Bioactive peptides extracted from hydrolyzed animal byproducts for dogs and cats

Abstract

Enzymatic hydrolysis produces bioactive peptides and hypoallergenic ingredients for pet food. Animal byproducts are the main sources of bioactive peptides and hydrolyzed proteins for dogs and cats. Prebiotic, antioxidant, anti-inflammatory, immunological, and antihypertensive effects of the bioactive peptides have been studied in dogs and cats. Hydrolyzed ingredients improve the palatability of extruded diets for dogs and cats. Worldwide, 350 million tons of meat are produced annually; 35% is chicken, 30% is pork, 20% is beef, and 15% is meat from other species. Milk and egg production are also important, estimated at 900 and 90 million tons, respectively (World Food and Agriculture—Statistical Yearbook 2022, 2022). Approximately 40% of the total animal live weight produced globally is inedible for human consumption, such as skin, blood and its elements, horns, hooves, viscera, bones, hair, and feathers. Thus, most of these inedible parts for humans are used to produce, via the rendering process, animal byproduct meal (ABPMs) for livestock and pet feeding (e.g., meat and bone meal, poultry byproduct meal, blood meal, animal plasma, and feather meal). About 9 million tons of ABPMs and fats are used annually to produce dry food for dogs and cats (Alexander et al., 2020). Feeding animals with these ingredients optimizes the surplus of food for human consumption, which is considered adequate in the food recovery hierarchy. Therefore, rendering is important because it recycles slaughterhouse waste into nutritious natural ingredients to meet animal nutritional requirements. Rendering yields ingredients with variation in quality, sometimes low nutrient bioavailability, and low added value. However, these ingredients usually contain essential amino acids in high concentrations and peptides with possible functionalities in animal health. Thus, the industry has increased the production and use of protein hydrolysates from these byproducts to find better alternatives for using ABPMs (He et al., 2013). More recently, the pet food industry has employed these ingredients, focusing on their nutritional and functional properties for dogs and cats. In addition to the greater bioavailability of amino acids, some benefits of protein hydrolysates are the release of bioactive peptides (BPs) that have antioxidant, antimicrobial, immunomodulatory, tissue repair, antihypertensive, and glycemic control effects (Zóia Miltenburg et al., 2021). Many of these functional properties are still being studied in companion animals. The present review focuses on the productive aspects of BPs and functional proteins, their applications, and the health benefits already identified in dogs and cats. Protein hydrolysis is the process of breaking the peptide bonds between amino acids and proteins. Protein hydrolysates are classified according to their degree of hydrolysis and contain free AAs, small peptides, and large peptides. The proportions of each hydrolysate product depend on protein source, water quality, protease type, and microbe species. Proteins and peptides have different molecular weights; in general, polypeptides with a molecular weight of >8,000 Daltons (≥72 AA residues) are referred to as proteins, and those with a molecular weight of <8,000 Daltons are peptides. In animal production, high-quality protein is not hydrolyzed. Only animal byproducts, such as brewer’s byproducts, plant ingredients containing anti-nutritional factors, and low-digestible proteins are hydrolyzed to produce peptides for animal feeding (Hou et al., 2017). Fish, chicken, and beef meat byproducts are the most common animal material in protein hydrolysate production. Hydrolysis can alter the properties of proteins in three different ways: reducing the molecular weight, increasing the number of ionizable molecules, or exposing hydrophobic groups. These properties are considered in food formulation because they are directly responsible for their functionality in food processing and the bioavailability of their components (He et al., 2013). Chemical, enzymatic, and microbial methods can produce hydrolyzed peptides of animal proteins. Choosing a technique for protein hydrolysis depends on the raw material, and a combination of methods can be implemented. For example, feathers, bristles, horns, beaks, or wool proteins contain keratin and are mostly hydrolyzed via acid or alkaline treatment associated with microbial keratolytic enzymes. Other animal (casein, whey, gut, and meat) raw materials are usually subjected to enzymatic hydrolysis or microbial fermentation. Hydrolysis can last 4 to 48 h, depending on the material’s resistance and the method. Chemical methods for protein hydrolysis include acid and alkaline hydrolysis treatments. In acid hydrolysis, raw materials are commonly treated with concentrated hydrochloric acid (HCl); however, sulfuric acid can be used as well. The most important factors that influence acid hydrolysis are the concentration and type of acid (hydrochloric acid or sulfuric acid), temperature (250 to 280 °F), pressure (32 to 45 psi), hydrolysis length (2 to 8 h), protein concentration and resistance of the raw material ingredient. These factors, individually and combined, affect the quality of the product. In the pet food industry, most acid-hydrolyzed proteins are used as flavor enhancers. Despite the low cost, acid hydrolysis may destroy some essential amino acids such as tryptophan, methionine, cystine, and cysteine. Furthermore, glutamine and asparagine are converted to glutamic and aspartic acids, respectively (Pasupuleti and Demain, 2010). Protein alkaline hydrolysis requires agents such as Calcium, Sodium, or Potassium Hydroxide to be submitted to high temperatures (>100 °C; Dai et al., 2014). However, lower temperatures (27 to 55 °C) and shorter processing lengths (4 to 8 h) are often desirable to obtain peptides in the food industry (Pasupuleti and Demain, 2010). After alkaline hydrolysis, the product is dehydrated, pasteurized, or spray-dried. Alkaline hydrolysis is a common method for processing low-digestible proteins, such as feathers used in feather meal production and certain keratin material. The alkaline hydrolysis increases the digestibility and the content of free amino acids in the ingredient; however, it has a few disadvantages. Degradation of some amino acids like serine, threonine (Pasupuleti and Demain, 2010; Hou et al., 2017), cystine, methionine, and lysine has been observed (Papadopoulos et al., 1985; Kim et al., 2002). In enzymatic hydrolysis, different proteolytic enzymes are used, the most common are papain, pepsin, protease complexes, pancreatin, trypsin, chymotrypsin, alkalases, thermolysin, and numerous other bacterial and fungal enzymes (Korhonen and Pihlanto, 2006). Depending on the purpose of hydrolysis, the enzymes (papain and pepsin, papain and actinase E, or trypsin alone) may be used alone or combined. Choosing the enzyme depends on the protein source, the degree of hydrolysis, and the other traits, such as bioactive properties, desired for the final product. Another type of hydrolysis is microbial, a type of enzymatic hydrolysis that occurs during a fermentative process. In the presence of substrate, microorganisms release proteases to break down complex nutrients in the medium and to take advantage of them in metabolism (Smid and Lacroix, 2013). The fermentative process can also produce new proteins, peptides, and free amino acids from microbial metabolism, which can improve the palatability, digestibility, and functionality of these ingredients (López-Pérez and Viniegra-González, 2016; Sandhu et al., 2017). The overall procedure for hydrolysate production is described in Figure 1. The process can last 4 to 48 h, depending on the time and hydrolysis method. Peptide production processes from animal and plant proteins. These procedures can be modified for peptide production depending on the protein source and product specifications. Adapted from the study by Hou et al. (2017). Hydrolysis improves the nutrient bioavailability and sensory attributes and releases BPs. Thus, producing protein hydrolysates from industrial byproducts is advantageous because it enhances these materials. For this reason, the production of hydrolysates from animal protein, including byproducts such as ruminant and pig leather, chicken feet, skin and intestine, liver, trachea, bones, blood, and plasma from different species has been widely studied to improve the usage of waste generated in meat production for human consumption. Thus, the bioactivity of these peptides as antioxidants, antihypertensives, antihyperglycemics, and anti-inflammatory agents has been studied. Dry food for dogs and cats contains protein derived from industrial byproducts of meat production for humans, which have been included in formulations up to 32% (Alexander et al., 2020). These ingredients are rich in essential and nonessential amino acids; however, most of these proteins are subjected to high temperatures during rendering, which impairs their digestibility and amino acid bioavailability. Animal protein meal industries have increased the production of hydrolyzed meals to improve the processing of these ingredients, especially via enzymatic hydrolysis, which yields peptides with bioactive properties. BPs are small molecules (0.5 to 5 kDa) ranging from 3 to 50 amino acid residues linked together (tripeptides and oligopeptides) in different combinations and arrangements, with various biological roles (Hou et al., 2017). Endogenous peptides are produced by different glands and cells in the body. On the other hand, exogenous peptides can be provided via food, dietary additives, and medications (Lorenzo et al., 2018). The digestive process of BPs is facilitated by greater exposure to endogenous digestive enzymes. After food intake, proteins are first hydrolyzed by pepsin I n the acidic environment of the stomach (pH 1.5 to 3). However, pepsin is an endopeptidase and does not break down protein into free amino acids. Therefore, products of pepsin reaction are partially hydrolyzed proteins and large peptides that will be further hydrolyzed by enteric (enterokinase) and pancreatic endo- (trypsin, chymotrypsin) and exo-peptidases (carboxypeptidases) in the intestine. After this step, free amino acids, small peptides (di- and tri-peptides), large peptides (oligopeptides), and, eventually, proteins that were not significantly hydrolyzed by pepsin and pancreatic enzymes (hair, feathers, and others resistant to endogenous enzymatic digestion) pass through the intestine. During the passage through the small intestine, they contact the brush border, and the membrane digestion phase begins. Peptidases in the brush border hydrolyze peptides into free amino acids, di- and tri-peptides, or oligopeptides. These molecules can then be absorbed via one of the four absorption pathways (Figure 2), hydrolyzed inside the enterocyte (intracellular digestion), or pass into the bloodstream, reaching target organs as peptides or free amino acids. Pathways of peptide absorption in the small intestine. Adapted from the study by Miner-Williams et al. (2014). 1) paracellular diffusion, 2) transcellular passive diffusion, 3) transcytosis, 4) carrier-mediated transport. When ingested, peptides play roles in metabolism, interfering with inflammatory responses, blood pressure, weight control, and glycemic response. The activity of BPs depends on the electrostatic charge, chain size, amino acid sequences in the peptide chain, and molecular surface hydrophobicity. There are four pathways by which BPs are taken up: paracellular diffusion, transcellular passive diffusion, transcytosis, and carrier-mediated transport, which depend on chemical and physical properties (Amigo and Hernández-Ledesma, 2020). Paracellular diffusion occurs by increasing the permeability of the junctions among enterocytes (tight junctions). In transcellular passive diffusion, the uptake depends on peptide hydrophobicity and charge neutrality. Transcytosis is a process of endocytosis by which peptides are absorbed by the cell with energy expenditure and subsequently processed inside the enterocyte. The last pathway, carrier-mediated transport, occurs via facilitated diffusion or secondary active transport. The PEPT1 pathway is the most common and is responsible for absorbing high amounts of the hydrolyzed peptides during digestion in mammals and birds (Miner-Williams et al., 2014). Many BPs, such as the antihypertensive peptides extracted from pigs (RPR, KAPVA, and PTPVP), can resist the intestinal membrane protease action and reach their targets intact (Escudero et al., 2012); however, there are no estimates on the amount of BPs that escape gastrointestinal and enterocyte hydrolysis. Due to the abundant expression and high capacity of the peptide carrier (PEPT1) in the small intestine, the rate of luminal absorption of peptides is higher than that of free amino acids (Vij et al., 2016). However, PEPT1 does not transport large peptides; its role is limited to di- and tri-peptides, regardless of the amino acid sequence. Before being transported to the bloodstream, BPs can influence the gut health. For instance, LKPT peptide stimulates the GLP-1 secretion in the ileum, by enteroendocrine cells (Theysgeur et al., 2020). As the GLP-1 influences central nervous system to decrease the permeability in the colon (Funayama et al., 2023), the consumption of BPs can favor the gut health in animals with chronic enteropathies as suggested by Meineri et al. (2022). In addition to the intestinal health effects some animal BPs possess during digestion, other post-absorption effects have also been investigated. The scientific literature has reported studies performed to assess the effects of BPs as antioxidants (Hou et al., 2017), antihypertensive (Zóia Miltenburg et al., 2021), antimicrobial (Vidal et al., 2022), anti-inflammatory, and anxiolytics. Depending on their physicochemical characteristics, BPs may have both immunostimulatory and immunosuppressive activities. In humans, egg-derived peptides showed an immunostimulatory effect in chemotherapy patients (Mine and Kovacs-Nolan, 2006). In addition, peptides hydrolyzed from bovine sarcoplasmic proteins showed cytotoxic effects against breast cancer cells and inhibited gastric cell proliferation (Jang et al., 2008). Despite their relevance, these effects have not yet been studied in dogs and cats. Protein hydrolysates have been extensively used in hypoallergenic diets for dogs and cats because they do not stimulate immune reactions due to their low molecular weight; thus, hypersensitivity symptoms are reduced (Olivry et al., 2017). Osteoarthritis is a degenerative joint condition that affects humans and dogs as well. Dogs are mainly affected by joint dysfunctions and hydrolyzed collagen peptides have been extensively used to develop products for pet food that benefit joints; however, these effects are still to be proven. Schunck et al. (2017) observed improved chondrocyte function using hydrolyzed collagen in dogs. In this study, the authors observed an improvement in the biosynthesis of type II collagen and elastin compared to the control group. In another study (Ruff et al., 2016), hydrolysates from the eggshell and membrane improved the mobility of dogs with arthritis. On the other hand, chicken liver hydrolysate did not modify any immunological parameters in healthy dogs, especially those related to more intense and allergic inflammatory responses (Pinto et al., 2022). Many benefits are attributed to the hydrolyzed collagen, such as increasing bone strength and density, decreasing the extracellular matrix and the markers of joint degeneration, inhibiting inflammatory cytokines, and improving joint stability. The antioxidant activity of BPs also depends on peptide chain, amino acid composition, and hydrophobicity. Most antioxidant peptides range from 4 to 16 amino acid residues with molecular weights ranging from 0.4 to 2 kDa (Zaky et al., 2022). Enzymes alter the antioxidant effects of BPs during hydrolysis. Saiga et al. (2003) observed greater antioxidant activity using the linolenic acid peroxidation system when peptides were obtained using actinase E compared to papain in the hydrolysis of porcine myofibrillar proteins. In another study, collagen from pig skin was hydrolyzed using three different enzymes to obtain antioxidant peptides (Li et al., 2007). However, these peptides showed in vitro antioxidant activity, which may not correspond to the expected in vivo responses. Dietary BPs must resist the intense thermal processing of pet food and digestion to be absorbed and reach target cells. For this reason, in vivo studies are important. Antioxidant effects were not observed in a study with adult dogs (Pinto et al., 2023) fed diets formulated with hydrolyzed chicken liver as the main protein source (24%, 32%, and 40% crude protein). Like other antioxidant compounds, BPs quench or eliminate free radicals and synergize with other antioxidant compounds. The advantage is that they are also nutrients and act as antioxidant additives. Recently, Hu et al. (2020) assessed the effects of two plant hydrolysates (hydrolyzed corn gluten meal and hydrolyzed distiller’s dried grains with solubles using neutrases or alkalases) on the oxidation of corn and fish oils and reported a similar effect compared to the synthetic antioxidant butylated hydroxytoluene. This result brings new perspectives on using hydrolysates as BPs are classified according to their of action into to amino acid in lysine or and that are commonly abundant in hydrophobic residues and and 2013). The of action of from that of Like other BPs, depend on the composition, and amino acid to be on their which can be or or et al., However, possess a higher than effect and play a immune role et al., 2020). resistance to is a human and animal health thus, may be a to the of microorganisms by electrostatic them reaching the and reducing the of resistance et al., 2018). Furthermore, hydrolyzed animal proteins can improve the intestinal health of containing hydrolyzed meat proteins have been in the treatment of chronic enteropathies in dogs and can the gut The antihypertensive peptides obtained from protein especially from animal protein, are of of the most studied BPs in humans and animals is enzyme affects one in three adult More than take and 4 of 5 are not However, in especially such as dogs and of are and are used for other for chronic and the of I to which and increases blood an important role in functional and bioactive properties of peptides. residues and hydrophobic residues antihypertensive BPs with high activity in the function et al., 2018). such as and have As a such as BPs to blood pressure, are and 2007). Most studied in humans is the hydrolysis of protein, and egg to produce antihypertensive BPs. In companion protein hydrolysates to is for patients from and chronic in more Miltenburg et al. compared the effects of feeding cats two diets containing or hydrolyzed poultry byproduct meal and observed that hydrolyzed meal reduced activity by These authors also assessed in vitro effects on the activity and observed and of for the hydrolyzed and in hydrolysis and peptide and improved peptide bioavailability in animals. 2 is an that affects of the human and has a similar in the such as and have been used in to and other studies have that animal protein hydrolysates are a source of BPs with action to control blood pressure and glycemic with similar activity to those in and an in vitro digestive system for dogs, et al. (2020) reported that protein hydrolysate has a activity compared to the no in vivo study that assessed the capacity of protein hydrolysates is et al. reported increased secretion and reduced plasma secretion in and healthy cats and glycemic when or These peptides have two possible of the first is related to the GLP-1 and the to their effect on the This result that benefit glycemic in protein hydrolysates be used for this Figure 3 the main of protein hydrolysates containing BPs in and attributed to bioactive peptides produced from protein hydrolysates of animal The of protein hydrolysates has been reported to in dogs and cats. However, the of more palatability studies have et al. and Miltenburg et al. reported that poultry byproduct hydrolysates were diets containing poultry byproduct meal by dogs and The pet food industry has increased the production of hydrolyzed ingredients, digestibility and palatability as as the Furthermore, studies with hydrolyzed ingredients on especially antioxidant, blood pressure control, and However, scientific on the effects of BPs on these is still The increasing number of in will to new especially those dogs and cats. is an of the Animal at the of in in from the of is in Animal with on and is a and in two linked to the of also has in a Food in the The main that is are as methods in of animal by product meals and animal fats as for and for the of food is a in Animal with an on and at the of in in Animal from the of do at a Food as with formulation and food for dogs and cats. The main are the quality of animal byproduct meals and animal fats as ingredients for pet of ingredients and for dogs and and In extracted from the animal byproduct meals and their effects on the health. is a in Animal at the of with an on and at the of in is in Animal from the of are in sensory aspects of extruded with different of food for dogs and production of and dry containing or dried animal This was for by the of Animal The in this are those of the and do not the or of the of Animal the or the The authors no or of

Topics & Concepts