# Mixing and fermenting chickpea and almond milk analogues to produce probiotic yoghurt analogues

Charles Silande, Andrea Hickisch, Andrea Strube, Nathalie Daniel, Marie-Bernadette Maillard, Séverine Chevalier, Gwénaële Henry, Ingrid Aguiló-Aguayo, Gwénaël Jan, Valérie Gagnaire

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## ► To cite this version:

Charles Silande, Andrea Hickisch, Andrea Strube, Nathalie Daniel, Marie-Bernadette Maillard, et al. Mixing and fermenting chickpea and almond milk analogues to produce probiotic yoghurt analogues. Food Research International, 2025, 221, pp.117576. 10.1016/j.foodres.2025.117576. hal-05293648

HAL Id: hal-05293648

https://hal.inrae.fr/hal-05293648v1

Submitted on 3 Oct 2025

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Mixing and fermenting chickpea and almond milk analogues to produce probiotic
yoghurt analogues

Charles Silande¹, Andrea Hickisch², Andrea Strube², Nathalie Daniel¹, Marie-Bernadette Maillard¹, Séverine Chevalier¹, Gwénaële Henry¹, Ingrid Aguiló-Aguayo³, Gwénaël Jan¹ and Valérie Gagnaire¹*

¹, INRAE Institut Agro, UMR Science and Technology of Milk and Eggs (STLO), F35000 Rennes, France

², Fraunhofer Institute for Process Engineering and Packaging IVV, 85354 Freising, Germany

³, Institute of Agrifood Research and Technology (IRTA), Postharvest, Fruitcentre, Catalonia, 25003 Lleida, Spain

Corresponding author: Valérie Gagnaire; valerie.gagnaire@inrae.fr INRAE, Institut Agro, STLO, 65 rue de Saint Brieuc, F35000 Rennes.

Abstract

The search for sustainable and healthy foods has led to the development of various fermented plant-
based products as an alternative to fermented dairy products. These fermented products must have
satisfactory sensory properties, meet nutritional requirements, and contain live and active probiotic
bacteria, that can exert beneficial health effects. In our study, we developed mixes of almond and
chickpea milk analogues (MA), with a protein content of approx. 3%, close to the protein content of
dairy milk. Two lactic acid bacteria strains, *Streptococcus thermophilus* CIRM-BIA772 and
*Lactobacillus helveticus* CIRM-BIA100, with known technological and probiotic properties, were used
to ferment the different MAs and to produce fermented yogurt analogues (YA). After fermentation,
bacterial population, carbohydrates and organic acid content, proteolysis, amino acid profile,
instrumental texture analysis, sensory properties and *in vitro* immunomodulation were measured. The
changes in composition of the almond and chickpea MA and the mixes made thereof resulted in different
capabilities of the two strains to grow with higher growth shown in the YA mixes. In turn, the strains
produced various amounts in lactic acid, peptides and free amino acids, including GABA. The samples
with higher almond content exhibited significantly lower apparent viscosity (222 vs. 937mPas at
ŷ = 50 s⁻¹) and a greater tendency toward syneresis compared to those with higher chickpea content (54%
vs. 5%). The plant-based aroma was consistent across samples as well as the yoghurt-like attribute that
can help acceptation by consumers. Finally, after fermentation, the YAs diversely reduced the
proinflammatory response induced by lipopolysaccharide of *E. coli*, with higher reduction of the IL8
secretion in 100 %chickpea and in the mixes with high chickpea content. These results provide valuable
insights into how the composition of the raw material blends and the fermentation conditions can be
modulated to control the sensory, textural, nutritional and health properties.

Keywords: 1 to 7: fermentation, probiotic, lactic acid bacteria, almond, chickpea, sensory, immunomodulation
# 1 Introduction

We are currently experiencing a growing demand for plant-based milk analogues for reasons related to lifestyle, health, diet and sustainability (Harper et al., 2022; Henn et al., 2022; Richardson et al., 2023; Willett et al., 2019). These new products should exhibit enhanced nutritional and health-promoting properties, while keeping good taste and flavour to ensure consumer acceptance (Drigon et al., 2023) and limiting the presence of deleterious components such as alkaloids, phytate, tryptic inhibitors and some carbohydrates responsible for bloating (Guillon & Champ, 2002; Harper et al., 2022).

Legumes (soybean, pea, chickpea, lupine, faba bean, lentils, etc) with high protein content, cereals (wheat, corn, barley, oat, etc), pseudo cereals (buckwheat, quinoa, chia, amaranth, etc), oilseeds (e.g. sunflower, hemp seed) and nuts (e.g. almond, cashew, pistachio, etc) are currently widely used as plant-based protein resources (Fang et al., 2024). Many legumes offer promising functional properties such as solubility, emulsifying or foaming capacity (Grasso et al., 2022). However, their flavour, often described as beany, green, grassy, can restrict their application. In contrast, nuts and cereals are often described as more neutral and pleasant in flavour, but have more limited functionality (Hernández-López et al., 2022).

Such flavour and techno-functionality defects can be overcome by fermenting and mixing plant-based resources. A wide range of traditional fermented liquid and solid plant-based food products exists worldwide (Tamang et al., 2016, 2020). Fermentation leads to improved organoleptic, nutritional, and health-promoting properties of plant-based resources (Montemurro et al., 2021). It may transform vegetable compounds into edible and bioactive ones, or reduce anti-nutritional compounds or undesired flavours including hexanal (Harper et al., 2022). Among microorganisms encountered in plant-based fermented foods, lactic acid bacteria (LAB) are widely represented. From a technological point of view, they ferment carbohydrates into acids, hydrolyse proteins into peptides and free amino acids, and produce metabolites leading to the typical flavour of fermented foods, through various enzymes (Canon et al., 2020; Engels et al., 2022; Fischer et al., 2022; Fritsch et al., 2015; Lacou et al., 2016; Liu et al., 2010; Thierry et al., 2015). In turn, acidification of the fermented plant-based products limits the growth of undesirable microorganisms, as pathogens and spoilage microorganisms, leads to the protein aggregation and gel formation. Proteolysis can change the final texture and flavour. From a healthy perspective, probiotic properties have been shown in various fermented products (Marco et al., 2021). LAB may indeed improve carbohydrate tolerance, reduce the levels of toxic and anti-nutritive factors, increase the bioavailability of bioactive compounds such as peptides, vitamins, free amino acids, organic acids and cofactors (Marco et al., 2021). They may exert positive action on the gut homeostasis through various mechanisms affecting the composition or function of the microbiota and by modifying the host's immunological responses, evidenced from *in vitro* to *in vivo* models and even in clinical studies (Illikoud et al., 2022). Co-culture of *streptococci* and *lactobacilli* were shown to give promising positive interactions, as exemplified and widely exploited in yoghurt, leading to enhanced final sensorial, nutritional and healthy properties (Canon et al., 2020).

Using mixes of plant-based resources may avoid technological defects such as phase separation, due to protein aggregation, and subsequent low texture quality (Grasso et al., 2020), when plant-based milk analogues (MAs) are acidified to produce yoghurt-type products. It allows varying the type of protein composition and thus viscoelastic gel properties to improve the final texture, while increasing the overall nutritional value, all without adding any gelling agents (gums, starches, pectin and agar). Plant species that can be used in that aim are widely present in the Mediterranean area, including chickpea and almond. Chickpea present some advantages in terms of high levels of protein, various carbohydrates and mono or polyunsaturated fats (Grasso et al., 2022; Özcan, 2023). Almond MA are widely produced and some MA based on chickpea or lupin or mixes thereof were shown to have a protein content with 3.2 g/100 mL equivalent to that of dairy milk, and are highly digestive (Duarte et al., 2022). The ability to be fermented in the form of milk or yoghurt analogues (YAs) has recently been demonstrated for chickpea (Mefleh et al., 2022; Zhang et al., 2022).

In our study, we aimed to develop mixes of almond and chickpea MA, with a protein content of approx. 3%, with desired sensory, textural, nutritional and immunomodulatory properties.
## 2 Material and methods

### 2.1 Preparation of milk analogues (MA)

#### 2.1.1 Chickpea MA

The chickpea seeds, cultivar PGI Garbanzo de Escacena (Andalusia, Spain), were dehulled with an underflow peeler (Streckel & Schrader GmbH, Hamburg, Germany) and classified in an air-lift system (Alpine Hosokawa AG, Augsburg, Germany). Afterwards, the seeds were washed with tap water and soaked overnight in cold tap water with 2% (w/w) hydrogen peroxide (ratio 1:2, seeds:water). After soaking, the excess water was removed and the soaked seeds were rewashed. Then, the soaked seeds were cooked in fresh water (ratio 1:6, dry seeds:water; the ratio refers to the initial weight of the dry seeds prior to soaking) at 85 °C for 45 min and 100% humidity in a steamer (iCombi Classic, Rational Aktiengesellschaft, Landsberg am Lech, Germany). The excess water was removed again and the cooked seeds were milled at speed 10 for 10 min (Thermomix TM6, Vorwerk Deutschland Stiftung & Co. KG, Wuppertal, Germany) with water (ratio 1:3, dry seeds:water). To degrade starch, the slurry was: 1) heated to 90 °C and hydrolysed for 1 h by stirring with 0.03% α-amylase (Optizym HiTaA 17105 L, SternEnzym GmbH & Co. KG, Ahrensburg, Germany) and 2) after cooling to 50 °C hydrolysed for 1 h with 0.05% gluco-amylase (Optizym GA 14400 L, SternEnzym GmbH & Co. KG). The slurry was filtered with a sieve (125 µm, NEXOPART GmbH & Co. KG). At this step, the protein content of the MA was approx. 1% (w/w). To enrich the protein content to 3% (w/w), chickpea protein isolate G910 (ChickP Protein Ltd, Israel) was added. Then 1.5% (w/w) rapeseed oil (Rapso, VOG AG, Linz, Austria) was added to the MA, emulsified with an Ultra Turrax T25 (Janke & Kunkel IKA Labortechnik, Staufen, Germany) at 10,000 rpm for 10 min at 50 °C and homogenised with a high-pressure homogeniser at 250bar and 50bar at 50°C (APV-2000, SPX FLOW Inc., Charlotte, USA). Finally, the MA was subjected to an ultra-high temperature (UHT) heat treatment at 140°C for 15s with a tubular heat exchanger (HT 220 HTST/UHT System, Omwe, Schalkwijk, Netherlands) followed by storage at 1°C.

#### 2.1.2 Almond MA

The peeled nuts (Local Landraces Marcona) were cold pressed with an oil press (OP 700 Emilio, Rommelsbacher GmbH, Dinkelsbühl, Germany). The almond press cake (11.5% (w/w)) was milled for 1min at speed 10 and 30min at speed 6 (Thermomix TM6) by adding water (88% (w/w)) and sucrose (0.5% (w/w)). After milling, the almond MA processing was comparable to that of the chickpeas including emulsification, homogenisation and UHT treatment. No rapeseed oil or protein isolate was added as the slurry already achieved a protein content of approx. 3% (w/w) and retained sufficient residual almond oil content.

### 2.2 Preparation of the mixed MA with different almond-chickpea ratios

The almond and chickpea MAs were mixed at different ratio under sterile conditions: 100% almond (100A), 75% almond-25% chickpea (75A), 50% almond-50% chickpea (50A), 25% almond-75% chickpea (25A) and 100% chickpea (100CP) and stored at 1°C until use for fermentation.

### 2.3 Bacterial strains and culture conditions

Two LAB strains: *Streptococcus salivarius* subsp. *thermophilus* CIRM-BIA772 (St772) and *Lactobacillus helveticus* CIRM-BIA100 (Lh100) were provided by the International Centre for Microbial Resources-Food Associated Bacteria (CIRM BIA, Centre International de Resources Microbiennes, bactéries d'intérêt alimentaire, Rennes, France, https://collection-cirmbia.fr/). Their main characteristics are presented in Table 1.
<table><caption>**Table 1.** Main characteristics of the two lactic acid bacteria strains used in this study.</caption><thead><tr><th rowspan="2">Genus</th><th rowspan="2">Species</th><th rowspan="2">Strain name</th><th rowspan="2">Strain code</th><th rowspan="2">Isolated from</th><th colspan="8">Carbohydrate utilisation<fnref n="1" /></th><th colspan="2">Proteolysis<br>(mM eq. Met)<fnref n="1" /></th><th colspan="2">Cytokine induction<fnref n="1" /></th></tr><tr><th>Gal</th><th>Glu</th><th>Fru</th><th>Mal</th><th>Suc</th><th>Raf</th><th>Sta</th><th>Starch</th><th>Soy protein isolate</th><th>IL-10</th><th>IL-12</th><th>IL-10/IL-12 ratio</th></tr></thead><tbody><tr><td>Lactobacillus helveticus</td><td></td><td>CIRM-BIA100</td><td>Lh100</td><td>artisanal lactic starter (for Gruyère de Comté cheese making), Franche-Comté, France</td><td>1</td><td>1</td><td>0.5</td><td>0.5</td><td>0</td><td>0</td><td>0</td><td>0.62</td><td>582.89</td><td>1.00</td><td>582.89</td></tr><tr><td>Streptococcus thermophilus</td><td></td><td>CIRM-BIA772</td><td>St772</td><td>lîle-de-France, France</td><td>0</td><td>1</td><td>0</td><td>0</td><td>1</td><td>0</td><td>0</td><td>0</td><td>-0.16</td><td>522.14</td><td>57.55</td><td>9.07</td></tr></tbody></table>

The strains were provided by the CIRM BIA, Centre International de Ressources Microbiennes, bactéries d'intérêt alimentaire, Rennes, France, (https://collection-cirmbia.fr/)(https://doi.org/10.15454/g6v6-yn54).

LAB strains were first reactivated from frozen (−80°C) glycerol stocks. St772 was cultivated at 2% v/v on M17-glucose broth (Difco™, Becton Dickinson, MD, USA), supplemented with 5 g/L glucose for 8h at 43°C. Lh100 was cultivated at 2% v/v on Man, Rogosa and Sharpe (MRS, Difco™) broth, at 43°C for 24h. Both strains were cultivated twice on these broths prior to be used as starter in the MAs. All YAs with the different almond to chickpea MA ratios (see 2.2 section) were prepared as follows: after the second preculture, each LAB strain was centrifuged (8000 g × 10 min, 25°C) and the broth supernatant discarded. The bacterial pellet was washed once in sterile physiological water and resuspended in the sterile almond/chickpea MAs to inoculate each strain at 2.5% v/v and incubation at 43°C in triplicate. Fermentation was stopped when the pH dropped to 4.5 i.e. within about 5h overall. For rheological and syneresis measurements, as well as sensory analysis, the inoculated samples, i.e. YAs, were stored at 1°C for 24h before measurement. For the biochemical analyses, aliquots were prepared and stored at -20°C until further uses.

LAB population levels were determined by colony forming unit (CFU) counting (Baron et al., 2006), after incubation at 43°C for 48h for St772 strain on M17-glucose-agar (Difco™), without anaerobiosis, and for Lh100 on MRS-agar (Difco™) with anaerobic incubation using anaerobiosis generators (ATCO Biocult, ATCO, Bretteville sur Odon, France) and in the presence of an oxygen indicator (Anaerotest®, Merck, Germany).

## 2.4 Biochemical analyses

### 2.4.1 Protein and dry matter content of the MAs

The protein content was calculated based on the nitrogen content, which was determined using a Nitrogen Analyzer FP 528 (Leco Corporation, Germany) according to the Dumas combustion method (AOAC 968.06). A factor of N × 5.8 was used to calculate the protein content. The dry matter was analysed according to the AOAC method 925.10 in a TGA 601 thermogravimetric system (Leco Corporation) at 105 °C.

### 2.4.2 pH

pH was measured at T0 for all matrices and a dedicated pot per MA was used to monitor the pH kinetics throughout fermentation to ensure that fermentation time was stopped at pH 4.5, with a 30min interval at the start of fermentation and a 15min interval for the final measurements (pH meter cyberscan pH110, Eutech instruments, Thermo Fisher Scientific, Germany).

<footnote n="1">¹The carbohydrate utilisation, the proteolysis and the immunomodulation potential expressed as cytokine induction referred to the publication of Illikoud et al (2023). The carbohydrate utilisation was by Api strip measurements. The ability of one strain to hydrolyse the proteins in the medium was determined by the ΔNH₂. ΔNH₂ = Conc. eq Met (sample) - Conc. eq Met (non-inoculated control). The results were expressed as mM equivalent Methionine, which was used as</footnote>
### 2.4.3 SDS PAGE

Proteins contained in the different almond to chickpea MA ratios, prior and after fermentation, were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with a 4–15% Criterion™ TGX™ Precast Midi Protein Gel (Bio-Rad, Marnes-la-Coquette, France) under reducing conditions according the protocol of Laemmli (1970). Samples were mixed with the sample buffer (1:1) (1.2M Tris-HCl, pH 6.8, 4% (w/v) SDS (Bio-Rad, Hercules, CA, USA), 1% (v/v) DTT (Sigma-Aldrich, Saint Quentin Fallavier, France). 20% (v/v) glycerol (Prolabo, VWR International, West Chester, PA, USA) and 0.02% (w/v) bromophenol blue (Sigma-Aldrich), heat-treated at 100°C × 10 min and centrifuged at 10,000 g × 10 min at 20°C. 10µL of supernatants were loaded per lane and 5µL of Precision Plus unstained protein standards (10–250 kDa, Bio-Rad) were used per gel, and run onto a Criterion cell according to the Bio-Rad's instructions. The migrated gels were fixed with 10% acetic acid and 40% ethanol solution, stained with the Bio-Safe Coomassie stain (Bio-Rad), scanned with ChemiDoc MP Imaging System (Bio-Rad) and analysed by ImageLab (BioRad).

### 2.4.4 Proteolysis

Overall proteolysis was measured at the end of the YA fermentation. The samples were deproteinised by half-diluting samples with 4% (w/w) trichloroacetic acid (VWR Chemicals, Rosny sous Bois, France). The peptides and amino acids were quantified in triplicates using the o-phthal dialdehyde (OPA) method of Church et al. (1983), adapted to microplate. The results were expressed as mg NH₂/L with Methionine, used as a standard. The relative contribution of peptides was determined as follows:

$$[\text{proteolysis OPA}]-[\text{free amino acids}*]= [\text{peptides}]$$

*the total content of the free amino acids analysed in 2.4.5 section was transformed in mg NH₂/L.

### 2.4.5 Free amino acid content

Free amino acid content of the YAs, fermented or not, was determined after centrifugation 8,500 g × 20min at 20°C. Samples were deproteinized by ultrafiltration on vivaspin 20 centrifugal concentrator columns at 6,000 g × 15min at 20°C (10 kDa MW cut-off, Sartorius, Palaiseau, France) followed by precipitation with 0.23M sulfosalicylic acid final concentration (Sigma-Aldrich) for 1 h at 4°C, and centrifuged at 10,000 g × 10min at 20°C to pellet the proteins. The supernatants were filtered through a 0.2-µm pore size membrane (Sartorius) and diluted three times with 0.2M lithium citrate buffer (pH 2.2) prior to injection. Amino acids were analysed using cation exchange chromatography on a Biochrom 30 AA analyzer (Biochrom Ltd, Cambridge, United Kingdom) according to Spackman et al. (1958) with lithium citrate buffers as eluents and the ninhydrin as a post-column reaction system.

### 2.4.6 Carbohydrate and organic acid analyses

Carbohydrates and organic acids were analysed on ultrafiltrated samples as described above. Lactate and pyruvate were quantified by high performance liquid chromatography on an Aminex A6 ion-exchange column (Bio-Rad, Hercules, CA, United States) according to Le Boucher et al. (2016). Fructose, galactose, glucose, maltose, mannose, raffinose, stachyose, sucrose and xylose were quantified by cation exchange chromatography ICS-3000 Dionex (Thermo Electron SA, Courtaboeuf, France) fitted with CarboPac PA1 (4 × 250mm) analytical column (preceded by a corresponding guard column 50 × 4mm) according to Aburjaile et al. (2016). Carbohydrate standards (Sigma-Aldrich) were prepared at 0.5, 2.5, 5, and 10mg/L (linearity range).

## 2.5 Rheological analyses and syneresis

Samples were gently stirred for 10s before measuring with a rheometer using cone-plate geometry (Physica MCR 301, CP40, diameter: 40mm, cone angle: 0.3°, gap size: 0.4mm, Anton Paar, Ostfildern-Scharnhausen, Germany). A pre-shearing was implemented with a shear stress (τ) of 0.1Pa at 10°C for 60s.

Rotational measurements were performed by increasing the shear rate (γ̇) at a linear ramp from 0 to 500s⁻¹ in 3min (upward flow curve), followed by a 5 min holding time and a linear decrease of γ̇ from 500 to 0s⁻¹ in 3min (downward flow curve). The apparent viscosity ηa (at 50s⁻¹) and the area between the upward and downward flow curve (A_H) referred to the hysteresis loop was calculated with RheoCompass™ Software version 3.0× (Anton Paar).
Oscillatory measurements with a shear strain (γ) amplitude sweep were performed to determine the linear viscoelastic range (LVR). The γ was varied in a logarithmic ramp from 0.01 to 100% at a constant angular frequency (ω) of 10rad/s. Subsequently, a small strain frequency sweep was carried out at a constant γ of 0.05% within the LVR. ω from 100rad/s to 0.1rad/s was recorded. Three parameters were calculated: i) yield point γ<sub>y</sub> indicates structural change when the slopes of curves changed (tolerance: ± 5 %), ii) flow point γ<sub>f</sub>, at intercept point of G' and G'' (Mezger, 2012) and iii) the elastic G', storage G" and complex modulus G*

Syneresis (water-holding capacity) was measured by the centrifugation method. Briefly, the MAs were filled in 50mL centrifuge tubes (Greiner Bio-One, Kremsmünster, Austria), which were weighed prior to filling, inoculated, incubated and cooled for 24h as described in Section 2.3. Full tubes were centrifuged at 877 g at 20°C for 10min. The separated water was collected and weighed. Syneresis was calculated as the percentage of exceeded water in relation to the yoghurt sample (Amatayakul et al., 2006).

## 2.6 Sensory analyses

Sensory characterisation of YAs made from almond and chickpea MAs followed DIN 10969 standards for descriptive sensory analysis. The study involved two sessions: a one-hour session for attribute selection and definition, focusing on retronasal aroma, taste, and mouthfeel/texture, followed by a 30-min session, involved panellists individually rating the intensity of these attributes on a 4-point scale (0 = not perceptible to 3 = strongly perceptible). Assessments were conducted by a panel of eight trained people (six women, two men), experts in sensory evaluation of plant-based products, in sensory analysis test rooms, minimising external influences in accordance with DIN ISO 8589:2007 standards. Samples were served in glass containers with three-digit random codes and maintained at 6 - 8°C and randomized to reduce order effects. The sensory evaluation was conducted in accordance with the Declaration of Helsinki (2013). This type of low-risk research involves trained adult panellists and is not subjected to an ethics committee approval. All participants were fully informed about the study's purpose and procedures, gave verbal consent, and could withdraw at any time. No personal data was collected, and participants' rights and privacy were protected throughout the study.

## 2.7 Immunomodulation test on Human epithelial intestinal cells

### 2.7.1 HT-29 cell challenging

HT-29 cells were first cultivated in T-25 flasks in complete Dulbecco modified Eagle medium (DMEMc, Dominique Dutscher, Brumath, France) supplemented with 10% (v/v) fetal calf serum (PAN-Biotech GmbH, Aidenbach, Germany), 100U/mL penicillin and 100µg/mL streptomycin sulphate (Sigma-Aldrich) at 37°C with 5% CO₂. Trypsin (0.05%)/EDTA (0.2%) (Gibco, Saint Aubin, France) was used to release adherent cells for subculturing. HT-29 cells were seeded in 24-well microtiter plates at a density of 10⁵ cells/well in volume of 1mL. The growth medium was changed every two days until complete confluence, i.e. 10⁶ cells per well in 1 mL volume. Twenty-four h before cell challenging, the culture medium was discarded and the cells were washed with PBS pH 7.4 (Dominique Dutscher). One mL of fresh DMEMc containing 10ng/mL of lipopolysaccharide (LPS) from *Escherichia coli* 0111: B4 (Sigma Aldrich) was added to cells composing the inflammatory group. For the non-inflammatory group, fresh DMEMc containing sterile distilled water was added. The two groups of cells were incubated in the presence of 10µL of each YA described above for 24h in 5% CO₂ at 37°C. Inflammatory and non-inflammatory controls, with non-fermented MA, were included. All samples and controls were performed in triplicate. The percentage of HT-29 cell viability after the different stimulation conditions was checked by trypan blue staining, and the cell viability was not affected (1.2 10⁶ cells/well). After 24h of HT-29 cell stimulation, the plates were centrifuged (800 g × 10min, 4°C), the supernatant was collected and transferred to 96-well plates containing 5µL/well of protease inhibitor (Sigma-Fast). The 96-well plates were immediately transferred to -20°C and then to -80°C for further ELISA assays.

### 2.7.2 Cytokines quantification

Levels of IL-8 cytokine production in the HT-29 cell supernatants were quantified, in triplicate, in 96 well plates (Nunc Immuno MaxiSorp, Thermo Electron LED) using commercially available ELISA kits (BD Biosciences, Pont de Claix, France) in accordance with the manufacturer's instructions.
Absorbance was read at 450nm using microplate spectrophotometer system (Bio teck instruments, Inc, Winooski, USA). Quantified data presented is the total concentration (pg/mL) of IL-8 produced.

## 2.8 Statistical Analyses

Analyses of variance (ANOVA) were performed using the FactoMineR package of R software (R Core Team, 2013) to determine whether the variation of MA composition, the fermentation period or interaction of both, significantly influenced lactic acid bacterial cell counts, biochemical composition (residual sugars, organic acids, proteolysis) and immunomodulation followed, in case of significant results by a post hoc Fisher's least significant difference test using the agricolae R package.

Sensory data were collected using RedJade Sensory Analysis Software (RedJade Sensory Solutions, LLC, USA) and analysed using XLSTAT-Sensory® software (Addinsoft, Paris, France). The statistical analyses included an analysis of variance (ANOVA), followed by a Duncan post hoc test ($\alpha$ = 0.05), and a principal component analysis (PCA). PCA was performed on the mean scores of the significant sensory attributes, based on the correlation matrix of standardised data. The first two principal components were retained for interpretation as these explained the largest proportion of variance.

# 3 Results

## 3.1 *Variable LAB growth according to the ratio of the almond and chickpea MAs*

The population level of the two LAB strains was determined before and after fermentation of almond and chickpea MAs and the mixes thereof, when the pH dropped to 4.5, as shown in Figure 1. The different MAs were inoculated with an average population level of 4.65 $10^6 \pm 1.09 \ 10^6$ and $1.22 \ 10^7 \pm 1.45 \ 10^6$ colony forming unit (CFU) /mL for St772 and Lh100 strains, respectively. Limited growth of St772 was observed in the almond YAs (100A) and in mixes 75A (Figure 1A). With increasing chickpea ratio, higher populations of St772 were reached at the end of fermentation time, in the 50A, 25A and 100CP, leading to a maximum population of $5.23 \ 10^7$ CFU/mL in the 25A but with no significant differences between YA. Limited growth of LH100 was observed in the almond and chickpea YAs (100A and 100CP) (Figure 1B), while higher populations were reached in all mixes, with a significant increase in 75A, 50A and 25A, respectively, leading to $10^8$ CFU/mL at the end of fermentation.
<figure><img src="image_3.png" /></figure>

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

**Figure 1.** LAB starter growth in yoghurt analogues (YAs) with various almond and chickpea ratios. Growth of (A) *Streptococcus salivarius* subsp. *thermophilus* CIRM-BIA772 and of (B) *Lactobacillus helveticus* CIRM-BIA100 was determined in plant-based milk analogues (MAs). MAs were 100A: 100% almond; 75A: 75% almond-25% chickpea; 50A: 50% almond-50% chickpea; 25A: 25% almond-75% chickpea; and 100CP: 100% chickpea. T0: matrices prior to fermentation; TF: matrices at the end of the fermentation time when pH dropped to 4.5. The results are expressed as mean ± SD of triplicate biological determinations and two technical replicates. Letters (a,b,c) indicate statistical differences between the means observed for the types of matrices and the time of fermentation at p values <0.001.

## 3.2 Carbohydrate utilisation and organic acid production in the almond and chickpea YAs

The initial overall content in carbohydrates globally varied between 8.3g/L for the almond MA (100A) and 16.4g/L for the chickpea MA (100CP) and with a gradual increase in the mixed MAs between both values (Figure 2A, T0). In almond MAs, sucrose was the main carbohydrate (87.2%). The other carbohydrates were raffinose 6%, glucose 2.5%, stachyose 2.4% and fructose 1.4%. Chickpea MA contained mainly glucose (70.2%), followed by 24.3% maltose, 2.5% sucrose, 1.4% fructose and less than 2% raffinose and stachyose. The high glucose and maltose content may result from the hydrolysis of starch by amylases during the chickpea MA preparation.
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** Concentrations of carbohydrates (A) and organic acids, (B) lactate and (C) pyruvate in YAs with various almond and chickpea ratios.</figcaption></figure>

The carbohydrates sucrose, glucose, maltose, raffinose, stachyose and fructose are expressed as a cumulative graph.

The almond and chickpea mixes were inoculated with *Streptococcus salivarius* subsp. *thermophilus* CIRM-BIA772 and *Lactobacillus helveticus* CIRM-BIA100. The various almond and chickpea ratios in the MAs are 100A: 100% almond; 75A: 75% almond-25% chickpea; 50A: 50% almond-50% chickpea; 25A: 25% almond-75% chickpea; and 100CP: 100% chickpea; T0: matrices prior to fermentation; TF: matrices at the end of the fermentation time when pH dropped to 4.5. The results are expressed as mean ± SD of triplicate biological
determinations. Letters (a to e) indicate statistical differences between the means observed for the types of matrices
× the time of fermentation at p values <0.001 for sucrose, glucose and maltose; P<0.01 for fructose, no statistical
difference for raffinose, and p values <0.001 for organic acids lactate and pyruvate.

Sucrose and maltose were the most consumed carbohydrates in all YAs (Figure 2A TF). This was shown in 25A and 100CP YAs, in line with the ability of St722 to hydrolyse sucrose and of Lh100 to hydrolyse maltose (Table 1). Glucose was highly consumed in 100A (-88%, estimated by ([Glu]~TF~-[Glu]~T0~)/[Glu]~T0~). In the other YAs, its consumption was counterbalanced by its release via sucrose (Glu-Fru) and maltose (Glu-Glu) hydrolysis, i.e.-0.6% in 75A up to +30.6% in 100CP. In this latter, glucose was above 11g/L, close to 100% of the overall carbohydrate content. Fructose was more produced from the sucrose hydrolysis than consumed by the bacterial strain Lh100, the only strain capable of using it (Table 1) in the different matrices. It was still high in 100A, compared to 100CP, with +71.1% and +6.0%, respectively. Raffinose and stachyose remained unchanged, in line with ST and LH inability to use them (Table 1).

LAB produced lactic acid from carbohydrates (Figure 2B), 4.8g/L in 100A and 3.2g/L in 100CP. Its concentration was higher in 75A and 50A, with 5.4 and 5.3g/L respectively. This is consistent with the bacterial populations reached in the various YAs, higher in almond-chickpea mixes than in 100A and 100CP. Pyruvate lies at the centre of LAB metabolism and serves as precursor of lactic acid production. It was also higher in 75A and 50A YA than in 100A and 100CP (Figure 2C).

3.3 Proteolysis in the almond and chickpea YAs

SDS-PAGE protein profiles of almond (100A) and chickpea (100CP) (Figure 3A) agreed well with those
described by Mandalari et al (2014), and Di Francesco et al (2024), respectively. Protein profiles
changed according to the ratio of almond and chickpea MAs used in the various mixes (Figure 3A).
After fermentation, bands corresponding to vicilin subunits of chickpea proteins greatly decreased (-
76% for 50A at TF for example) and even disappeared, as well as part of bands below the albumin
molecular mass. Contrastingly, the main almond protein, prunin, remained quite unchanged (<10%
intensity variation).
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /><figcaption>**Figure 3.** Distinct proteolytic indices in YAs with various almond and chickpea ratios: (A) modifications of the protein profile evidenced by SDS-PAGE; (B) peptides and free amino acids, expressed as a cumulative graph.</figcaption></figure>

The total amount of free NH₂ has been separated into the part related to free amino acids and the one related to peptides. The peptides were calculated by difference between total OPA values and free amino acid-related NH₂ groups, converted in OPA response using the free amino acid analysis.

The almond and chickpea mixes were inoculated with *Streptococcus salivarius* subsp. *thermophilus* CIRM-BIA772 and *Lactobacillus helveticus* CIRM-BIA100. The various almond and chickpea ratios are 100A: 100% almond; 75A: 75% almond-25% chickpea; 50A: 50% almond-50% chickpea; 25A: 25% almond-75% chickpea;
and 100CP: 100% chickpea; T0: matrices prior to fermentation; TF: matrices at the end of the fermentation when pH dropped to 4.5. The results regarding peptides and free amino acids are expressed as mean ± SD of triplicate biological determinations and three technical replicates. Letters (a to e) indicate statistical differences between the means observed for the types of matrices and the time of fermentation at p values <0.001.

Proteolysis was confirmed by changes in the overall content of free amino acids and peptides, as shown in Figure 3B. The almond MA 100A at T0, was mainly composed of an 80/20 free amino acids/peptides ratio, while the chickpea MA 100CP had a 50/50 ratio and the mixes a gradual ratio between both values. After fermentation, the free amino acid content decreased only in 100A, confirming use of nitrogen compounds already present in almond MA and not from the prunin protein (Figure 3A). Contrastingly, in 100CP and in the different mixes, a higher production of both peptides and free amino acids was observed. Proteolysis was maximal in 50A, with 262.1 and 582.3mg NH₂/L of peptides and free amino acids, respectively (Figure 3B).

Almond MA contained high amounts of asparagine (Asn), Glutamic acid (Glu), γ-aminobutyric acid (GABA), Leucine (Leu), valine (Val), proline (Pro), alanine (Ala) and arginine (Arg) (Table 2), in agreement with the amino acid composition of prunin protein. The chickpea MA contained three times less free amino acids than the almond MA, i.e. 168.2 vs 510.3mg/L (Figure 3B) and was mainly composed of Arg, tryptophan (Trp), Glu, cysteine (Cys), Leu and Val (Table 2). After fermentation, GABA, that can be synthesised from Glu by various LAB, increased in all YAs with higher final concentration in 100A, 75A and 50A, and lower amount in 25A and 100CP (Table 2). Regarding the essential amino acid category completed with Cys and tyrosine (Tyr) that can be synthesised from Met and Phe respectively (Table 2), there was at least maintenance of the initial concentration or an increase in their content after fermentation, with highest significant value in the 50A TF. An exception is for Met that was not present in 100CP YA anymore, but the Cys concentration may compensate this absence. The other amino acids were also significantly highly represented in the 50A after fermentation (Table 2).

<table><caption>**Table 2.** Release of free amino acids (FAA) in yoghurt alternatives with various almond and chickpea ratios with acidic amino acids and GABA, essential amino acids + Cys and Tyr, and other amino acids. The free amino acid content is expressed in mg/L.</caption><thead><tr><th rowspan="2"></th><th colspan="2">100A</th><th colspan="2">75A</th><th colspan="2">50A</th><th colspan="2">25A</th><th colspan="2">100CP</th></tr><tr><th>T0<br>Mean<sup>1</sup> ± SD</th><th>TF<br>Mean ± SD</th><th>T0<br>Mean ± SD</th><th>TF<br>Mean ± SD</th><th>T0<br>Mean ± SD</th><th>TF<br>Mean ± SD</th><th>T0<br>Mean ± SD</th><th>TF<br>Mean ± SD</th><th>T0<br>Mean ± SD</th><th>TF<br>Mean ± SD</th></tr></thead><tbody><tr><td colspan="11"><strong>Acidic FAA+ GABA</strong></td></tr><tr><td>Asp</td><td>33.2 ± 0.6<sup>c2</sup></td><td>11.1 ± 1.5<sup>b</sup></td><td>25.2 ± 0.6<sup>d</sup></td><td>1.6 ± 0.1<sup>a</sup></td><td>17.7 ± 0.0<sup>c</sup></td><td>1.5 ± 0.0<sup>a</sup></td><td>9.6 ± 0.2<sup>b</sup></td><td>1.3 ± 0.1<sup>a</sup></td><td>1.7 ± 0.1<sup>a</sup></td><td>1.5 ± 0.1<sup>a</sup></td></tr><tr><td>Asn</td><td>163.0 ± 1.5<sup>b</sup></td><td>116.4 ± 1.4<sup>g</sup></td><td>123.4 ± 2.2<sup>g</sup></td><td>97.6 ± 1.8<sup>f</sup></td><td>85.4 ± 0.1<sup>c</sup></td><td>77.4 ± 4.5<sup>d</sup></td><td>44.5 ± 0.5<sup>c</sup></td><td>20.5 ± 1.7<sup>b</sup></td><td>4.1 ± 0.3<sup>a</sup></td><td>8.1 ± 0.5<sup>a</sup></td></tr><tr><td>Glu</td><td>84.0 ± 1.0<sup>e</sup></td><td>3.3 ± 0.2<sup>a</sup></td><td>65.5 ± 1.3<sup>f</sup></td><td>20.9 ± 1.6<sup>c</sup></td><td>47.8 ± 0.1<sup>c</sup></td><td>35.8 ± 2.5<sup>d</sup></td><td>29.0 ± 0.2<sup>d</sup></td><td>17.7 ± 3.9<sup>bc</sup></td><td>10.4 ± 0.4<sup>ab</sup></td><td>14.6 ± 0.7<sup>bc</sup></td></tr><tr><td>Gln</td><td>5.9 ± 0.3<sup>ab</sup></td><td>1.0 ± 0.2<sup>a</sup></td><td>5.0 ± 0.2<sup>ab</sup></td><td>35.2 ± 2.1<sup>cd</sup></td><td>3.8 ± 0.2<sup>ab</sup></td><td>56.6 ± 8.0<sup>d</sup></td><td>2.5 ± 0.1<sup>ab</sup></td><td>18.9 ± 4.2<sup>bc</sup></td><td>1.5 ± 0.1<sup>a</sup></td><td>15.9 ± 0.8<sup>ab</sup></td></tr><tr><td>GABA<sup>3</sup></td><td>23.9 ± 0.3<sup>c</sup></td><td>65.7 ± 1.6<sup>g</sup></td><td>17.9 ± 0.2<sup>d</sup></td><td>41.1 ± 1.0<sup>f</sup></td><td>12.4 ± 0.1<sup>c</sup></td><td>19.7 ± 1.5<sup>d</sup></td><td>6.6 ± 0.1<sup>b</sup></td><td>12.4 ± 0.1<sup>c</sup></td><td>0.7 ± 0.1<sup>a</sup></td><td>8.4 ± 1.0<sup>b</sup></td></tr><tr><td colspan="11"><strong>Essential FAA + Cys and Tyr</strong></td></tr><tr><td>Cys<sup>4</sup></td><td>3.2 ± 0.4<sup>a</sup></td><td>2.9 ± 0.4<sup>ab</sup></td><td>4.4 ± 0.2<sup>ab</sup></td><td>4.3 ± 0.1<sup>b</sup></td><td>5.9 ± 0.5<sup>c</sup></td><td>5.4 ± 0.6<sup>c</sup></td><td>7.0 ± 0.4<sup>cd</sup></td><td>7.1 ± 0.1<sup>cd</sup></td><td>7.6 ± 0.5<sup>d</sup></td><td>8.0 ± 0.4<sup>d</sup></td></tr><tr><td>Met</td><td>1.7 ± 0.1<sup>e</sup></td><td>1.3 ± 0.1<sup>cde</sup></td><td>1.6 ± 0.1<sup>de</sup></td><td>1.6 ± 0.0<sup>de</sup></td><td>1.3 ± 0.0<sup>cd</sup></td><td>0.4 ± 0.1<sup>b</sup></td><td>1.0 ± 0.1<sup>bc</sup></td><td>0.8 ± 0.0<sup>b</sup></td><td>0.9 ± 0.1<sup>bc</sup></td><td>0.0 ± 0.0<sup>a</sup></td></tr><tr><td>Ile</td><td>7.3 ± 0.3<sup>f</sup></td><td>1.4 ± 0.1<sup>a</sup></td><td>6.2 ± 0.2<sup>ef</sup></td><td>1.6 ± 0.0<sup>a</sup></td><td>5.1 ± 0.0<sup>de</sup></td><td>4.1 ± 0.2<sup>bc</sup></td><td>3.8 ± 0.0<sup>cd</sup></td><td>1.9 ± 0.7<sup>ab</sup></td><td>2.2 ± 0.3<sup>ab</sup></td><td>2.0 ± 0.1<sup>ab</sup></td></tr><tr><td>Leu</td><td>6.2 ± 0.3<sup>ab</sup></td><td>3.5 ± 0.4<sup>a</sup></td><td>6.3 ± 0.4<sup>ab</sup></td><td>6.2 ± 0.3<sup>ab</sup></td><td>6.4 ± 0.1<sup>ab</sup></td><td>14.6 ± 0.9<sup>b</sup></td><td>6.5 ± 0.1<sup>ab</sup></td><td>6.2 ± 1.1<sup>ab</sup></td><td>6.4 ± 0.2<sup>ab</sup></td><td>6.5 ± 0.3<sup>ab</sup></td></tr><tr><td>Val</td><td>12.4 ± 0.2<sup>bc</sup></td><td>4.8 ± 0.2<sup>a</sup></td><td>10.7 ± 0.4<sup>bc</sup></td><td>8.3 ± 0.2<sup>abc</sup></td><td>9.0 ± 0.1<sup>abc</sup></td><td>17.3 ± 1.0<sup>c</sup></td><td>7.4 ± 0.3<sup>ab</sup></td><td>8.9 ± 0.9<sup>abc</sup></td><td>6.7 ± 0.2<sup>ab</sup></td><td>9.3 ± 0.2<sup>abc</sup></td></tr><tr><td>Phe</td><td>11.9 ± 0.3<sup>ef</sup></td><td>12.1 ± 0.5<sup>ef</sup></td><td>9.9 ± 0.2<sup>def</sup></td><td>8.9 ± 0.2<sup>cde</sup></td><td>8.2 ± 0.2<sup>bcd</sup></td><td>14.4 ± 0.7<sup>f</sup></td><td>6.3 ± 0.1<sup>abc</sup></td><td>7.9 ± 0.7<sup>bcd</sup></td><td>4.5 ± 0.2<sup>a</sup></td><td>5.5 ± 0.4<sup>ab</sup></td></tr><tr><td>Thr</td><td>4.5 ± 0.1<sup>e</sup></td><td>2.8 ± 0.4<sup>bcd</sup></td><td>3.8 ± 0.1<sup>cde</sup></td><td>3.6 ± 0.2<sup>cde</sup></td><td>3.3 ± 0.1<sup>bcd</sup></td><td>5.1 ± 0.3<sup>de</sup></td><td>2.6 ± 0.1<sup>abcd</sup></td><td>1.7 ± 0.4<sup>ab</sup></td><td>2.3 ± 0.0<sup>abc</sup></td><td>1.0 ± 0.2<sup>a</sup></td></tr><tr><td>Lys</td><td>8.2 ± 0.2<sup>cd</sup></td><td>1.1 ± 0.1<sup>a</sup></td><td>7.4 ± 0.2<sup>bcd</sup></td><td>2.7 ± 1.3<sup>ab</sup></td><td>6.6 ± 0.1<sup>bcd</sup></td><td>13.8 ± 1.1<sup>de</sup></td><td>5.8 ± 0.1<sup>abcd</sup></td><td>9.6 ± 1.9<sup>cde</sup></td><td>5.1 ± 0.2<sup>abc</sup></td><td>14.7 ± 0.1<sup>e</sup></td></tr><tr><td>His</td><td>4.6 ± 0.1<sup>bcd</sup></td><td>6.8 ± 0.3<sup>de</sup></td><td>3.5 ± 0.2<sup>abc</sup></td><td>8.3 ± 0.3<sup>ef</sup></td><td>3.1 ± 0.1<sup>ab</sup></td><td>11.0 ± 0.7<sup>f</sup></td><td>2.4 ± 0.2<sup>ab</sup></td><td>6.6 ± 0.6<sup>cde</sup></td><td>1.7 ± 0.2<sup>a</sup></td><td>5.7 ± 0.1<sup>cd</sup></td></tr><tr><td>Trp</td><td>6.3 ± 0.2<sup>a</sup></td><td>4.1 ± 0.2<sup>b</sup></td><td>8.5 ± 1.1<sup>d</sup></td><td>5.0 ± 1.1<sup>ab</sup></td><td>9.9 ± 0.3<sup>de</sup></td><td>6.3 ± 0.4<sup>bc</sup></td><td>12.2 ± 0.4<sup>f</sup></td><td>8.4 ± 0.6<sup>cd</sup></td><td>15.9 ± 0.3<sup>g</sup></td><td>10.6 ± 0.6<sup>ef</sup></td></tr><tr><td>Tyr</td><td>5.7 ± 0.2<sup>f</sup></td><td>2.1 ± 0.1<sup>b</sup></td><td>5.0 ± 0.2<sup>ef</sup></td><td>1.9 ± 0.0<sup>b</sup></td><td>4.3 ± 0.0<sup>de</sup></td><td>2.9 ± 0.1<sup>bc</sup></td><td>3.5 ± 0.1<sup>cd</sup></td><td>0.3 ± 0.6<sup>a</sup></td><td>2.8 ± 0.1<sup>bc</sup></td><td>0.0 ± 0.1<sup>a</sup></td></tr><tr><td colspan="11"><strong>Others FAA</strong></td></tr><tr><td>Ala</td><td>23.0 ± 0.4<sup>ab</sup></td><td>14.6 ± 0.6<sup>ab</sup></td><td>18.3 ± 0.6<sup>ab</sup></td><td>70.0 ± 3.3<sup>c</sup></td><td>13.5 ± 0.2<sup>ab</sup></td><td>98.6 ± 2.3<sup>c</sup></td><td>8.6 ± 0.2<sup>a</sup></td><td>39.9 ± 3.5<sup>b</sup></td><td>3.9 ± 0.2<sup>a</sup></td><td>24.7 ± 2.3<sup>ab</sup></td></tr><tr><td>Arg</td><td>28.2 ± 0.8<sup>a</sup></td><td>41.0 ± 1.1<sup>ab</sup></td><td>40.2 ± 1.1<sup>ab</sup></td><td>76.4 ± 1.1<sup>de</sup></td><td>51.9 ± 0.5<sup>bc</sup></td><td>105.7 ± 2.5<sup>ef</sup></td><td>63.5 ± 0.2<sup>cd</sup></td><td>92.8 ± 1.2<sup>fg</sup></td><td>78.9 ± 4.3<sup>ef</sup></td><td>105.3 ± 1.6<sup>g</sup></td></tr><tr><td>Gly</td><td>10.9 ± 0.2<sup>cde</sup></td><td>9.7 ± 0.3<sup>bcd</sup></td><td>8.5 ± 0.3<sup>abc</sup></td><td>15.9 ± 0.7<sup>de</sup></td><td>6.2 ± 0.2<sup>abc</sup></td><td>22.3 ± 1.6<sup>c</sup></td><td>3.9 ± 0.1<sup>abc</sup></td><td>5.7 ± 1.3<sup>abc</sup></td><td>1.4 ± 0.0<sup>a</sup></td><td>2.7 ± 0.4<sup>ab</sup></td></tr><tr><td>Pro</td><td>48.8 ± 1.4<sup>f</sup></td><td>53.1 ± 0.3<sup>g</sup></td><td>36.5 ± 0.8<sup>e</sup></td><td>48.8 ± 0.5<sup>f</sup></td><td>25.1 ± 0.1<sup>d</sup></td><td>40.0 ± 0.3<sup>c</sup></td><td>12.9 ± 0.4<sup>c</sup></td><td>21.6 ± 0.7<sup>d</sup></td><td>1.4 ± 0.1<sup>a</sup></td><td>8.4 ± 0.4<sup>b</sup></td></tr><tr><td>Ser</td><td>9.4 ± 0.1<sup>c</sup></td><td>8.1 ± 0.4<sup>bc</sup></td><td>7.3 ± 0.1<sup>bc</sup></td><td>16.4 ± 0.5<sup>de</sup></td><td>5.4 ± 0.4<sup>abc</sup></td><td>22.6 ± 0.5<sup>c</sup></td><td>3.4 ± 0.2<sup>ab</sup></td><td>14.6 ± 0.9<sup>d</sup></td><td>1.7 ± 0.1<sup>a</sup></td><td>14.4 ± 1.0<sup>d</sup></td></tr></tbody></table>

## 3.4 Composition, rheology and syneresis

Almond-based (100A) and chickpea-based (100CP) MA showed comparable protein contents, while the dry matter content was slightly higher in 100CP compared to 100A (Table 3).

<footnote n="1">¹The results are expressed as mean ± SD of triplicate determinations</footnote>

<footnote n="2">²Superscript letters indicate statistical differences in the same row between the means observed for the types of matrices × the time of fermentation at p values <0.05 except for Tyr with p value<0.1.</footnote>

<footnote n="3">³Cys has non-significative difference of the matrix × time of fermentation, but there was significative difference of the matrix at p value <0.001 as shown in the table.</footnote>

<footnote n="4">⁴GABA: γ-amino butyric acid</footnote>

<footnote>The various almond and chickpea ratios in the yoghurt alternatives are 100A: 100% almond; 75A: 75% almond-25% chickpea; 50A: 50% almond-50% chickpea; 25A: 25% almond-75% chickpea; and 100CP: 100% chickpea; T0: matrices prior to fermentation; TF: matrices at the end of the fermentation time when pH dropped to 4.5.</footnote>
<table><caption>**Table 3.** Dry matter and protein content of almond-based (100A) and chickpea-based (100CP) milk alternatives. Values are given as mean ± standard deviation.</caption><thead><tr><th>Sample</th><th>Dry matter [%]</th><th>Protein [g/100g]</th></tr></thead><tbody><tr><td>100A</td><td>6.8 ± 0.05<sup>a</sup></td><td>2.87 ± 0.05<sup>a</sup></td></tr><tr><td>100CP</td><td>7.0 ± 0.03<sup>a</sup></td><td>2.93 ± 0.04<sup>a</sup></td></tr></tbody></table>

Different letters indicate significant differences with P<0.05.

Three different rheological tests were carried out to evaluate the influence of the initial raw materials on the final textural properties of the YAs (Table 4 and Figure 4). All samples showed shear thinning behaviour and ηa decreased rapidly with an increasing ẏ at the beginning of the measurement and then decreased slowly, remaining almost constant (Table 4). Samples with a higher almond content had a much lower ηa than those with a higher chickpea content, ranging from 222 to 937mPas for 100A and 100CP, respectively at ẏ = 50s⁻¹.

<table><caption>**Table 4.** Mean values of the rheological parameters and extent of syneresis of all samples</caption><thead><tr><th rowspan="2">Parameter</th><th colspan="5">Samples</th></tr><tr><th>100A</th><th>75A</th><th>50A</th><th>25A</th><th>100CP</th></tr></thead><tbody><tr><td colspan="6"><strong>Rotational</strong></td></tr><tr><td>ηa (ẏ = 10 s<sup>-1</sup>) [mPas]</td><td>803 ± 160<sup>a</sup></td><td>781 ± 61<sup>a</sup></td><td>1113 ± 82<sup>b</sup></td><td>1702 ± 114<sup>c</sup></td><td>2322 ±</td></tr><tr><td>ηa (ẏ = 50 s<sup>-1</sup>) [mPas]</td><td>222 ± 36<sup>a</sup></td><td>300 ± 24<sup>b</sup></td><td>457 ± 35<sup>c</sup></td><td>689 ± 48<sup>d</sup></td><td>937 ±</td></tr><tr><td>ηa (ẏ = 100 s<sup>-1</sup>) [mPas]</td><td>126 ± 20<sup>a</sup></td><td>168 ± 12<sup>b</sup></td><td>245 ± 18<sup>c</sup></td><td>364 ± 24<sup>d</sup></td><td>487 ±</td></tr><tr><td>ηa (ẏ = 200 s<sup>-1</sup>) [mPas]</td><td>73 ± 10<sup>a</sup></td><td>89 ± 6<sup>b</sup></td><td>124 ± 8<sup>c</sup></td><td>178 ± 10<sup>d</sup></td><td>235 ±</td></tr><tr><td>A<sub>H</sub> [Pas<sup>-1</sup>]</td><td>2244 ± 419<sup>a</sup></td><td>3007 ± 260<sup>b</sup></td><td>4250 ± 439<sup>c</sup></td><td>6528 ± 542<sup>d</sup></td><td>9072 ±</td></tr><tr><td colspan="6"><strong>Amplitude sweep</strong></td></tr><tr><td>G' (yield point) [Pa]</td><td>98 ± 16<sup>a</sup></td><td>94 ± 10<sup>a</sup></td><td>126 ± 19<sup>b</sup></td><td>223 ± 16<sup>c</sup></td><td>325 ±</td></tr><tr><td>G'' (yield point) [Pa]</td><td>25 ± 3<sup>a</sup></td><td>25 ± 2<sup>a</sup></td><td>33 ± 4<sup>b</sup></td><td>61 ± 3<sup>c</sup></td><td>89 ±</td></tr><tr><td>γ<sub>y</sub> (yield point) [%]</td><td>3.4 ± 1.7<sup>a</sup></td><td>2.6 ± 1.6<sup>a</sup></td><td>2.4 ± 1.9<sup>a</sup></td><td>3.5 ± 1.3<sup>a</sup></td><td>3.5 ±</td></tr><tr><td>G', G'' (flow point) [Pa]</td><td>NA</td><td>8.3 ± 0.9<sup>a</sup></td><td>14.3 ± 2.3<sup>b</sup></td><td>32.4 ± 2.4<sup>c</sup></td><td>47.9 ±</td></tr><tr><td>γ<sub>f</sub> (flow point) [%]</td><td>NA</td><td>81.7 ± 8.3<sup>a</sup></td><td>56.9 ± 6.4<sup>b</sup></td><td>41.8 ± 3.6<sup>c</sup></td><td>42.9 ±</td></tr><tr><td colspan="6"><strong>Frequency sweep</strong></td></tr><tr><td>G' (ω = 10 rad/s) [Pa]</td><td>128 ± 50<sup>b</sup></td><td>90 ± 9<sup>a</sup></td><td>140 ± 7<sup>b</sup></td><td>243 ± 17<sup>c</sup></td><td>349 ±</td></tr><tr><td>G'' (ω = 10 rad/s) [Pa]</td><td>30 ± 12<sup>b</sup></td><td>22 ± 2<sup>a</sup></td><td>34 ± 2<sup>b</sup></td><td>60 ± 4<sup>c</sup></td><td>86 ±</td></tr><tr><td>Extent of syneresis [%]</td><td>53.6 ± 6.2<sup>a</sup></td><td>43.2 ± 4.7<sup>b</sup></td><td>25.3 ± 3.2<sup>c</sup></td><td>9.4 ± 1.5<sup>d</sup></td><td>4.3 ±</td></tr></tbody></table>

Superscript letters indicate statistical differences in the same row (p < 0.05). ηa = apparent viscosity; A<sub>H</sub> = hysteresis loop area; ẏ = shear rate; G' = storage modulus; G'' = loss modulus; γ = shear strain; ω = angular frequency.
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 4:** A) Hysteresis loops, B) Amplitude sweeps and C) Frequency sweeps of YA 100A, 50A and 100CP at the end of the fermentation when pH dropped to 4.5.
100A: 100% almond; 50A: 50% almond and 50% chickpea, 100CP: 100% chickpea.</figcaption></figure>

Thixotropic properties were assessed via the $A_H$, a measure of regeneration after shear-induced structure breakdown (Table 4 and Figure 4B). Larger areas were observed in YAs with higher chickpea ratios, with a linear increase of $A_H$ ranging from 2244 to $9072 \text{Pas}^{-1}$ for 100A to 100CP respectively (Table 4) in accordance with the almond-chickpea ratios.

Amplitude sweeps were performed to characterise samples according to their $\gamma_y$ and $\gamma_f$ (Table 4 and Figure 4). Initially, G' and G'' remained constant with increasing $\gamma$ (Figure 4). Interestingly, G' and G'' were higher in YAs with increased chickpea content (100CP, 25A, 50A), indicating more pronounced viscoelastic properties than in samples with increased almond content (100A, 75A). However, G' values of the various samples did not increase linearly to the varying content of the raw materials; e.g. replacing 25% almond with chickpeas (100A, 75A) showed no statistically significant difference in G' and G''. No significant differences were found for the $\gamma_y$, marking the end of the linear viscoelastic region, among the different samples. The $\gamma_f$, indicating gel collapse, occurred at lower $\gamma$ in chickpea-rich samples. In contrast, in almond-rich samples, G'' curve bends stronger (Figure 4) and consequently G' crossed at higher $\gamma$. At 100A, no crossover point could even be detected.

Frequency sweeps showed that elastic properties dominated in the linear viscoelastic region, as all samples showed $G' > G''$ across the entire measuring range, resulting in linear curves (Table 4 and Figure 4) corresponding to soft fluid gels. Chickpea-rich samples showed significant higher G' and G'' values, indicating a higher gel rigidity. However, the increase was not linear: 100A and 75A showed no statistically significant difference, G' and G'' only increased significantly in 50A.

Regarding the susceptibility to syneresis (Table 4), 100A showed higher syneresis (~54%), than 100CP with only ~4% syneresis, and a linear decrease in the mixes enriched in chickpea.

## 3.5 Sensory analysis

The sensory properties of the YA samples were assessed using twelve attributes across three sensory dimensions: taste (sour and bitter), retronasal flavour perception (including watery, yoghurt-like, nutty, cereal-like, and plant-based notes) and texture or mouthfeel (such as thin, medium viscous, gritty, blunt/occupying, and mouth drying sensations). Six attributes varied significantly among YAs (Table 5 and Figure 5): sour, watery (neutral flavour, diluted taste), cereal-like aroma, thin, medium viscous, and gritty texture.
<table><caption>**Table 5.** Results of ANOVA (mean values) and post-hoc analysis (Duncan test) of the sensory attributes of fermented YA gels.</caption><thead><tr><th rowspan="2">Attributes</th><th colspan="5">Samples</th></tr><tr><th>100A</th><th>75A</th><th>50A</th><th>25A</th><th>100CP</th></tr></thead><tbody><tr><td>Sour*</td><td>1.4<sup>a</sup></td><td>2.8<sup>c</sup></td><td>2.8<sup>c</sup></td><td>2.1<sup>b</sup></td><td>1.6<sup>ab</sup></td></tr><tr><td>Cereal-like*</td><td>0.6<sup>a</sup></td><td>0.6<sup>a</sup></td><td>1.4<sup>A</sup></td><td>1.8<sup>b</sup></td><td>1.9<sup>b</sup></td></tr><tr><td>Thin*</td><td>2.5<sup>c</sup></td><td>2.4<sup>c</sup></td><td>1.6<sup>b</sup></td><td>1.0<sup>ab</sup></td><td>0.5<sup>a</sup></td></tr><tr><td>Medium viscous*</td><td>0.3<sup>a</sup></td><td>0.1<sup>a</sup></td><td>0.9<sup>ab</sup></td><td>1.4<sup>b</sup></td><td>2.4<sup>c</sup></td></tr><tr><td>Gritty*</td><td>1.9<sup>b</sup></td><td>0.4<sup>a</sup></td><td>0.1<sup>a</sup></td><td>0.0<sup>a</sup></td><td>0.0<sup>a</sup></td></tr><tr><td>Watery (neutral flavour)*</td><td>2.4<sup>b</sup></td><td>2.0<sup>b</sup></td><td>1.3<sup>a</sup></td><td>1.0<sup>a</sup></td><td>0.8<sup>a</sup></td></tr><tr><td>Yogurt-like</td><td>2.0</td><td>2.0</td><td>2.1</td><td>2.3</td><td>1.9</td></tr><tr><td>Nutty</td><td>0.5</td><td>0.8</td><td>1.3</td><td>1.3</td><td>1.3</td></tr><tr><td>Plant-based</td><td>1.3</td><td>1.6</td><td>1.4</td><td>1.6</td><td>1.5</td></tr><tr><td>Blunt/occupying</td><td>2.1</td><td>1.5</td><td>1.1</td><td>1.5</td><td>1.1</td></tr><tr><td>Bitter</td><td>0.5</td><td>0.8</td><td>0.8</td><td>0.5</td><td>0.9</td></tr><tr><td>Mouth drying</td><td>2.0</td><td>1.8</td><td>1.8</td><td>1.6</td><td>1.9</td></tr></tbody></table>

Significant attributes are marked with an asterisk (*), while non-significant attributes are printed in italics. (Different letters indicate significant differences for the respective attribute; significance level α ≤ 0.05).

<figure><img src="image_13.png" /><figcaption>**Figure 5:** Results of Principal Component Analysis (PCA) performed on the significant descriptors, showing the distribution of YA fermented gel samples along components 1 and 2. Key sensory attributes include sourness, watery (neutral flavour, diluted taste), cereal-like aroma, thin, medium viscous, and gritty texture. 100A: 100% almond; 75A: 75% almond-25% chickpea; 50A: 50% almond-50% chickpea; 25A: 25% almond-75% chickpea; and 100CP: 100% chickpea.</figcaption></figure>

The attribute 'sour' was most pronounced in samples 75A and 50A, and significantly higher than in 100A and 100CP (Table 5). 'Watery' was most notable in 100A, suggesting a diluted flavour, while
100CP had the lowest score at 0.8, indicating a more balanced taste. Cereal-like aroma was most significant in 100CP, compared to 75A and 100A, indicating a more pronounced cereal-like aroma in the 100CP sample (Table 5).

The ‘thin’ attribute was significantly higher in 100A and 75A while 100CP showed the lowest values, indicating a more fluid texture. Medium viscosity was strongest in 100CP, which was significantly higher than the scores for 75A and 100A. Although gritty texture scores were generally low, 100A had the highest score at 1.9 than in other samples with very low scores (Table 5).

Plant-based and yoghurt-like attributes were perceived consistently across samples: the plant-based note at relatively low intensities, indicating it was not a dominant characteristic, and the yoghurt-like impression at a moderate level. (Table 5). No significant difference was found for bitter, nutty, yoghurt-like, blunt/occupying, plant-based, and mouth drying.

The significant attributes were identified by PCA along the first two components. PC1 explained the largest proportion of variance, separating samples with a pronounced cereal-like aroma and medium viscosity (such as 100CP) from samples with a watery flavour and thin texture (notably 100A). PC2 contrasted sourness and gritty texture, indicating that 75A and 50A were perceived as sourer and less gritty, while 100A showed the opposite pattern. Although some grouping tendencies can be observed (e.g. chickpea-rich samples are positioned closer to each other along PC1), the limited sample size and overlap between samples prevent clear definition of distinct clusters. Instead, PCA illustrates general sensory tendencies that differentiate almond-rich formulations from chickpea-rich ones.

## 3.6 2.6 Immunomodulation properties

Immunomodulatory properties of the different almond and chickpea YAs were tested *in vitro*, on cultured HT29 human intestinal epithelial cells, both in the presence and in the absence of proinflammatory lipopolysaccharide (LPS) from *E. coli*. The concentrations of IL-8, involved in the inflammatory process (Figure 6) was monitored as a read-out. Unstimulated HT29 cells secreted low amounts of IL8, i.e. 387.5 ± 29.3pg/mL, in the absence of LPS. All non-inoculated MAs, almond, chickpea and mixes thereof increased IL-8 production by unstimulated cells to an average value of 5213.2 ± 545.5pg/ mL) (Figure 6, without LPS). This IL-8 secretion was decreased by 41%, 67% and 55% in the 50A TF, 25A TF and 100CP TF, respectively, upon fermentation.

<figure><img src="image_14.png" /></figure>

**Figure 6.** Immunomodulation properties of the YAs with various almond and chickpea ratios, expressed as interleukin 8 concentration variation determined by ELISA. IL-8 concentrations were measured in cell supernatants of HT-29 cells incubated with or without addition of pro-inflammatory lipopolysaccharide from *E. coli* (LPS, 10 ng/mL) prior to addition of YAs. The various almond and chickpea ratios in MAs are 100A: 100% almond; 75A: 75% almond-25% chickpea; 50A: 50% almond-50% chickpea; 25A: 25% almond-75% chickpea; and 100CP: 100% chickpea; T0: matrices prior to fermentation; TF: matrices at the end of the fermentation when pH dropped to 4.5. Cell control: HT29 without incubation with YAs. The results are expressed as mean ± SD of triplicate biological determinations and two technical replicates. Letters (a to d) indicate statistical differences
between the means observed for the types of matrices and the time of fermentation at p values <0.001 for the data
without addition of LPS, and p value <0.01 with LPS

The LPS proinflammatory stimulus, as expected, increased the basal level of inflammation as shown by
the high IL-8 secretion by LPS stimulated HT29-cells, 14,961.9± 1515.5 pg/mL (Figure 6, with LPS).
No effect of MAs was observed on inflammation, as this IL-8 response was of the same order of
magnitude for all MAs. However, after fermentation, this LPS-induced pro-inflammatory response was
significantly repressed in the YAs 50A, 25A TF and 100CP TF, by 29%, 42% and 54% reduction of
IL8, respectively.

4 Discussion

To develop sustainable and healthy plant-based fermented products, a number of criteria need to be met, such as: i) satisfactory sensory properties, ii) adequate nutritional values, including essential amino acids, and iii) the presence of live and active probiotic bacteria that can exert beneficial health effects. We therefore chose almond and chickpea MAs and different almond:chickpea ratios to assess whether mixing plant-based resources and fermenting them by two LABs could help to obtain desired sensory, textural, nutritional and immunomodulatory properties in YAs.

Regarding fermentation, we used the strain screening pipeline developed in a previous study (Illikoud et al., 2023) to preselect the two strains used in this study, *Streptococcus thermophilus* CIRM-BIA772 (St772) and *Lactobacillus helveticus* CIRM-BIA100 (Lh100), which showed interesting technological and probiotic properties. Figure 1 shows that both strains grew better in almond-chickpea mixes to reach populations above 10⁸ CFU/mL, leading to more than 10¹⁰ CFU in a YA portion of 100 g. These almond-chickpea mixes actually combined the different types of carbohydrates present in almond MA, *i.e.* sucrose, and in chickpea MA, *i.e.* mainly glucose and maltose due to starch hydrolysis during chickpea MA preparation. This allowed growth of the two LAB strains without any carbohydrate competition since St772 uses preferentially sucrose and Lh100 maltose as shown in Table 1, and glucose that can also be used by both strains was largely in excess in the mixes (Figure 2). The resulting acidification to pH 4.5, shall enhance the YA preservation and, together with high populations of St772 and Lh100 in the mixed YAs, shall prevent growth of spoilage or pathogenic microorganisms. Acidification further improved texture of the different almond and chickpea YAs (Table 4 and Figure 4). Fermentation by LAB also enhanced the overall sensory quality of the YAs since the attribute "yoghurt-like" was rated at 2 out of 3, regardless of the matrix. This is relevant for consumer acceptance, as a familiar taste may ease the transition to plant-based diets. Fermentation with LAB is a good lever to modulate the organoleptic properties of plant-based products by reducing typical plant-based notes (Canon, Maillard, et al., 2022; Zhang et al., 2022).

LAB strains improved nutritional qualities through the release of peptides and free amino (Figure 3B and Table 2). The various nitrogen compounds present in almond and chickpea also gave rise to higher content in peptides and free amino acids in the mixed YAs (Figure 3B) that may enhance bioavailability, especially regarding essential amino acids (Table 2). The higher amount of free amino acids and peptides in almond MA, compared to chickpea MA (Figure 3B), most likely may enhance initiation of growth for both strains before production of cell-wall proteases in the mixed YAs, answering LAB auxotrophies (Kunji et al., 1996). The chickpea proteins, namely vicilin subunits and albumin, were more hydrolysed, (Figure 3A), in contrast to prunin from almond, allowing better LAB growth in the mixed YAs. Of the two strains, Lh100 was proteolytic (Table 1) in agreement with other *L. helveticus* strains on various substrates (Liu et al., 2010; Panayotova et al., 2024; Sadat-Mekmene et al., 2013) whereas St772 failed to do so (Table 1), like most *S. thermophilus* strains (Markakiou et al., 2020). Proteolysis of chickpea proteins allowed metabolic interactions between Lh100 and St772 as is the case in yoghurt between *L. delbrueckii* subsp. *bulgaricus* and *S. thermophilus* species (Courtin et al., 2002; Markakiou et al., 2020). Peculiar amino acid and peptides provided by the strain Lh100 may enhance positive interactions (Canon, Briard-Bion, et al., 2022). Among the bioactive molecules produced during fermentation, GABA has notably antioxidant and sensory activity (Edalatian Dovom et al., 2023; Linares et al., 2016). LAB produce GABA as a defence mechanism to maintain their viability under acidic conditions, through the α-decarboxylation of glutamic acid (Santos-Espinosa et al., 2020). In our study, the production of GABA varied among the YAs (Table 3) with a maximal production in the almond YA,
100A (65mg/L at TF), related to the initial high content of free glutamic acid. In line with this, GABA was also produced in mixes, yet not (or very few) in chickpea (100CP) in which bacterial growth was more limited (Figure 1, and Table 2).

Finally, the LAB strains, previously selected for their immunomodulatory properties observed *in vitro* in human PBMCs (Illikoud et al., 2023), led to fermented products able to reduce *in vitro* inflammation in HT-29 cultured HIEC (Figure 6). LPS is a pro-inflammatory endotoxin produced by Gram-negative bacteria such as *E. coli*. It activates Toll-like receptor TLR4, triggering signalling cascades that activate the TRIF (Toll/IL-1R domain-containing adaptor-inducing IFN-β)-dependent pathway (Ciesielska et al., 2021). As a consequence, activation of the transcription nuclear factor κB (NF-κB) leads to enhanced secretion of IL-8. This induction was prevented in the presence of fermented mixed 50A and 25A YA, by an anti-inflammatory effect that remains unknown. However, probiotic bacteria were previously shown to mitigate the inflammatory response via Janus kinase/signal transduction and activator of transcription (JAK/STAT) and inflammatory signalling pathways (Aghamohammad et al., 2022), downregulating the JAK genes and TIRAP, IRAK4, NEMO, and RIP genes in the NF-κB pathway. Accordingly, LPS triggers excessive intestinal epithelial barrier permeability, while probiotics may protect its tightness (He et al., 2025).

Mixing almond and chickpea MAs was also an advantage in terms of improving texture and sensory properties (Table 4 and 5). The rheological tests illustrated that chickpea-rich samples exhibited a markedly higher ηα (235mPas for 100CP vs. 73mPas for 100A, at 200s⁻¹), indicating a denser and more viscous gel structure than almond-rich samples. In comparison, commercial products showed even higher viscosities (230-750mPas), likely due to stabilisers such as pectin, modified starch or gums. The 100CP YA (226mPas) approached the ηα of dairy yoghurt (240mPas), highlighting chickpea as a promising base. Higher protein content in some commercial products (soy and dairy) may further explain their higher ηα (Grasso et al., 2020). Thixotropic measurements showed increased A_H with higher chickpea content, indicating greater structural recovery after shear in contrast to almond YAs. This lower A_H value of almond YAs was also found by comparison with soy YAs and was interpreted by a greater resilience of almond YAs to structural deformation (Devnani et al., 2022). It would be the case if upward curves were in the same height, which was not evident in our study (Figure 4A). Regarding the viscoelastic properties of the samples, the chickpea-dominant samples showed higher G' and G'' reflecting enhanced gel rigidity and elasticity. However, this effect was not linear: while 100A and 75A showed no significant changes, a 50% substitution of chickpea in the formulation (50A) resulted in a significant increase of G' and G''. This non-linear trend suggests a threshold effect, reached only with a certain concentration of chickpea proteins. The wide range of G' and G'' values highlights the potential to tailor product texture to consumer preferences, as the rigidity and elasticity of YAs can directly influence mouthfeel and overall acceptability (Gupta et al., 2022).

Despite similar protein contents, almond proteins formed weaker gel networks, resulting in lower water retention (Table 4). This can probably be attributed to the varying gelling properties of chickpea and almond proteins during acid-induced gelation of the YA, which remains still less well understood especially for almond proteins. Acid-induced gelling properties have actually been mainly studied for soy globulins (i.e., 11S glycinin and 7S β-conglycinin), which are comparable to chickpea globulins (11S legumin, 7S vicilin) in terms of molecular characteristics (Chang et al., 2012; Grasso et al., 2022). Glycinin and conglycinin form strong gels, as acidification leads to protein unfolding, exposing hydrophobic and free sulfhydryl groups that facilitate extensive intermolecular interactions (He et al., 2024).

Sensory data reflect similar trends particularly regarding viscosity and water-holding capacity. The texture-related attributes, particularly thinness and medium viscosity, demonstrated clear compositional dependencies. Higher almond content was associated with a thinner texture, while chickpea-rich samples were perceived as more viscous. The slightly more pronounced gritty texture in 100A may be attributed to residual almond particulates or insufficient protein matrix formation, indicating less homogeneous microstructural integration and highlighting the need for optimized processing to improve smoothness. Despite comparable final pH values across all samples, sourness was differently perceived, particularly in almond-chickpea mixes. While 100A and 100CP showed the lowest sourness scores, 50A and 75A mixes were perceived as significantly sourer. This trend suggests that factors beyond pH – such as
buffering capacity, matrix interactions, or the distribution of organic acids – may amplify sourness perception. Similar observations have been reported in other complex food matrices, where interactions between minerals, proteins, and acids modulate taste intensity (Da Conceicao Neta et al., 2007; Engel et al., 2000).

## 5 Conclusion

Combining almond and chickpea MAs provides a balanced approach for optimising vegetable YAs. Chickpea MA contributes to increased viscosity and gel strength, while almond MA reduces the intensity of plant-based notes like cereal-like aroma. This was made also possible by selecting appropriate starter strains of bacteria to produce YA with enhanced technological, rheological, sensory and probiotic properties. This work opens new avenues for the development of plant-based functional products for target populations, in the context of the growing incidence of ailments linked to gut inflammation. Such products may be used as part of a preventive diet or as functional food supplements.

## 6 Acknowledgements

This research was funded by the LOCALNUTLEG project (Developing innovative plant-based added-value food products through the promotion of LOCAL Mediterranean NUT and LEGUME crops) and financed by PRIMA (Partnership for Research and Innovation in the Mediterranean Area) funded by the European programme H2020. The PRIMA programme is an Art. 185 initiative supported and funded under Horizon 2020, the European Union's Framework Programme for Research, and Innovation. Project identifier: Grant Agreement n° 2033.

**Data availability.** The data are available on the LOCALNUTLEG repository database with the following references: Gagnaire, Valérie; Jan, Gwénaël; Silande, Charles; Hickisch, Andrea, 2025, "WP2-3.3 Yogurt-type fermented probiotic gels", https://doi.org/10.57745/DPY89U, Recherche Data Gouv, V1, UNF:6:TekmS055X2kmC9VNqhXCsg== [fileUNF]

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