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# Meat by-products as a source of bioactive peptides and functional ingredients: Regulatory and safety barriers to valorization

Mohammed Gagaoua, Arun Das, Yu Fu, Amira Leila Dib, Pramod Kumar Nanda

► To cite this version:

Mohammed Gagaoua, Arun Das, Yu Fu, Amira Leila Dib, Pramod Kumar Nanda. Meat by-products as a source of bioactive peptides and functional ingredients: Regulatory and safety barriers to valorization. Current opinion in green and sustainable chemistry, 2024, 47, pp.100910. 10.1016/j.cogsc.2024.100910. hal-04502991v2

HAL Id: hal-04502991

https://hal.inrae.fr/hal-04502991v2

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ELSEVIER

# Meat by-products as a source of bioactive peptides and functional ingredients: Regulatory and safety barriers to valorization

Mohammed Gagaoua<fnref n="1" />, Arun K. Das<fnref n="2" />, Yu Fu<fnref n="3" />, Amira Leila Dib<fnref n="4" /> and Pramod Kumar Nanda<fnref n="2" />

<figure><img src="image_3.png" /></figure>

Proposals for sustainable use of meat industry waste and by-products have seen remarkable growth in the past decade. This study aims to shed light on the often-overlooked realm of meat by-products, positioning them as an invaluable source of bioactive peptides and functional ingredients. It emphasized in the first part the main strategies for the valorization of meat industry by-products into diverse bioactive peptides, and then it introduced in the second part the diverse and current methods of identification and characterization of bioactive peptides and protein hydrolysates. While the promise of these macromolecules is immense, the study focuses and takes an in-depth look in the third part at the regulatory and safety barriers hindering their efficient valorization. By addressing regulatory and safety concerns, this review aims to pave the way for a more sustainable and responsible utilization of meat by-products, ensuring not only the economic viability of the meat sector but also fostering a holistic and safe approach towards enhanced food and animal production sustainability.

## Addresses

Corresponding authors: Gagaoua, Mohammed (mohammed.gagaoua@inrae.fr); Nanda, Pramod Kumar (npk700@gmail.com)

**Current Opinion in Green and Sustainable Chemistry** 2024, 47:100910

This review comes from a themed issue on **Bioactive compounds from agri-food byproducts and wastes (2024)**

Edited by Khaoula Khwaldia and Abdennacer Boulila

Available online 13 March 2024

For complete overview of the section, please refer the article collection - **Bioactive compounds from agri-food byproducts and wastes (2024)**

https://doi.org/10.1016/j.cogsc.2024.100910

2452-2236/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Keywords

Meat industry by-products, Peptide bioactivities, Regulatory barriers to valorization, Safety barriers to valorization, Meat industry sustainability.

## Introduction

In recent decades, the surge in proposals for sustainable utilization of meat industry waste and by-products has marked a significant advancement in the field [1]. In fact, the meat industry, while producing 345 million metric tons of meat, also produces/generates significant quantities of by-products (approximately 155 million metric tons), including blood, skin, bones, trimmings, organs, viscera, feet, hoofs, horns, and skulls, among other materials, during the slaughtering and meat processing processes [1,2]. Therefore, it is essential to handle and dispose of these materials in an environmentally friendly manner. The by-products are potential sources for several molecules, among which peptides and protein hydrolysates can be used for several purposes. However, there are several technical steps and challenges in utilizing meat by-products for the recovery of bioactive peptides and functional ingredients [3,4]. This can be, for instance, illustrated by the bibliometric map analysis, which highlights the diversity of themes addressed around the recovery of bioactive peptides and/or protein hydrolysates from meat industry by-products (Figure 1). In fact, the biological activities of peptides derived from animal by-products are influenced by factors like the source of the proteins (animal, organ, status of the biological samples that can be fluid (blood) or tissue), peptide structure, amino acid sequence, molecular weight, processing conditions employed, etc [4,5]. Moreover, achieving commercial success is currently limited due to challenges of extraction, separation (fractionation), and purification conditions, low bioavailability, insufficient clinical trials determining safety and efficacy, and regulatory restrictions [6]. The conventional processes (fermentation, aging, homogenization, etc.) employed to generate peptides result in lower efficacy. On the other hand, fermentation and enzymatic methods are confronted with challenges in commercialization, as the processes of fermentation and enzymatic hydrolysis are unpredictable due to the variable efficiency of enzymes on substrates, processing time, cost, yield, etc.” [7]. Emerging technologies for the conversion of protein-rich by-products into peptides require expensive equipment, time-consuming procedures, knowledge of data analysis, etc. Hence, *in vitro* and *in vivo* studies, along with studies on the bioavailability, toxicity, allergenicity, and synergistic effects of peptides, are required before claiming nutritional, functional, and therapeutic effects [8].

<footnote n="1">¹ PEGASE, INRAE, Institut Agro, 35590, Saint-Gilles, France</footnote>

<footnote n="2">² Eastern Regional Station, ICAR-Indian Veterinary Research Institute, Kolkata, 700 037, India</footnote>

<footnote n="3">³ College of Food Science, Southwest University, Chongqing, 400715, China</footnote>

<footnote n="4">⁴ Laboratoire de la Gestion de la Santé et Productions Animales, Institut des Sciences Vétérinaires El-Khoub, Université Constantine 1 Frères Mentouri, Constantine 25000, Algeria</footnote>
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<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>

VoSViewer bibliometric map analysis of the 60 most frequent keywords from the articles in the field of meat by-products valorization for the production of bioactive peptides and protein hydrolysates (Scopus database search, 2020–2023). The size of the circles correlates with the number of occurrences. Keywords in the same cluster (same color) frequently appear in the same articles and create a specific group related to a thematic (e.g. source, bioactivity, methods, identified characteristics, etc.). The lines highlight the degree of relationship among the keywords and topics. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

While the potential of these macromolecules is immense, navigating their valorization is a complex task due to regulatory barriers. In this context, this paper places a spotlight on the regulatory and safety barriers that impede their efficient valorization. In fact, these barriers, rooted in diverse regional standards and safety protocols, pose challenges to the efficient utilization of these valuable by-products. From stringent approval processes to varying interpretations of safety guidelines, the regulatory landscape often impedes the streamlined incorporation of meat-derived peptides and protein hydrolysates into diverse industries. Understanding and addressing these regulatory hurdles is crucial for unlocking the full potential of these by-products, promoting sustainability, and harnessing the economic and nutritional benefits they offer to diverse sectors. Ultimately, this overview aims to pave the way for a more sustainable and

responsible approach to the utilization of meat by-products, ensuring economic viability, and fostering holistic and safe practices in food and animal production sustainability.

## Main strategies for valorization of meat by-products

### Generation and extraction methods of bioactive peptides

The current methods applied to generate protein hydrolysates comprise acid-base hydrolysis, microbial fermentation, and enzymatic treatments. Among these, acid-base hydrolysis methods are normally employed in industrial production. However, the corrosiveness of acidic and alkaline solutions poses a risk of damaging pipelines or reaction tanks, thereby imposing significant limitations on their industrial applications [9]. In fact, microbial fermentation imposes stringent requirements
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on starter culture, and the culture process for bacteria is both lengthy and intricate. In comparison with the traditional acid-base hydrolysis method, enzymatic hydrolysis is commonly used with several advantages, including mild reaction conditions, uniform protein hydrolysates, and the ability to minimize secondary degradation of peptides during enzymatic hydrolysis [10,11].

The main preparation processes of bioactive peptides are illustrated in Figure 2, which include raw material pretreatment, enzymatic hydrolysis, separation and purification, bioactivity evaluation, peptide identification, and activity verification. Firstly, raw material pretreatment involves procedures such as bone grinding, freeze-drying of viscera, and homogenization. Additional pretreatment methods include blood centrifugation and decolorization. These processes contribute to the degradation of the structure of the samples, thereby facilitating the entry of enzymes and thereby initiating protein hydrolysis. Enzymatic hydrolysis typically occurs in both batch reactors and continuous reactors. Upon reaching the desired degree of hydrolysis, heat treatment is normally employed to inactivate

the enzyme, thereby terminating the enzymatic hydrolysis reaction [12]. The commercial food-grade enzymes commonly used to extract bioactive peptides from meat by-products include papain, pepsin, alcalase, neutrase, flavorzyme, trypsin, etc [5].

The resultant protein hydrolysate is subsequently subjected to centrifugation for the removal of lipids and impurities, recovering the supernatant. The degree of hydrolysis and bioactivity of the obtained samples are further assessed. Subsequently, the most active components are further separated and purified using chromatography, membrane filtration, electrodialysis, etc [12]. For instance, reversed-phase high-performance liquid chromatography (RP-HPLC) involves the purification of peptides based on hydrophobicity. Membrane filtration (e.g. ultrafiltration) is commonly used for separating peptides on the basis of molecular weight. Electrodialysis can be employed for the rapid separation of peptides according to positive and negative charges. In addition, to optimize the screening process, in silico digestion using computer-assisted strategies has been used to identify bioactive peptides from meat by-

<figure><img src="image_5.png" /><figcaption>**Figure 2**</figcaption></figure>

flow chart summarizes the main steps allowing the generation of bioactive peptides from meat by-products, along with the methods and strategies used for their characterization. LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry, FTIR: Fourier transform infrared spectroscopy, NMR: nuclear magnetic resonance.
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<table><caption>Table 1
A nonexhaustive list of bioactive peptides from meat by-products.</caption><thead><tr><th>Bioactivity</th><th>Peptide sequence</th><th>Source</th><th>Enzymes used and operating conditions [temperature in °C and time in min or hours]</th><th>Results</th><th>Refs.</th></tr></thead><tbody><tr><td rowspan="10">Antioxidant capacity</td><td>N/A</td><td>Bovine bone collagen</td><td>Papain [65 °C, 20h]</td><td>The amount of Thiobarbituric acid reactive substance (TBARS) was 0.836 mg MDA/kg of meat.</td><td>[14]</td></tr><tr><td>TSKYR</td><td>Bovine hemoglobin</td><td>Pepsin [pH 3.5, 23 °C, 30min]</td><td>The amount of TBARS was 1.75 mg MDA/kg of meat.</td><td>[21]</td></tr><tr><td>N/A</td><td>Bovine meat trimming</td><td>Papain [60 °C, 2h]</td><td>The highest antioxidant activity was 3.22 g of Trolox equivalent antioxidant capacity (TEAC).</td><td>[31]</td></tr><tr><td>N/A</td><td>Goat viscera</td><td>Hydrolysis using alcalase [60 °C] and further hydrolysis using brauzyn [70 °C, 2h]</td><td>The scavenging rates of ABTS radical and DPPH radical were 70.65% and 42.24%, respectively.</td><td>[10]</td></tr><tr><td>QTALVELLK, SLHTLFGDELCK, MPCTEDYLSLILNR</td><td>Sheep plasma</td><td>Alcalase [55 °C, 6h]</td><td>The amount of Ferric reducing antioxidant power (FRAP) was 0.335 mM FeSO<sub>4</sub>/µg.</td><td>[49]</td></tr><tr><td>LEDGLK, IDDVLK</td><td>Sheep abomasum</td><td>Papain [46 °C, 3.8h]</td><td>The IC<sub>50</sub> values of DPPH radical scavenging activity were 0.58 mg/mL.</td><td>[22]</td></tr><tr><td>N/A</td><td>Porcine skin collagen</td><td>Hydrothermal processing [210 °C, 2100 kPa, 10min]</td><td>The scavenging activity of the ABTS free radical reached 86% at 5 mg/Mi. The reducing power was 0.269 at 700 nm at 10 mg/mL.</td><td>[32]</td></tr><tr><td>SY, PN, GS, KP, AOHR</td><td>Porcine bone collagen</td><td>Papain [55 °C, 3h, 1:1 (w/w)]</td><td>The DPPH scavenging activity reached 62.9% at 50 mg/mL.</td><td>[50]</td></tr><tr><td>YDQLPEPRKPIE</td><td>Porcine blood</td><td>Hydrolysis using papain pepsin [pH 2, 37 °C, 1h] and further hydrolysis using trypsin [pH 7.0, 37 °C, 2h]</td><td>The HRAS value was 66.89%, the RAS value was 89.37%, and the DPPH free radical scavenging rate was 38.32% at 5%. The cells exhibited the highest activity of 91.44% (the concentration of H<sub>2</sub>O<sub>2</sub> was 700 mM) at 0.1 mg/mL.</td><td>[23]</td></tr><tr><td>HLDYYLGK<br>DTYIRQPW<br>MYPGIAD</td><td>Duck meat (liver)</td><td>Enzyme hydrolysis using alcalase (pH 8, 50 °C) papain (pH 7, 50 °C), and neutrase (pH 7, 50 °C)</td><td>The ABTS radical-scavenging activities of HLDYYLGK, DTYIRQPW, and MYPGIAD had lower IC<sub>50</sub> values than the glutathione (GSH) group, with 29.13 ± 0.49 µM, 36.23 ± 1.86 µM, and 26.40 ± 0.59 µM, respectively.</td><td>[51]</td></tr><tr><td rowspan="3">DPP-IV inhibitory activity</td><td>GPVG, FGPGP, APGGAP, GPPGPT, GPVGPPG</td><td>Bovine collagen</td><td>Papain and protemax [50 °C, 4h]</td><td>The lowest IC<sub>50</sub> value of DPP-IV was 3.04 mg/mL.</td><td>[16]</td></tr><tr><td>HR, YR, HLP</td><td>Bovine hemoglobin</td><td>Papain [65 °C, 24h]</td><td>The lowest IC<sub>50</sub> value of DPP-IV was 0.99 mg/mL.</td><td>[25]</td></tr><tr><td>GPAGPIGPV, GPAGPOGFPG</td><td>Sheep skin collagen</td><td>Alcalase and neutrase [37 °C, 10min]</td><td>The lowest IC<sub>50</sub> value of DPP-IV was 67.12 µg/mL.</td><td>[24]</td></tr></tbody></table>
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<table><tbody><tr><td rowspan="3">Antihypertensive activity</td><td>HR, YR, HLP</td><td>Bovine hemoglobin</td><td>Papain [65 °C, 24h]</td><td>The lowest IC<sub>50</sub> of ACE-I was 0.19 mM, and the IC<sub>50</sub> of renin was 7.09 mM.</td><td>[25]</td></tr><tr><td>TPYPCV, VVYPWR, FLCT</td><td>Red blood cells</td><td>Pepsin [pH 2, 37 °C, 2h] and Trypsin [pH 7.5, 37 °C, 2h]</td><td>The lowest IC<sub>50</sub> value of ACE-I was 2.58 Mm.</td><td>[26]</td></tr><tr><td>N/A</td><td>Porcine liver</td><td>Autolysis [pH 4.8]</td><td>The IC<sub>50</sub> value of ACE-I was 0.81 g/L.</td><td>[52]</td></tr><tr><td rowspan="3">Antimicrobial activities</td><td>TSKYR</td><td>Bovine hemoglobin</td><td>Pepsin [pH 3.5, 23 °C, 30min]</td><td>MIC were between 1 and 9 µM against four tested strains: <i>Micrococcus luteus</i>, <i>Listeria innocua</i>, <i>Escherichia coli</i>, and <i>Salmonella enteritidis</i>.</td><td>[21]</td></tr><tr><td>N/A</td><td>Porcine blood</td><td>Trypsin [pH 7, 50 °C, 2h]</td><td>When the addition level was 64 mg, the hydrolysates had a great resistance to pathogenic microorganisms (<i>Bacillus cereus</i>). The inhibitory zone diameter was 2.55 cm.</td><td>[27]</td></tr><tr><td>N/A</td><td>Porcine placenta</td><td>Papain [pH 6.5, 50 °C, 20 min]</td><td>The inhibitory zone diameter was 10.7 mm for <i>Staphylococcus aureus</i> and 7.8 mm for <i>Escherichia coli</i>.</td><td>[28]</td></tr><tr><td rowspan="2">Anti-inflammatory activities</td><td>GPAGPSGPAGK, GPAGPSGPAGKDGR, GPSGPQGIR, GPAGPQGPR, GEAGPAGPAGPAGPR, GEGGPQGPR</td><td>Yak bone collagen</td><td>Hydrolysis using a mixture of alcalase, neutrase and flavourzyme [55 °C, 4h]</td><td>When the dosage of peptide was 100 nmol/mL, the inhibition rate of 1L-1β reached 88.95%. The inhibition rates of TNF-α, IL-6, and NO were 60.23%, 63.98%, and 60.11%.</td><td>[29]</td></tr><tr><td>N/A</td><td>Bovine lung</td><td>Hydrolysis using papain [pH 6.5 65 °C] and further hydrolysis using pepsin [pH 2.0 37 °C] and alcalase [pH 9.5, 60 °C, 24h]</td><td>When the hydrolysate concentration was 0.05%, IL-6 decreased by 53% and IL-1β decreased by 52%.</td><td>[30]</td></tr><tr><td rowspan="2">Anti-tyrosinase activity</td><td>N/A</td><td>Bovine meat trimming</td><td>Papain, 60 °C, 2h</td><td>The inhibitory activity of tyrosinase was 55.6% at 10%.</td><td>[31]</td></tr><tr><td>N/A</td><td>Porcine collagen</td><td>Hydrothermal processing [210 °C, 2100 kPa, 10 min]</td><td>The inhibition rate of tyrosinase activity was 33% at 0.5%.</td><td>[32]</td></tr><tr><td>Calcium supplement</td><td>GPSGLPGERG, GAPGKDGVRG</td><td>Sheep bone collagen</td><td>Alcalase [50 °C, 2h] and neutrase [50 °C, 2h]</td><td>The calcium binding rate was 89%.</td><td>[15]</td></tr><tr><td>Anti-aging activity</td><td>N/A</td><td>Porcine collagen</td><td>Hydrothermal processing [210 °C, 2100 kPa, 10 min]</td><td>At a concentration of 0.5%, the inhibition rates of tyrosinase, collagenase, and elastase were 33%, 48.98%, and 22.22%.</td><td>[32]</td></tr></tbody></table>
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products, which among its steps involves virtual
screening and prediction [13]. To date, the bioactivities
of meat by-products have been comprehensively inves-
tigated, resulting in the identification of several novel
peptide sequences with antioxidant, dipeptidyl pepti-
dase (DPP)-IV inhibitory, antihypertensive, antimicro-
bial, anti-inflammatory, tyrosinase inhibitory, calcium-
chelating, and anti-aging activities (Table 1).

Overview of the current methods for the identification
and characterization of bioactive peptides and protein
hydrolysates

The characterization and identification of bioactive
peptides and protein hydrolysates mainly include two
aspects. First, the characterization of the secondary
spatial structure of short bioactive peptides using
Fourier transform infrared spectroscopy (FTIR), circular
dichroism, Raman spectroscopy, X-ray diffraction, and
nuclear magnetic resonance (NMR). Second, the iden-
tification of the amino acid sequence of peptides
normally employs mass spectrometry. Aubry et al.
identified the secondary structure of peptide samples
through a scanning electron microscope and infrared
absorption spectrum [14]. Hu et al. measured the
conformational changes in the secondary structure of
peptide-calcium chelate by circular dichroism, FTIR,
and X-ray diffraction [15]. Fourier transform infrared
spectroscopy was employed to investigate the relative
content of secondary structures, and nuclear magnetic
resonance spectroscopy can be further used for both
qualitative and quantitative analysis of the composi-
tional structure [16].

Mass spectrometry, in the context of proteomics and
peptidomes, is the most common method for the iden-
tification of peptide sequences [17,18]. Currently, a
diverse array of mass spectrometry techniques exist,
encompassing various ion sources, protein cleavage
modes, and mass analyzers tailored for the structural
analysis of bioactive peptides. The ion sources comprise
electron ionization (EI), chemical ionization (CI), elec-
tro-spray ionization (ESI), atmospheric pressure chemi-
cal ionization (APCI), laser diode (LD), and matrix-
assisted laser desorption ionization (MALDI) [19].

The main mass spectroscopic decomposition methods of
protein samples include collision-induced dissociation
(CID), high-energy collision dissociation (HCD), elec-
tron capture dissociation (ECD), and electron transfer
dissociation (ETD), electron-activated dissociation
(EAD), ultraviolet photodissociation (UVPD). There are
numerous mass analyzers, encompassing single-focusing
mass spectrometry, double-focusing mass spectrometry,
time-of-flight, ion traps, triple quadrupole, and Fourier
transform ion cyclotron resonance analyzers. The char-
acteristics of fragments and mass results acquired from
the mass spectrometer are juxtaposed with the theoret-
ical peptide spectra derived from an *in silico* digested

protein database. This process facilitates the identifica-
tion of the target peptide sequence [20].

Diversity of peptide bioactivities in meat by-products

Antioxidant activity

The peptides from meat by-products, such as blood,
bone, and viscera, have been reported to exhibit potent
antioxidant capacity. Przybylski et al. isolated the
bioactive peptide (TSKYR) from bovine hemoglobin
through hydrolysis with porcine pepsin [21]. The anti-
oxidant activity of the bioactive peptide generated by
porcine pepsin was assessed, revealing a TBARS value of
1.75 MDA/kg of meat when the supplementation level
of the bioactive peptide was 0.5%. Notably, it exhibited
comparable antioxidant effects to the widely utilized
antioxidant butylated hydroxytoluene (BHT). Liu et al.
hydrolyzed sheep abomasum using papain, resulting in
the isolation of the peptide LEDGLK IDDVLK, with an
IC50 value of 0.58 mg/mL for DPPH radical scavenging
activity [22]. Zhan et al. employed porcine plasma as the
raw material, subjecting it to simulated *in vivo* digestion
using pepsin and trypsin [23]. The obtained bioactive
peptide YDQLPEPRKPIE exhibited notable antioxi-
dant properties, with an ABTS (2,2'-azino-bis(3-ethyl-
benzothiazoline-6-sulfonic acid) value of 89.37%, and a
DPPH (2,2-diphenyl-1-picrylhydrazyl) value of 38.32%.
The observed antioxidant effect was superior to that of
glutathione. The antioxidant activity may be related to
the C-terminal amino acid in the peptide sequence,
which is composed of acidic or basic amino acid residues,
such as Cys, Met, Tyr, and Ser.

Dipeptidyl peptidase-IV inhibitory activity

DPP-IV inhibitory activity has been found in bovine he-
moglobin, collagen, and sheep collagen-derived peptides.
Among them, the DPP-IV IC50 of sheep collagen is the
lowest, which suggests the collagen-derived peptide has
the strongest specific binding to DPP-IV and DPP-IV
inhibitory activity. Furthermore, papain was the most
effective protease to generate potent DPP-IV inhibitory
peptides from bovine hemoglobin and sheep skin.
Notably, the sheep skin hydrolysate exhibited the high-
est DPP-IV inhibitory activity, achieving an *in vitro*
inhibitory rate of 58%. A series of peptide sequences with
DPP-IV inhibitory activities were identified, including
GPAGPIGPV, GPAGPOGFPG, GPVG, FGPGP, and
APGGAP [16,24]. It is worth noting that the inhibitory
activity is closely related to the presence of a pro residue
at the C-terminal of the peptide.

Antihypertensive activity

ACE-I inhibitory activity of peptides has been found in
protein hydrolysates of meat by-products, such as blood
and red blood cells. For example, Lafarga et al. found
that after papain-treated hydrolysis, bovine hemoglobin
had high ACE and renin inhibitory activities [25]. The
values of ACE-I IC50 and renin IC50 were 0.19 mM and
<table><caption>**Table 2**</caption><caption>Summary of the European Union (EU)/USDA/FSSAI regulations on safety aspects of animal by-products and derived products.</caption><thead><tr><th>Regulations</th><th>General Principles</th></tr></thead><tbody><tr><td>(EC) TSE Regulation No 999/2001</td><td>Total ban on using any remains of animals (by-products) in feed for livestock.</td></tr><tr><td>EC Regulation No 852/2004</td><td>Sets out the hygiene requirements with respect to storage, handling, disposal/elimination of all food waste, non-edible by-products, and refuse.</td></tr><tr><td>EC Regulation No 183/2005</td><td>Feed business operators, other than primary producers, are required to store and transport feed under certain hygienic conditions.</td></tr><tr><td>Regulation (EC) No 1069/2009, and legislation (Regulation (EU) No 142/2011)</td><td>Provides a definition of animal by-products and categories (1, 2, and 3), requirements for compulsory treatment processes, permitted options for disposal, storage, labeling, transport, trade, or future use of animal by-products.<br>Category 1: Material with high specific risk, as well as animal by-products containing some specifies substances and environmental contaminants.<br>Category 2: Animal by-products with high risk, infected or contaminated carcasses, as well as materials declared unfit for human consumption.<br>Category 3: Includes inedible and free from infection carcass materials, as well as animal materials that are fit but not intended for human consumption for commercial reasons.</td></tr><tr><td>Commission Decision 2009/719/EC</td><td>Revised monitoring programs allow member states to test for the presence of TSEs in specific target groups of animals, such as emergency slaughtered animals and fallen stock.</td></tr><tr><td>Regulation (EU) 68/2013, Preambles 2 to 5</td><td>Feed ingredients should comply with the applicable restrictions for chemical and microbiological safety.</td></tr><tr><td>Commission Regulation (EU) No 1924/2006, No 353/2008, No 1169/2009</td><td>The European food safety authority (EFSA's) regulation on nutrition and health claims: functional food products need to be justified scientifically through clinical trials.</td></tr><tr><td>Regulation (EU) 2021/1372 amending Regulation (EC) No 999/2001</td><td>Use of processed animal proteins of poultry origin in pork feed and pig PAPs in poultry feed; use of ruminant collagen and gelatin in feed for nonruminant farmed animals.</td></tr><tr><td>FDA - Code of Federal Regulations (CFR) 21 (part 507)</td><td>Facilities that are not producing human food but are producing by-products for use as animal food are required to register and comply with all of 21 CFR part 507, unless they meet the criteria for an exemption.</td></tr><tr><td>Food Safety and Standards (Food Product Standards and Food Additives) Amendment Regulations, 2020 under sub-regulation 2.5.2</td><td>Milk and meat-producing animals except poultry, pigs, and fish shall not be fed feed containing meat or bone meal including internal organs, blood meal, and tissues of bovine or porcine origin materials, except milk and milk products.</td></tr></tbody></table>

7.09 mM, respectively. Compared with seaweed protein hydrolysate (IRLIIVLMPILMA) only having renin-inhibiting activity, bovine hemoglobin with ACE-I and renin-inhibiting activity exhibited higher antihypertensive properties [25]. The ACE inhibitory activity of the obtained peptides (TPYPCV, VVYPWR, and FLCT), generated through the hydrolysis of porcine red blood cells with pepsin and trypsin, resulted in an ACE IC50 value of 2.58 µL. In comparison to α-bovine hemoglobin in bovine blood, the extract from porcine red blood cells exhibits enhanced ACE inhibitory activity. Nevertheless, when contrasted with the ACE-targeting drug Lisinopril (IC50 = 0.2 nm), the ACE inhibitory activity of the porcine red blood extract is comparatively weak [26].

## Antimicrobial peptides

Peptides derived from bovine hemoglobin and porcine serum albumin have been documented to demonstrate antibacterial properties. The antibacterial spectrum of bovine hemoglobin is broader than that of porcine serum albumin, exhibiting inhibition against two Gram-positive bacteria (*Micrococcus luteus* and *Listeria innocua*) and two Gram-negative bacteria (*Escherichia coli* and *Salmonella enteritidis*) [21]. The inhibitory effect of porcine serum albumin-derived peptides on *Bacillus cereus* has been identified. TSKYR, a pentapeptide obtained by hydrolysis of bovine hemoglobin by pepsin, exhibited good antibacterial activity (MIC, 1.9 µM) [21]. Jin et al. found that porcine plasma albumin was hydrolyzed by trypsin to produce antibacterial peptides with a potent antibacterial
effect against *B. cereus* [27]. The antibacterial effects were as follows: at a sample dosage of 64 mg, the diameter of the antibacterial zone was 2.55 cm, surpassing the antibacterial effect of antimicrobial peptides (2.5 mg). Porcine placenta-derived peptides exhibited notable antibacterial efficacy against *Staphylococcus aureus* and *E. coli*, with antibacterial zone diameters of 10.7 mm and 7.8 mm, respectively [28].

## Antiinflammatory activity

Nitric oxide (NO), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) are commonly observed inflammatory factors. The prevention of excessive production of inflammatory factors can mitigate the risk of inflammatory diseases such as atherosclerosis and metabolic syndrome. Bioactive peptides with inhibitory effects on inflammatory factors have been identified in both yak bone and bovine lung. Peptides, including GPAGPSGPAGK and GPSGPQGIR, produced by the combined enzymatic hydrolysis of yak bone using alcalase, neutrase, and flavourzyme, can effectively regulate the NK-κB signaling pathway and NO production to inhibit the inflammatory response [29]. Bovine lung underwent hydrolysis with alcalase, and the resulting hydrolyzed products exhibited noteworthy antiinflammatory activity in RAW264.7 macrophages. This activity was marked by a substantial reduction in the production of IL-6, IL-1β, and NO [30].

## Other bioactivities

In addition to the aforementioned common biological activities, bioactive peptides also exhibit other bioactivities including, tyrosinase inhibition, calcium-binding, antiaging, and antifatigue activities. Tedeschi et al. reported that following papain hydrolysis, the hydrolysate derived from calf fleshing meat demonstrated heightened tyrosinase inhibitory activity, reaching 55.6% at a 10% addition level [31]. This activity was comparable to the antityrosinase activity observed in a substance extracted from *Scomber japonicus* [31], which can be used as a food preservative or as an additive in the production of food packaging. Choi et al. found that when the amount of protein hydrolysate obtained by thermal hydrolysis of porcine skin collagen was 0.5%, the inhibitory activity of tyrosinase was 33%, the inhibitory activity of collagenase was 49%, and the inhibitory rate of elastase was 22% [32]. Sheep bone collagen-derived peptides (GPSGLPGERG and GAPGKDGVRG) obtained by the proteolytic action of alcalase and neutrase exhibited high calcium-binding capacity (89%), which can be used as calcium supplements in food and drugs.

Three dipeptide isomers of aspartic acid (Lα, Dα, and Lβ), resulting from the oral administration of pig liver hydrolysate, exhibited anti-fatigue effects by activating AMP-activated protein kinase (AMPK) in the liver at low concentrations [33]. In comparison to 0.1% ascorbic acid (1 mg/mL), the inhibition rates of

collagenase and elastase were similar to those of ascorbic acid, with the exception of lower antityrosinase activity. The hydrophobic amino peptide (Pep-NH₂) present in the protein hydrolysate of porcine liver, when administered through intraperitoneal injection to mice, demonstrated an enhancement in exercise activity after forced walking. Moreover, there are certain disease-related functional properties, such as the potential treatment of alcoholic liver disease and cardiovascular disease. The liver-protecting peptide isolated from porcine liver through alcalase-treated hydrolysates exhibited the capability to activate ethanol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). The sequence NTLPHPTAP was found to bind to ALDH, and the formation of the complex ADH/ALDH-NTLPHPTAP extended the overall structure of the enzyme, facilitating its enhanced binding to the substrate [34].

## Regulatory barriers to valorization, challenges and opportunities

### Current regulations and restrictions related to meat by-products valorization

The threat of transmission of contagious diseases like swine fever, epidemics of foot and mouth disease, and the spread of zoonotic infections, including transmissible spongiform encephalopathies (TSEs) (Bovine spongiform encephalopathy (BSE), mad cow disease), while utilizing animal by-products has raised public health concerns. This has prompted various regulatory bodies, including the EU, to implement restrictions and regulations on the use, storage, disposal, export, and import of the types and volumes of meat by-products (Table 2). In order to safeguard animal and human health, animal by-products that are classified under “Category 3” and in the lowest risk group are now permitted for food and feed applications (EU Regulation 1069/2009). The purpose is to prevent the transmission of animal diseases, contaminants, etc. that could occur in processed feed or valorized products, including bioactive peptides/protein hydrolysates with functional applications.

The functional and nutritional claims of bioactive peptides/protein hydrolysates are also governed by regulatory guidelines. In Europe, the dietary or therapeutic values of bioactive peptides are evaluated by the EC (EC 1924/2006 regulation) [35]. Before declaring any novelty, the characterization of the peptides, animal and human trials, and other safety issues are harmonized and revealed by the EFSA. In the United States, functional claims are monitored by the FDA [36], and the marketed products must have a disclaimer that can be delivered by the U.S. Food and Drug Administration. However, for therapeutic/drug claims, FDA preapproval is necessary. In Japan, validation of functional foods is governed by FOSHU and FNFC guidelines, framed in 1991 under JMHLW. After
clinical verification and obtaining legal approval, pro-
ducers get the FOSHU label on their packaged prod-
ucts. In India, functional values of food products are
monitored by the FSSAI under the FSS Act, 2006. For
this, food products need at least 15 years and a
maximum of 30 years of safe and efficient usage in India
and other countries, respectively. In China, bioactive
peptide-based food supplements are legalized by the
CFDA (http://www.sfdachina.com/info/88-1.htm), and
products are marketed with the Blue Hat logo. Besides,
prior permission is required before conducting *in vitro*
and *in vivo* clinical trials due to safety issues, etc.

Opportunities for regulatory improvement and
innovation

Different countries have their own laws and regulations for the utilization of animal by-products. These differences in processing, value addition, characterization, and marketing pose challenges for global trade (Figure 3). Various process and product innovations characterized by valorized bioactive peptides are underway. Some meat-derived peptides are bitter in taste [3], which is an issue for their commercial rejections. Innovative tools like iBitter-Fuse and VirtualTaste can identify and mask the bitterness of peptides by blocking bitter taste receptor

activity [8]. Furthermore, the use of endo- and exo-
peptidases, γ-glutamyl transpeptidase from *Bacillus
amyloliquefaciens*, or glycation of protein hydrolysates can
reduce the bitterness to a great extent [37,38]. Another
innovative concept is the *in silico* hydrolysis simulation
model, which can help in predicting known protein se-
quences and cleavage sites of chemicals or enzymes to
extract new generation bioactive components [39].
Likewise, encapsulation of bioactive peptides can over-
come the limitations of nutraceutical and commercial
applications by improving their bioactivity, stability, sol-
ubility, food matrix interaction, sensory properties, etc.
[37,40].

Safety barriers to valorization

Microbial safety considerations: risks associated with
meat by-products and control measures ensuring
safety

Animal by-products or derived products, if contami-
nated and insufficiently processed, can be a source of
microbial (parasitic, bacterial, and viral) hazards. In
order to reduce the microbiological risks associated with
animal by-products and protect environmental, animal,
and human health, microbial safety assessment is
required at every stage along the continuum from farm-

<figure><img src="image_6.png" /><figcaption>Figure 3</figcaption></figure>

Challenges and opportunities for the valorization of meat by-products for bioactive peptides and the regulatory framework.
to-fork [41]. Identification of potential hazards and implementation of control measures by combining physical and chemical methods and enforcing safety regulations and food safety standards can help in mitigating the risk of contamination. Routine screening of animal by-products, feed, and bioactive peptides by endorsement of traceability is also a radical step to mitigate the epidemiological risks [42]. During the processing of animal by-products, Hazard Analysis and Critical Control Point (HACCP) need to be implemented at every step of production and processing to overcome the risks of microbiological hazards. Further, valorization of meat by-products should be restricted to materials belonging to Category 3 only to mitigate the challenges of BSE and other specified risk materials [3].

## Chemical contaminants and residues

Veterinary drugs, including antibiotics, anthelmintic, and nonsteroidal anti-inflammatory drugs (NSAIDs), are often used to enhance production and treat diseases in animals. However, the benefits of drug administration are not without associated risks, as the drug residues or metabolites may accumulate within the tissues and meat byproducts of animals. Likewise, the sources of chemical contaminants (pesticides, heavy metals, additives, toxins, etc.) contracted by animals could be water, the environment, feed, and fodder.

As the goal is to produce safe and high-quality bioactive peptides and functional ingredients, it is important to screen animal by-products through residue monitoring programs and implement quality control measures. First of all, the drug residues and chemical contaminants in animal by-products should be within the set tolerances or maximum residue limits set by regulatory bodies [43]. For this, regular screening and testing of samples to detect and quantify veterinary drugs and chemical residues is needed. Samples not fulfilling the standards and guidelines set by regulatory bodies may be discarded in order to ensure the safety and quality of the products. Additionally, it is possible to significantly decrease residues and contaminant levels in animal products and by-products by regulating the usage of veterinary drugs (strictly following dose, duration, and withdrawal periods), adopting good veterinary practices, reducing exposure to chemical contaminants, and offering high-quality feed and water to animals [44].

## Monitoring and mitigation strategies

Control strategies should include the adoption of good animal husbandry practices and waste management [45], as well as the screening and detection of potential hazards and contaminants through monitoring and surveillance programs. Further, proper use of veterinary drugs, adherence to withdrawal periods, monitoring of feed

quality, etc. are also recommended to minimize emerging contaminants. It is mandatory to have toxicological investigations in vitro in both animal and human model systems before opening the marketing channels [46]. As the synergistic/additive effects of bioactive peptides could be suppressed by other peptides, their reactive natures or modes of action need to be addressed before adding them to any food formulations [47]. In this regard, polyphenols, if introduced, could offer synergistic effects and solutions by forming peptide-polyphenol complexes, enhancing the safety and functionality of peptides [48]. Furthermore, regulatory societies should come up with daily recommended intake levels of bioactive peptides and their dose-response effectiveness on human health. Environment-friendly, advanced scientific validation is required to mitigate these health-related threats.

## Concluding remarks

From the above discussion, this study highlights the importance of meat by-products as a source of bioactive peptides and functional ingredients while depicting the research gaps and areas for further exploration from regulatory and safety perspectives. Clearly, there is an opportunity to bring uniformity to the evaluation of animal by-products, especially bioactive peptides, through the collaborative efforts of various stakeholders and policymakers: i) in developing a comprehensive regulatory framework; ii) updating food safety standards; and iii) implementing robust traceability systems and labeling requirements. Above all, collaboration among regulatory bodies, industry stakeholders, and research institutions is crucial in sharing knowledge to establish best practices for improving the safety of meat by-products valorization. Further, human clinical trials must be done to substantiate the health claims before the introduction of bioactive peptide-based products into the market. Finally, a successful valorization of animal by-products requires the implementation of several key measures with proper monitoring and mitigation strategies, as these small fractions of peptides with lower molecular weights are more likely to show cytotoxicity, allergenicity, mutagenicity, etc.

## CRediT roles

**Mohammed Gagaoua:** Conceptualization, Data curation, Investigation, Methodology, Resources, Supervision, Visualization, Writing — original draft, Writing — review & editing.
**Arun K. Das:** Data curation, Investigation, Methodology, Writing — original draft.
**Yu Fu:** Data curation, Investigation, Methodology, Visualization, Writing — original draft, **Amira Leila Dib:** Data curation, Investigation, Writing — review & editing.
**Pramod Kumar Nanda:** Data curation, Investigation, Methodology, Writing — original draft.
## Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

Data will be made available on request.

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