from LD + S could produce significant quantities of gas under the abovenamed circumstances within 24 h (Table 4).
<table><caption>**Table 6.** Gas production (mbar) at 37 °C within 24 h.</caption><thead><tr><th>Sample</th><th>Number</th><th>&lt;100 mbar</th><th>&gt;800 mbar</th></tr></thead><tbody><tr><td>LD - S</td><td>n = 32</td><td>19 (59.4%)</td><td>13 (40.6%)</td></tr><tr><td>LD + S</td><td>n = 8</td><td>7 (87.5%)</td><td>1 (12.5%)</td></tr><tr><td>total</td><td>n = 40</td><td>26 (65.0%)</td><td>14 (35.0%)</td></tr></tbody></table>

Gas production less than 100 mbar was demonstrated in 5/5 C. lambica, 4/4 C. holmii, 3/3 Trichosporon spp., 3/3 G. silvicola, 2/2 C. pelliculosa, 2/2 C. rugosa, 1/1 Sap. suaveolens, 1/1 Candida spp., 1/1 C. valida, 1/1 P. manshurica, 1/1 C. spherica, 1/3 S. cerevisiae and 1/11 C. krusei. Yeasts that showed some signs of growth or grew particularly well at 37 °C within 48 h showed different reactions. Some isolates needed more than 24 h to produce high amounts of gas (Figure 3). Some isolates did not produce amounts greater than 100 mbar, even within a given 60-h period. In Figure 3 such yeasts are C. valida and C. lambica. Therefore, their curves in Figure 3 are so close to the x-axis they are hardly visable, just like the curve of the control (sterile SAB-bouillon without yeast isolate).

<figure><img src="image_23.png" /><figcaption>**Figure 3.** Cumulative pressure graph C. = Candida; C. krusei, LD - S = isolate of C. krusei from LD - S; C. krusei, LD + = isolate of C. krusei from LD + S.</figcaption></figure>

The highest correlation between yeast growth at 25 °C and 37 °C within 48 h (see Section 3.2) and gas formation was found in yeasts that did not grow at 37 °C at all. None of these yeasts (8/8) were able to produce gas during the 24-h incubation period at 37 °C.

# 4. Discussion

Increased numbers of yeasts in liquid feed for pigs has been the subject of some publications in previous years [10–12,16,26–28]. Some publications compared yeasts in liquid feed for pigs with different feed composition taken from different stables or with and without the addition of starter cultures. However, none have yet compared the yeasts in liquid swine diets with and without maize silage, with the identification results derived from two methods.

## 4.1. Identification

As in most other studies, the genus Candida (C.) was found most often in our research study. C. krusei was found most often in LD - S and in all samples as a whole, whereas C. lambica was found most often in LD + S. In addition to these two species, another 17 yeast species from six genera were also diagnosed in this study.
### 4.1.1. *C. krusei*

Overall, the most often found yeast in this study, *C. krusei*, was isolated from feed samples with the lowest and highest pH-values. From the literature, it is known to grow at low pH-values [29], ferment up to a pH-value of 3.6 [30] and can also form biofilms [31]. These properties are likely to be beneficial for yeast persistence in the liquid feed and the feeding system. In part, *C. krusei* is capable of pseudohyphae formation and mostly of growing at 37 °C, both characteristics that could contribute to HBS.

*C. krusei* is responsible for about 2% of yeast infections caused by *Candida* species in humans [32]. *Pichia kudriavzevii*, *Issatchenkia orientalis* and *Candida glycerinogenes* are proven to be the same yeast with collinear genomes 99.6% identical in DNA sequence. Under these names, the yeast is used for industrial-scale production of glycerol and succinate, and is also used to make some fermented foods [32]. The latter use in fermented foods also explains the frequent occurrence in liquid feed for pigs, which also has a low pH value (see Section 3.3).

### 4.1.2. *C. lambica*

The significantly higher presence of *C. lambica* in LD + S is possibly due to maize silage in the feed but could also be due to the lower storage temperature of the maize silage outdoors during winter [33]. As LD + S samples were gained from the institute's own research projects, it is known that animals did not develop HBS or any other disease and that they ate a lot more with the ad libitum feeding of LD + S in comparison to the previous feed intake with commercial feed (Jörling, 2017) [8]. This was observed, although the yeast content of the feed was temporarily more than $1 \times 10^8$ cfu/g feed (Jörling, personal observations, results of which have not yet been published). Olstorpe et al. [26] discovered that *Pichia fermentans* (*C. lambica*) was dominant in all their experiments. They assumed that *C. lambica* was able to improve palatability as it has been described to improve the flavor composition during wine- and cheese-making [26]. Presumably, the yeast species within the diet might be more important than the orientation values, which are the same for all yeasts when the hygiene status of the liquid diets is under debate.

### 4.1.3. Yeasts from Liquid Diets for Pigs

In the present study, mostly *C. krusei*, *C. holmii*, *C. lambica*, *S. cerevisiae*, *C. humilis* and *Geotrichum* spp. were identified (see Section 3.1, Table 1). Together, they accounted for 66.5% of all yeast isolates. Other species could not be identified (7.5%, Table 1) or were only detected in lower proportions (26%; see Section 3.1).

Middelhoven et al. [33] observed, in whole-crop maize ensiled for two weeks, similar yeast species compared to those found in LD + S. They predominately found *C. holmii*, *C. lambica*, *C. milleri* (current name: *C. humilis*), *Hansenula anomala* (current name: *Wickerhamomyces anomalis*, anamorph: *C. pelliculosa*) and *Saccharomyces dairensis* [33], whereby only the latter yeast did not occur in our study. The comparability of the yeast species in both studies could indicate that it is not so much the storage over winter but rather the substrate that influences the yeast occurrence.

The biochemical profiles of both yeasts are very similar and therefore sometimes misidentified [34]. Both yeasts are able to assimilate mostly glucose, lactose, glycerol, inositol and N-acetylglucosamine, while xylose is only metabolized from *C. lambica*. On the other hand, the next frequently identified yeasts, *C. holmii*, *C. humilis* and *S. cerevisiae*, cannot perform this metabolic function, with the exception of glucose. Instead, they metabolize galactose and raffinose, and, in part, trehalose (*C. holmii* and *C. humilis*), sucrose (*C. holmii* and partly also *S. cerevisiae*) as well as maltose (*S. cerevisiae*). This could suggest that the assimilative capacities of the yeasts are not essential for their presence or absence in different liquid feeds for pigs.

Likewise, many different yeasts were identified in studies on yeast determination from liquid feed samples for pigs, and different species dominated in the different feed samples [6,26–28].
Similarities to the isolated yeasts in the present study were observed in the studies by Olstorpe et al. [26], who examined liquid feeds based on a cereal grain mix and wet wheat distiller's grain with and without starter cultures. Without starter cultures, they observed *P. fermentans* (*C. lambica*), *C. pararugosa*, *C. rugosa*, *P. galeiformis* (current name: *P. mandshurica*), *T. asahii*, *Issatchenkia orientalis* (*C. krusei*, *P. kurdriavzevii*), *C. ethanolica* and *C. vini*. These yeasts were found in the present study as well, except the last three mentioned ones.

Olstorpe et al. [26] isolated *C. kefyr* from wheat-based liquid feed as the dominant yeast, which was isolated only once in the present study. This previous publication also found *C. krusei*, *C. pelliculosa* and *Pichia membranaefaciens* (*C. valida*). Plumed-Ferrer and Wright [35] most frequently observed *K. exigua* (*C. humilis*), *Debaromyces hansenii* and *Pichia deserticola* in fresh batches of liquid feed, of which only *K. exigua* was often observed in the present study. On the other hand, in this previous study, other yeasts were also isolated, such as *Pichia kurdriavzevii* (*C. krusei*), *S. exiguous* (*C. holmii*), *Pichia membranaefaciens* (*C. valida*) and *Wickerhamomyces anomalus* (*C. pelliculosa*), which were identified in the present study, too.

Significantly more morphologically different yeasts were observed in LD + S (see Section 3.1), which had lower pH-values than conventional feed and were stored outdoors during winter, which, as a consequence, were exposed to changing temperatures. These different colonial morphologies could be a result of changing environmental conditions, as explained in previous publications [36,37].

The genera *Geotrichum* and *Trichosporon* are classified as yeast or yeast-like organisms, but *Geotrichum* was formerly classified as a mold [38–40]. The colony morphology is very similar to other yeasts and therefore was described in many previous studies concerning yeasts in liquid swine diets [25,27,28], so that comparability with other studies is possible. *Saprochaete suaveolens*, formerly classified as *Geotrichum fragrans*, is also classified as a yeast or yeast-like organism (mycobank.org [41]). Hereafter, for the sake of simplicity, all genera are referred to as yeasts, even if the term yeast or yeast-like organism would be more accurate.

#### 4.1.4. Method Comparison

The present study compared different methods for identifying yeasts to find the best method for the chosen substrate and the yeasts contained in it. From previous studies [6,10,26,28,42], it was known that many tests for identifying yeasts from the environment produce fewer results than those from clinical material [43–45]. Additionally, different databases on which different test procedures are based also influence the obtained results [45,46].

For clinical samples consisting mainly of *Candida* spp., the method of MALDI-TOF outperformed the diagnosis capacities of the phenotypic tests by reducing the delay in results and improving the reliable identification rate at species level [43]. On the other hand, this method requires significantly higher acquisition costs for the equipment. Therefore, this method was compared with the ID 32 C test, which has virtually no purchase costs.

### ID32C

In our study, 5.2% less reliable results were observed with the ID 32 C test in comparison to MALDI-EX. Nevertheless, in individual cases, correct identification could only be made with this simple biochemical method (see Table 4). *C. rugosa* was twice identified with more than 99.5% accuracy as "very good identification", while MALDI-EX identified these two yeast isolates as *C. pararugosa*. The latter was not included in the ID32C-database. Considering the fact that several authors [44,45,47] proposed a lower identification score for the yeast identification with MALDI (see below), perhaps the MALDI results are the correct ones. In the case of *Geotrichum* spp. and *Saprochaete suaveolens*, the situation is similar. *Saprochaete suaveolens* was not included in the database of the ID32C test. Comparable to the finding in the present study, namely that *Rhodotorula* was better identified with ID32C, Olstorpe et al. [6] reported that, with the applied PCR fingerprinting, two *Rhodotorula glutinis* isolates were incorrectly classified as *Cryptococcus satoi* or *Pichia membranaefaciens*, but correctly identified with ID32C.
The ID32C test can be easily performed in any laboratory and does not require an expensive device. In addition to species identification, the biochemical test has the advantage of showing which enzymes can be produced by the respective yeast isolate. This, in turn, could allow or exclude opportunities for identifying which feed components could be metabolized by the yeast.

Selected Biochemical Reactions of the ID32C-Test

**Metabolization of N-Acetylglucosamine (NAG):** More yeasts from LD + S, even if not significant, were able to metabolize the amino sugar NAG. This is the monomeric constituent of chitin, which is one of the most abundant renewable resources found in nature [48]. The uptake of NAG into the yeast cell, its metabolites in the cell and conversion to cell wall formation have already been described for various yeasts [48]. The cell wall reinforced by NAG (chitin) offers protection against low pH-values in the environment [22]. Although the pH-values in the LD – S were not significantly lower than those of LD + S, the prolonged period of survival in silage (see below) may have led to the ability of yeasts to metabolize NAG.

**Metabolization of Lactic Acid (LAT):** More yeasts from LD + S were able to metabolize lactic acid. As this finding is not statistically significant, the ability to metabolize lactic acid obviously is not a prerequisite for yeasts in LD + S. Lactic acid bacteria are the predominant group of bacteria found in maize silages, and are able to multiply in liquid feed, lactic acid being a main product of their metabolism [49]. Maize silage used for LD + S in the present study was kept outdoors during winter and early spring until it was fed to the animals in late spring and early summer. Being able to use a substrate present in the environment is presumed to be an advantage for yeasts [50,51], which have to survive in these conditions for a long time.

**MALDI-DS:** The less time-consuming and less expensive MALDI-DS reduced the identification rate significantly ($p < 0.001$) by more than half compared to MALDI-EX (37.5% vs. 78.75% reliable identification). Thus, the use of this method is clearly limited, at least if different yeasts are to be identified from environmental samples. In contrast to bacteria, yeasts possess a thick and chitinous cell wall, which might lead to the difficulties encountered with the MALDI-DS method [52].

**MALDI-EX:** In our study, 78.75% of the 80 different yeasts could be identified by MALDI-EX and 75.0% by the ID 32 C. While 11 isolates were not identified at all, six isolates achieved only probable results at the genus level (see Table 4). An incorrect diagnosis was observed only once, mistakenly identifying *P. occidentalis* instead of *C. krusei* (see Section 3.1.1).

A comparison of identification of 96 foodborne yeasts with MALDI-TOF and two conventional tests, of which one was ID 32 C, was made by Pavlovic et al. 2014 [53]. In their study, more yeast isolates could be identified with MALDI-TOF than with the ID32C test, too.

The identification rate of the different methods in the present study was comparable to those of others in which yeasts were isolated from the environment rather than from clinical material [6,54–56]. As already shown by these and other authors [1,57], none of the methods were capable of reliably detecting all yeast isolates from the liquid feed. Many authors attribute these differentiation failures to the background of the ID32C, MALDI-TOF and other commercially available tests, as these were developed and established for clinically relevant yeasts in human beings and not for yeasts in animal feed [1,45,57]. None of the available methods can be considered as the golden standard for the differentiation of yeasts from liquid feeds. With respect to the low examination costs, low workload, fast availability of results and available databases, which means the highest rate of correct identification, the different methods exhibit advantages and disadvantages.

Although MALDI-EX was the best method for gaining the most reliable identification results in this study, it has to be considered that results from this method are only as good as the underlying database [46]. Vlek et al. [46] identified 61.5% of their yeasts from human patients using the Bruker Daltonic database (BDAL), but improved their identification rate up to 86.8% by adding their database with the in-house database from the Centraalbureau voor Schimmelcultures (Central Bureau for Fungal Cultures) (BDAL + CBS in-house). This allows the assumption to be made that even more yeasts will
be identified with this method in the future, if correspondingly relevant data continue to be added, especially for the non-clinical yeasts found in the surroundings. An improvement in the identification results of 845 environmental yeasts by one third was also described by Augustini et al. [45] after developing a supplementary database.

Besides the databases as reason for missing reliable yeast identification, Augustini et al. [45] stated that identification scores <2.00 are not able to unequivocally affirm that the identification at species level is unreliable. They cited studies that showed identification results under 2.00, but with correct identifications. This observation was underlined in the studies by Tan et al. [44]. Repeating MALDI-TOF attempts in 10.2% of the yeast isolates, which had indicated spectral scores as being unacceptable on the first attempt (scores < 2.00), resulted in acceptable scores (>2.00). Most of these achieved a correct identification on the first attempt [44]. The authors concluded that lowering the identification score from <2.00 to <1.70 could reduce the repetition rate [44]. With a cut-off of <1.70, Lee et al. [50] also improved the identification rate of their 284 pathogenic yeasts from clinical samples compared to the required cut-off value of >2.00 [49]. When comparing the results of two different MALDI-TOF systems (Biotyper from Bruker and ASTA MALDI-TOF MS), Lee at al. [50] found that only 39.5% of the isolates with confirmed identification with molecular sequencing met the cut-off score in both systems. The majority of the isolates (58.6%) ranged between 1.70 and 2.00 when using the Bruker Biotyper and scores > 140 using ASTA MALDI-TOF.

Lee et al. [52] performed a formic acid extraction with a shorter protocol. Most of the yeasts obtained from samples of clinically infected humans were identified correctly, but the method failed to identify the slimy *Cryptococcus* spp. Considering the fact that in our study no *Cryptococcus* spp. were found, possibly this shorter, easier and inexpensive method could have provided as good results as MALDI-EX. On the other hand, different *Cryptococcus* spp. were isolated from liquid swine diets in studies by Olstorpe et al. [6]. Therefore, MALDI-EX seemed to be the best method to reliably identify as many yeasts species as possible.

Extending databases, lowering the identification score for yeasts as well as shorter protocols could improve the ratio of reliable results of environmental yeasts with MALDI-EX in the future, so that the results of this method could be highlighted even more.

Various molecular biological methods described in the literature were not included in this study, although previous authors achieved good results [27]. Gori et al. [27] had difficulties in separating the two most commonly occurring yeasts in their study with 26S rRNA sequencing: *C. humilis* (formerly named *C. milleri*; 58.4%) and *C. holmii* (*Kasachstania exigua*; 17.5%), together accounting for 75.9% of all results (n = 766 yeasts). They distinguished the two yeasts biochemically according to their sucrose and raffinose metabolism [27]. In the present study, *C. humilis* and *C. holmii* accounted together for 20% of all results (n = 16). In retrospect, it can be assumed that the 26S rRNA method would not have been advantageous in these cases.

## 4.2. *Temperature*

In the present study, clearly more than half of the yeasts (52.9%) grew better at 25 °C than at 37 °C or did not grow at 37 °C at all (23.5%). Those yeasts that did not grow at 37 °C at all will presumably not grow in the intestines of pigs, where the internal body temperature normally still exceeds 37 °C.

Considering only yeasts isolated from LD + S, there are even more isolates that prefer cooler temperatures (see Table 5). An explanation for these yeasts preferring lower temperatures than yeasts from LD - S could be the chosen time of sampling of LD + S in late spring and early summer in the two projects, when the liquid diets were composed. After harvesting the maize plants and making silage in the fall in the respective projects, this was stored outdoors during winter, where yeasts had to cope with low temperatures. Thus, yeasts may have adapted to these temperatures or died. Storing feed materials or liquid diets at cool temperatures possibly reduces the yeast species, which prefer 37 °C, and as a result have little or no impact on gut health.
The present results could also indicate an adaptation of the yeasts to their feed origin and storage temperature. These results were obtained directly after the cultivation of the yeasts from the respective feed (see Section 2.3). Therefore, yeasts had little or no opportunity to adapt to the new temperatures. This is in the broadest sense comparable with the climatic conditions during the long period between the fall and spring. On the other hand, the possibility to adapt would exist at warmer outside temperatures and in case of the pre-fermentation of the liquid feed (24 h, 38 °C), as is sometimes practiced, especially with controlled fermentation [58]. Suutari et al. [59] reported morphological changes in some yeasts that had to adapt to cooler or very warm temperatures in a bouillon. The investigations in this previous study on growth performance at different temperatures was made on agar plates. The possible easier adaptation to new temperature conditions in a bouillon could also be an explanation for the observations that some yeasts only produce gas at 37 °C after a longer period of time (see Section 3.4, C. humilis in Figure 3).

Margesin et al. [60] isolated yeasts and bacteria from cold-adapted habitats and classified 60% of the yeasts but only 8% of bacteria to be true psychrophils, which showed no growth above 20 °C, indicating that the remaining microorganisms are able to adapt to warmer temperatures. Yeasts that do not grow or grow very poorly at 37 °C are thought to have little or no effect on gut health [20]. As a result, the lack of or partly low clinical symptoms on farms with a high yeast load in the feed are explicable. Correspondingly, yeasts that did not grow at all or worse at 37 °C than at 30 °C were also found on yeasts obtained from swab samples from milking machines [55]. A large majority of them could not be recovered from the milk collected with these milking machines.

In both groups (LD – S and LD + S), 1.9 different yeasts were identified. On the other hand, significantly more different colonial morphologies of the yeasts were found in LD + S, possibly indicating that temperature could have an influence on morphology, as was also observed by Nadeem et al. [37].

### 4.3. pH-Value

In the present study, the LD + S had on average slightly higher pH-values than LD – S and they contained significantly more C. lambica. Whether this connection is accidental or related to the higher pH-value can only be suspected due to the small number of farms of origin. Lack of growth at 37 °C [34], a good smell/taste [26] but no described ability of biofilm formation, as found by the Olostorpe et al. [26], could mean that this yeast is expected to be less harmful as a feed contaminant and for gut health than other yeasts. On the other hand, some yeasts are known to adapt to pH-values, to temperature and to different media [37], so that the safety of C. lambica in liquid swine diets still needs to be tested.

For fungi as well as bacteria, one of the most important environmental conditions is ambient pH. Changes in external pH result in phenotypic, metabolic and physical changes of the microorganisms [22]. The low pH-values in liquid feeds, especially fermented ones or those containing silage compared to normal feed for pigs, in general favor yeasts. This is due to the fact that at pH-values < 5.0, many bacteria are not able to stay alive or to grow as fast as they do at higher pH-values [61]. Molds depend on oxygen, but yeasts are able to grow at low pH-values with and without oxygen [3]. Some yeasts are known to be able to adapt to low pH-values in their surroundings by forming a thicker cell wall with chitin (N-acetyl glucuronidase) [22]: the high buffering capacity in the cytosol, high H⁺-ATP-ase and/or high endogenous energy reserves of C. krusei [29]. Therefore, fermented liquid feeds, especially after controlled fermentation, always poses a certain risk of increased yeast content.

### 4.4. Gas Production

Quantitatively comparing gas production of different yeasts under standardized conditions with Ancom RF Gas Production System was, to the best of our knowledge, performed for the first time. Investigations in a bouillon, produced in accordance with European and US Pharmacopoeia guidelines, allows for a comparison of gas-producing yeasts irrespective of feed or water. The SAB-bouillon provides ideal conditions for yeasts and contains high amounts of glucose (20 g/L). However, the total
gas quantities measured do not describe quantities that would also be produced in the feed or in the animal, since the competing flora is always different, and feed is not composed like a bouillon or an agar for yeasts.

Different generation times, sizes and numbers of buds make it difficult to precisely calculate the yeast quantity with density determination or even with quantitative cell counting. Hence, the amounts of gas production were not precisely determined but categorized to two major groups, as described above. Additionally, not the exact yeast numbers per milliliter were determined but only the density by means of the McFarland standard. Exemplarily, for some samples with the density of McFarland 0.3, the yeasts were counted, resulting in 1–4 × 10⁵ cfu yeasts per mL. Thus, these yeast counts are just about acceptable regarding the requirements in liquid feed according to Kamphues et al. [17].

In the present study, slightly increasing gas pressures were also measured for yeasts that did not produce gas at the beginning of the experiment. This could be explained by the rising room temperature during processing to the 37 °C in the incubator.

The gas formation capacity of the yeasts differed very clearly between 888 mbar and 10,419 mbar. *C. kefyr* formed over ten times more gas than one of the *S. cerevisiae* isolates. Only one yeast isolate from LD + S was able to produce higher amounts of gas at 37 °C within 24 h. This was partly caused by its preference for cooler temperatures, as described above. The reason for the differing amounts of gas production of yeasts may to some extent be seen in the lack of oxygen produced in the Ancom Gas Production System, which is also found in the pig's colon. Some yeasts like *C. sphaerica*, *C. variabilis*, *C. kefyr*, *C. lambica*, *C. krusei*, *S. cerevisiae* and *C. pelliculosa* are known to metabolize glucose under anaerobic conditions; variable metabolization is expected from other yeasts like *C. valida*, *G. candidum* and *G. capitatum*, while *C. rugosa* and *Rhodotorula* spp. are mostly not capable of fermentation [61]. The latter cannot be expected to produce gas amounts under the conditions available in the present study as well as those found in the gastrointestinal tract of pigs. As such, they cannot be expected to cause a disease such as HBS. Comparing the growth of a yeast from liquid feed for pigs at 37 °C and 25 °C can give a good indication of whether a yeast is likely to cause HBS. However, it is not possible to make an accurate prediction because yeasts are partially capable of adapting to temperatures and some yeasts hardly ferment under anaerobic conditions despite growth at 37 °C. On the other hand, no high gas production within 24 h was observed in the present study when a yeast isolate did not grow at 37 °C. Presumably, those yeasts are not supposed to cause HBS. A test of growth at 37 °C would be easy to perform in every laboratory and could give a hint at whether a yeast would be able to grow in a pig's alimentary tract. Further studies will be needed to clarify which amount of gas production can generally be called high or low. Apart from this, it has to be considered that a yeast, even if it is not able to form a biofilm itself, may colonize the biofilm of the lines of the feeding system. Such yeasts could potentially be capable of adapting to warmer temperatures, especially in the summer months.

## 4.5. Summary

In several studies of liquid feed, samples for pigs' yeasts were identified, which were also found in the present study. The most commonly detected yeast in our study was *C. krusei*. This is the first study of liquid feed with and without maize silage. In liquid feed with maize silage (LD + S), significantly more *C. lambica* was found.

MALDI-EX provided the most reliable results (78.75%), but the ID 32 C-test, easy to perform in every laboratory, was sufficient for confirming 75.0% of the identified yeasts. Both tests together identified 88.75% of the yeasts because some yeasts were only reliably identified with one or the other test. The quicker MALDI-DS-method provided only 37.5% reliable results, this being significantly less than the other two methods. Thus, a formic acid/acetonitrile extraction (MALDI-EX) before analysis should be preferred.

Clearly more than half of all yeast isolates grew better at 25 °C than at 37 °C. Fourteen isolates showed no growth at all at 37 °C. Gas amounts produced by the different yeast isolates differed more than tenfold within a 24-h incubation period at 37 °C in SAB-bouillon measured with the Ancom
Gas Production System. Most of the tested *C. krusei* and *S. cerevisiae* but none of the tested *C. holmii*, *Trichosporon* spp., *G. silvicola* and *C. pelliculosa* were able to produce gas. While only one yeast from LD + S was able to produce gas within 24 h, more yeasts (40.6%) from LD - S were able to do so. None of the yeasts that did not grow on the SAB-agar at 37 °C were able to produce high amounts of gas within a 24-h incubation period at 37 °C in the bouillon, presuming that those yeasts could only slightly affect the animals' health.

Due to the fact that the majority of *C. krusei* isolates were able to grow at 37 °C, produce high amounts of gas, grow in low pH conditions and form biofilms, as is known from the literature, this yeast species seems to be predestinated to grow in liquid diets and to remain in a biofilm in the pipelines serving the liquid diet. Therefore, special interest should be given to this yeast species. The evaluation of yeast levels in liquid feed for pigs has so far only been determined on the basis of the number of yeasts per gram feed. Laboratory values alone could possibly incorrectly estimate the influence of yeasts on the health of the animals as either being too low or too high. Additional investigations are needed to further characterize the effect of each yeast species on pig health. Moreover, investigating the effect of having the storage temperature of the feed significantly below body temperature could be interesting.

**Author Contributions:** Conceptualization, B.K.; methodology, B.K.; software, B.K. and U.S.; validation, B.K., H.K., U.S. and C.V.; formal analysis, B.K. and C.V.; investigation, H.K. and B.K.; resources, J.K. and C.V.; writing—original draft preparation, B.K.; writing—review and editing, B.K., C.V. and U.S.; visualization, B.K. and C.V.; supervision, C.V.; project administration, B.K.; funding acquisition, J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This publication was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft) and the University of Veterinary Medicine Hannover, Foundation, Hannover, Germany within the funding program Open Access Publishing.

**Acknowledgments:** We would like to thank Frances Sherwood-Brock for proof-reading the manuscript to ensure correct English.

**Conflicts of Interest:** The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Article

# Dietary Supplementation of a Live Yeast Product on Dairy Sheep Milk Performance, Oxidative and Immune Status in Peripartum Period

Alexandros Mavrommatis<fnref n="1" />,*, Christina Mitsiopoulou<fnref n="1" />, Christos Christodoulou<fnref n="1" />, Dimitris Karabinas<fnref n="1" />, Valentin Nenov<fnref n="2" />, George Zervas<fnref n="1" /> and Eleni Tsiplakou<fnref n="1" />,*

Received: 10 November 2020; Accepted: 1 December 2020; Published: 3 December 2020

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**Abstract:** This study evaluated the dietary administration of *Saccharomyces cerevisiae* live yeast on milk performance and composition, oxidative status of both blood plasma and milk, and gene expression related to the immune system of lactating ewes during the peripartum period. Chios ewes were fed either a basal diet (BD) (Control, *n* = 51) or the BD supplemented with 2 g of a live yeast product/animal (ActiSaf, *n* = 53) from 6 weeks prepartum to 6 weeks postpartum. Fatty acid profile, oxidative, and immune status were assessed in eight ewes per treatment at 3 and 6 weeks postpartum. The β-hydroxybutyric acid concentration in blood of ActiSaf fed ewes was significantly lower in both pre- and postpartum periods. A numerical increase was found for the milk yield, fat 6% corrected milk (Fat corrected milk (FCM<sub>6%</sub>)), and energy corrected milk yield (ECM) in ActiSaf fed ewes, while daily milk fat production tended to increase. The proportions of C<sub>15:0</sub>, C<sub>16:1</sub>, C<sub>18:2n6t</sub>, and C<sub>18:3n3</sub> fatty acids were increased in milk of ActiSaf fed ewes, while C<sub>18:0</sub> was decreased. Glutathione reductase in blood plasma was increased (*p* = 0.004) in ActiSaf fed ewes, while total antioxidant capacity measured by 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) method was decreased (*p* < 0.001). Higher ABTS values were found in the milk of the treated group. The relative transcript levels of *CCL5*, *CXCL16*, and *IL8* were suppressed, while that of *IL1B* tended to decrease (*p* = 0.087) in monocytes of ActiSaf fed ewes. In conclusion, the dietary supplementation of ewes with *S. cerevisiae*, improved the energy utilization and tended to enhance milk performance with simultaneous suppression on mRNA levels of pro-inflammatory genes during the peripartum period.

**Keywords:** *Saccharomyces cerevisiae*; livestock; ewes; energy; antioxidant; cytokines

## 1. Introduction

Both meat and dairy products consumption are expected to increase in 2050 by 73 and 58%, respectively, compared to their 2010 levels [1,2], due to the rapid population growth rate. Ruminants' milk (67%) and meat (33%) cover 51% of proteins derived from the livestock sector and have a dominant role in food security, which is linked to how efficient animals utilize feed. Ruminants' feed efficiency depends upon the microbes residing within the rumen that ferment and transform feeds into volatile fatty acids (VFAs), proteins, and vitamins which are exploited by the host [3]. This multikingdom ecosystem's efficiency is dependent on various factors, the most prominent being that of diet. The improvement of the rumen microbiome habitat through the advancement

<footnote n="1">¹ Laboratory of Nutritional Physiology and Feeding, Department of Animal Science, School of Animal Biosciences, Agricultural University of Athens, Iera Odos 75, GR-11855 Athens, Greece; mavrommatis@aua.gr (A.M.); chr_mitsiopoulou28@hotmail.com (C.M.); c.christodoulou@aua.gr (C.C.); vdkarabinas@vkarabinas-sa.gr (D.K.); gzervas@aua.gr (G.Z.)</footnote>

<footnote n="2">² Phileo Lesaffre Animal Care, Marcq en Baroeul, 59700 Nord, France; info@phileo.lesaffre.com</footnote>

<footnote>* Correspondence: eltsiplakou@aua.gr; Tel.: +30-2105294435</footnote>
of feed efficiency technologies entails a fundamental stepping stone in the overall improvement of livestock systems sustainability and food security concerns.

In intensive farming systems, high genetic merit animals require higher amounts of concentrate to fulfil their energy and nutrient demands, resulting in metabolic imbalances in rumen function and their microbiome governance. Probiotic yeasts are currently popular and widely used in ruminant feeding systems, especially since some of them have been officially authorized as feed additives in Europe [4]. The main purpose for using such additives in ruminant diets is to prevent rumen flora disorders and disturbances [5]. Dietary supplementation with live yeast (LY), *Saccharomyces cerevisiae*, improves rumen function through several modes of action [6]. This improvement is related to the oxygen scavenging properties of yeast in rumen (anaerobiosis mechanism), which upgrades bacterial viability and therefore the animal production [6]. Amongst the favorable bacteria are cellulolytics, which through the increase in their activity enhance fiber digestion. Moreover, LY can also stabilize the ruminal pH [7], not only after feeding, but also during the peripartum period where animals often find themselves in a negative energy balance and are further sensitive to metabolic diseases. It has been proven that even a low-grade energy deficiency weakens the animals' antioxidant system, which fails to neutralize the formation of Reactive Oxygen Species (ROS) and triggers the pro-inflammatory response [8,9].

By improving ruminants' feed utilization and both energy and nutrient availability during the peripartum period, not only can milk performance and chemical composition be enhanced, but furthermore a downregulation in the immunostimulation response can be achieved through the limitation of lipomobilization metabolites [10,11]. Although LY supplementation in ruminants' diets is a well-established nutritional strategy, previous works have only focused on district parameters instead of a holistic approach. Specifically, except for milk performance [7,10–14], scarce information has been linked to the potential improvement of energy balance and oxidative status and therefore to the immune response under the influence of dietary yeasts inclusion in ruminants.

Taking into account the aforementioned information, the objective of this work was to evaluate the effect of LY *S. cerevisiae* (CNCM I-4407, 10¹⁰ CFU/g, ActiSaf; Phileo Lesaffre Animal Care, France) in dairy sheep during the transition and early lactation period (6 weeks prepartum and 6 weeks postpartum) on milk performance and composition, antioxidant status (determined by Glutathione transferase (GST), Glutathione reductase (GR), Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px), Catalase (CAT) and Lactoperoxidase (LPO) activities, antioxidant capacity with 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and Ferric reducing ability of plasma (FRAP) methods and oxidative stress indicators such as Malondialdehyde (MDA) and protein carbonyls (PCs)) on both milk and blood plasma and key-gene expression (CCL5, CXCL16, INFG, IL1B, IL2, IL6, IL8, IL10, TNF, NFKB) in monocytes and neutrophils which are associated with cytokine production.

## 2. Materials and Methods

### 2.1. Location and Environmental Conditions

The experiment was conducted from November 2019 to March 2020 on a commercial dairy sheep farm in the region of Chiliomodi in Korithia, Greece. This region has a typical Mediterranean climate with hot dry summers and relatively mild wet winters. During the experimental period, the mean temperatures in November, December, January, February, and March were 12.2, 8, 9.3, 11.1 and 13.1 °C, respectively. The selected farm represents the typical intensive dairy sheep production system of Greece.

### 2.2. Animals and Diets

Animals' housing, management, handling, and care complied with the latest European Union Directive on the protection of animals used for scientific purposes [15], while taking into account an extended experimental design report, the Bioethical Committee of Faculty of Animal
Science (currently known as the Agricultural University of Athens Ethical Committee in Research; FEK 38/A/2-3-2018, eide AUA) approved the experimental protocol. One hundred and twenty (120), 1- to 3-year-old dairy ewes (*Ovis aries*), of pure Chios breed, were physically selected from a flock of six hundred. At approximately 6 weeks before parturition, the ewes were divided into two homogenous groups based on their body weight (BW), number of parturition, and the milk yield from the previous year only for the case of multiparous ewes (2.1 ± 0.68 kg). Both groups had the same number of primi- (n = 20) and multiparous (n = 40) animals. More specifically, the ewes mean BW in the Control group (n = 60) was 61.5 ± 10.70 (SD) kg while in the ActiSaf group (n = 60) was 61.5 ± 11.02 (SD) kg. The Control group was fed a basal diet comprising of concentrate mix, alfalfa hay, and oat hay, while the ActiSaf group consumed the same basal diet supplemented with 2 g of *S. cerevisiae* LY/day/ewe (CNCM I-4407, 10¹⁰ CFU/g, ActiSaf; Phileo Lesaffre Animal Care, France) (Table 1). The animals were housed in two pens based on the dietary treatment. Both diets were isonitrogenous and isocaloric and were designed to meet ewes' requirements in the transition period and early lactation according to the flock fat (6%) corrected milk yield [16,17]. The animals were fed on a group basis while forages were offered separately from the concentrate in three equal portions after milking. Diet selectivity did not occur, no refusals of forages and/or concentrate were observed, and all animals had free access to fresh water. The experimental procedure lasted 6 weeks started from each ewes' parturition. After this, each ewe was returned to the commercial farm flock and the experiment ended when the final ewe had completed its 6th week on lactation. Since milk performance was recorded at the same time points, lactation stage had no effect on milk performance. Control ewes (n = 60) gave birth to 141 lambs (prolificacy = 2.35; 69 females and 72 males) while those of the ActiSaf (n = 60) gave birth to 142 (prolificacy = 2.36; 65 females and 77 males). In addition, since the experimental trial took place on farm-scale conditions, few ewes were unable to be exploited for data curation due to abortions (4), mastitis (10) or dystocia (2), hence the final number of subjects was re-adjusted to 51 and 53 for the Control and ActiSaf groups, respectively.

<table><caption>**Table 1.** Concentrates composition (g/kg), diet intake (g), daily nutrients intake (g/ewe), and feeds chemical composition and fatty acid profile (%).</caption><thead><tr><th rowspan="2">Ingredients (g/kg)</th><th colspan="2">Concentrates</th></tr><tr><th>Control</th><th>ActiSaf</th></tr></thead><tbody><tr><td>Maize grain</td><td>575</td><td>575</td></tr><tr><td>Wheat middlings</td><td>180</td><td>178</td></tr><tr><td>Soybean meal</td><td>220</td><td>220</td></tr><tr><td>Mineral and vitamin</td><td>25</td><td>25</td></tr><tr><td>ActiSaf</td><td>-</td><td>2</td></tr><tr><th colspan="3">Daily feed intake (g/day/ewe)</th></tr><tr><td></td><td>Prepartum</td><td>Postpartum</td><td>Prepartum</td><td>Postpartum</td></tr><tr><td>Oat hay</td><td>700</td><td>700</td><td>700</td><td>700</td></tr><tr><td>Alfalfa hay</td><td>700</td><td>700</td><td>700</td><td>700</td></tr><tr><td>Con. Mix</td><td>1000</td><td>2000</td><td>-</td><td>1000</td></tr><tr><td>ActiSaf Mix</td><td>-</td><td>-</td><td>1000</td><td>1000</td></tr><tr><th colspan="5">Nutrients intake (g/day/ewe)</th></tr><tr><td>Dry matter</td><td>2093</td><td>2956</td><td>2091</td><td>2953</td></tr><tr><td>Crude protein</td><td>318</td><td>480</td><td>318</td><td>481</td></tr><tr><td>Ether extract</td><td>46</td><td>69</td><td>44</td><td>66</td></tr><tr><td>NDF</td><td>835</td><td>991</td><td>835</td><td>992</td></tr><tr><td>ADF</td><td>562</td><td>626</td><td>561</td><td>624</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th colspan="5">Diet chemical composition (%)</th></tr><tr><th></th><th>Con. Mix</th><th>ActiSaf Mix</th><th>Alfalfa hay</th><th>Oat hay</th></tr></thead><tbody><tr><td>Dry matter</td><td>86.28</td><td>86.02</td><td>88.21</td><td>87.63</td></tr><tr><td>Organic matter</td><td>81.70</td><td>81.16</td><td>80.87</td><td>80.91</td></tr><tr><td>Crude protein</td><td>16.23</td><td>16.29</td><td>14.72</td><td>7.38</td></tr><tr><td>Ether extract</td><td>2.22</td><td>2.01</td><td>1.38</td><td>2.06</td></tr><tr><td>NDF</td><td>15.62</td><td>15.68</td><td>43.25</td><td>53.68</td></tr><tr><td>ADF</td><td>6.32</td><td>6.19</td><td>32.56</td><td>38.74</td></tr><tr><td>Ash</td><td>4.58</td><td>4.86</td><td>73.4</td><td>6.72</td></tr><tr><th colspan="5">Fatty acids composition (%)</th></tr><tr><td>C<sub>14:0</sub></td><td>0.1</td><td>0.1</td><td>0.6</td><td>5.6</td></tr><tr><td>C<sub>16:0</sub></td><td>14.3</td><td>14.3</td><td>23.9</td><td>3.7</td></tr><tr><td>C<sub>18:0</sub></td><td>3.9</td><td>3.9</td><td>3.1</td><td>19.5</td></tr><tr><td>cis<sub>.9</sub> C<sub>18:1</sub></td><td>36.1</td><td>36.1</td><td>3.1</td><td>16.2</td></tr><tr><td>C<sub>18:2</sub> n-6</td><td>42.2</td><td>42.2</td><td>21.3</td><td>54.0</td></tr><tr><td>C<sub>18:3</sub> n-3</td><td>1.7</td><td>1.7</td><td>41.9</td><td>1.2</td></tr></tbody></table>

NDF = Neutral detergent fiber; ADF = Acid detergent fiber.

## 2.3. Feed Samples Analyses

Samples of the alfalfa hay, oat hay, and concentrate were analyzed for organic matter (OM; Official Method 7.009), dry matter (DM; Official Method 7.007), and crude protein (CP; Official Method 7.016) according to the Association of Official Analytical Chemists (1984) using a Kjeldahl Distillation System (FOSS Kjeltec 8400, Demark). Neutral detergent fiber (NDF) and acid detergent fiber (ADF) expressed exclusive of residual ash according to the method of Van Soest using an ANKOM 2000 Fiber Analyzer (USA) as described by Tsiplakou et al. [18] (Table 1).

## 2.4. Milk Samples Collection

The sheep were milked three times per day (at 0700, 1300 and 2000 h) with a milking machine equipped with a digital milk meter and an electronic identification system (Sylco, Greece); thus, milk yield was recorded daily, and software (Sylco, Greece) was set to provide weekly averages. Milk samples were collected from each ewe weekly (at 7, 14, 21, 28, 35 and 42 days from parturition) for a 6-week period with sampling bottles (Sylco, Greece) of 200 mL appropriately for the milking parlor, to receive a representative sample of the milked quantity. Each of the milk samples from the mix of three subsamples was derived from each milking time (at 0700, 1300 and 2000 h) by taking 5% of the milked quantity.

## 2.5. Milk Chemical Composition

The milk samples were analyzed for fat, protein, lactose, total solids, and total solids no-fat by IR spectrometry (MilkoScan 120; FOSS, Hillerød, Demark) after proper calibration according to the methods of Gerber [19] and Kjeldahl [20].

## 2.6. Blood Metabolic Biomarker (B-HBA) Determination

Four weeks before the expected parturition, blood B-HBA was individually determined (before the morning feeding, 0700 h) once every three days until the lambing to ensure that 15 days before parturition a measurement would be recorded (Table S1). Two weeks postpartum, the sample collection for B-HBA was repeated. Blood ketone concentrations were measured using an electrochemical capillary blood monitoring device (FreeStyle Precision Neo, Abbott Laboratories Hellas S.A) with the corresponding individual foil-wrapped test strips for B-HBA. This method of B-HBA determination possesses 98.4% accuracy for the prediction of both toxemias' pregnancy and ketosis in Chios ewes [21]. After the insertion of a test strip into the device, a drop of blood was applied to the assigned spot,