Protein Sources as Functional Ingredients in Pig Diets
The increase in demand for protein sources for animal feeds, related to the increase in animal production worldwide and to the increase in competition of use for human food, puts emphasis on efficient use of available protein sources. First of all, protein sources for animal feeds are carriers of protein and (essential) amino acids and other nutrients for production animals. In addition, specific constituents of these ingredients and peptides released during the enzymatic digestion of constituent proteins in the digestive tract can exert other, non-strict nutritional effects on e.g. feed intake, nutrient digestion, intestinal microbiota, signaling of the immune system or, after absorption, on physiological and metabolic processes. We are only at the beginning of understanding these ‘functional’ effects of dietary protein sources, which could generate added value when included in animal feeds. The aim of the present paper is to provide a short overview and some examples on functional properties of dietary protein sources in diets for pigs.
Protein sources for animal feeds
Animal production is growing worldwide. This relates to the increasing demand for high quality food products associated with the increasing world population, growing to about nine billion people in 2050, and with the increasing wealth of the human population (FAO, 2009). As a result there is increasing competition for feed and food ingredients to be used as immediate ingredient for human foods or for inclusion in animal feeds. These factors enforce efficient and sustainable use of these resources.
Proteins and amino acids are essential nutrients for production animals, including pigs. For this reason, there is an increasing demand for protein sources for animal feeds throughout the world. The demand can be fulfilled by traditional plant protein sources, such as soya bean meal and other oil seed by-products, legume seeds, cereals and their protein rich by-products resulting from starch and ethanol production, and by animal protein sources such as milk derived products e.g. whey. Alternative protein sources, such as sea cultivated algae, seaweeds, and insects, however, get increasing attention. Van Krimpen et al. (2013) considered options for increasing protein production for animal feed in Europe (Table 1).
They concluded that the identified protein sources differ substantially in terms of environmental sustainability. Products with a low dry matter content, i.e. lucerne, leaves, aquatic proteins are considered to be less sustainable due to the high energy costs for drying. Within the category of oil seeds, European produced soybean meal seems to be the most promising alternative for soybean meal and soya beans imported from South America. Protein yield of soybean meal produced in Europe, however, should be further increased to make this crop economically feasible for the farmer. To achieve this, new varieties have to be developed with an ultra-short growing season. Among the grain legumes, peas seemed the most promising alternative for soybean meal. The protein yield is reasonably high, but should be further improved. In the long term, leaf proteins and aquatic proteins probably might contribute to provision of protein for animal feeds. Knowledge on the nutritional value and potential functional effects of the inclusion of some of these alternative protein sources in animal feeds, however, is scarce.
Nutritional and functional value of feed ingredients
Traditionally, feed ingredients, including protein sources are evaluated on their capacity to deliver digestible nutrients and energy in the form of protein, amino acids, carbohydrates, fats, minerals and vitamins (nutritional value). These values are used in diet formulation, in which the dietary supply of nutrients from ingredients is balanced against the known nutrient requirements of animals. Novel feed ingredients are generally also first evaluated on this basis.
Many feed ingredients, however, are also known for other effects on their consumer. They can relate to known or unidentified constituents which interfere with either nutritional value or with specific effects on e.g. feed intake, nutrient digestion, nutrient or organ metabolism, animal health or quality of the edible animal derived end product (Figure 1). Such effects can be considered positive (functional effects) or negative (antinutritional effects). The latter refer to constituents naturally present in ingredients (e.g. antinutritional factors) or to substances concentrated or formed during processing of feed ingredients (e.g. during heat or enzymatic processing).
Functional protein sources for food and feed
Functional food or feed ingredients have been defined as modified ingredients that provide health or other benefits beyond satisfying traditional nutrient requirements (Sanders, 1998, Biesalski et al., 2009). Functional ingredients contain bioactive compounds or properties with supposed positive effects on the digestive process, intestinal barrier function, intestinal and systemic health.
Feed ingredients of plant and animal origin are generally a set of nutrients in a complex matrix and physical structure. They consist of different anatomical components of the plant or animal and are carriers of nutrients, which, after hydrolysis and absorption in the GIT, become available for use in organs and tissues. Ingredients and constituents in diets can also have other “non-nutritional”, functional properties e.g. in relation to feed intake (palatability, satiety), passage rate through the GIT, pro- and antimicrobial properties, (anti-) oxidative effects, immune signaling and metabolic effects. Beside specific secondary metabolites, compounds that are not directly involved in the normal growth, development, or reproduction, in feed ingredients of plant origin, the carbohydrate, protein and fat fraction of feed ingredients seem most relevant in this respect. Bioactive compounds are extra nutritional constituents that typically occur at low concentrations in foods. They are intensively studied to evaluate their effects on health (Kris-Etherton et al., 2002), in particular in humans and in model animal species. Bioactive compounds may be present in feed ingredients of plant and animal origin as such, but can also be released during enzymatic digestion, in particular those of peptide and protein nature, which are released during the process of protein digestion in the GIT (Jansman, 2016).
An overview of legume proteins and their nutraceutical properties in man has been provided by Carbonara et al. (2015). Most of the efforts in this field are directed towards effects on human health and disease, however, it can be anticipated that similar effects of proteins from a variety of protein sources applied in animal feeds do exist and, once known, can be applied to develop functional feeds for animals supporting their development and health.
At least in animal feeds, bioactivity in legume seeds, was originally considered from a negative perspective. Various proteins and other naturally present constituents were identified with antinutritional properties (anti-nutritional factors) limiting nutrient digestion and absorption and/or intestinal barrier function (Lallès and Jansman, 1998). Examples are Protease-Kunitz and Bowman-Birk-inhibitors, α-amylase inhibitors (AAIs), lectins, storage proteins (7S and 11S globulins, prolamins, glutelins) from legume seeds (soybean, lupin) and cereal grains (wheat), have extensively been characterized. Epidemiological studies, however, identified a decreased occurrence of breast, colon and prostate cancer in legume-consuming populations. This triggered research of protease inhibitors, primarily soybean Bowman-Birk inhibitor (BBI), in the suppression of cancer promotion in vitro and in vivo (Birk, 1993). Anticarcinogenic, hypocholesterolemic, glucose and blood pressure-lowering, antioxidant and antimicrobial effects have later been documented for several legume proteins and peptides by in vitro and in vivo studies (Carbonara et al., 2015).
Plant foods studied for their bioactive peptide related activity in humans include soybean, oat, wheat, hemp seed, canola, flaxseed, and pulses (Lopez-Barrios et al., 2014). They mention that particularly, pulse seeds hydrolysates and bioactive peptides have been described with in vitro activities towards cancer, cardiovascular conditions, or their physiological effects on oxidative damage, inflammation, hypertension, and high cholesterol.
Bioactivity of proteins and peptides can be predicted from known protein amino acid sequences. Computer-assisted databases are available for predicting bioactive proteins and peptides located within a parent protein. Other databases can predict precursor protein of a bioactive peptide from a known amino acid sequence (Marambe and Wanasundara, 2012). Recently, Kar et al. (2016) elucidated potential bioactive properties of the protein fraction in some common and new protein sources for animal feeds using such an in silico omics approach. A fingerprint of the bioactivity for a number of protein sources was provided, showing that protein sources potentially do have blood pressure inhibiting (inhibition of angiotensin converting enzyme (ACE) results in an antihypertensive effect), anti-oxidative, antimicrobial, (metabolically) stimulating, inhibiting and regulating effects. To sustain the physiological effects, bioactive peptides must show stability against GI proteases and be absorbed through the enterocytes to the blood without being degraded by peptidases from the brush border and in serum. There is an exception with peptides that act in the GIT and do not have to be absorbed to exert their biological properties (Udenigwe and Aluko, 2012).
The potential in vivo effects of peptides and proteins formed during the process of protein digestion, in modifying and supporting health and functions of the GIT and systemic metabolism and health requires further exploration. Antimicrobial peptides are also found in mammalian tissues, in milk, eggs, plants, insects and microbiota and can be isolated or produced chemically or via microbial fermentation with potential for application in food and feed as modifier of intestinal microbiota and immune modulatory activity. Due to their broad spectrum of activity against several species of bacteria, fungi, protozoa, and viruses, such peptides may show beneficial effects on performance, nutrient digestibility, intestinal morphology as well as intestinal and faecal microbiota in pigs (Xiao et al., 2015). Peptides with antioxidative properties have also been recognized with potential to be used as nutraceuticals and alternatives for supplemented antioxidants in food and feed.
However, it is still considered a challenge to understand the role and determine the fate of dietary anti oxidative peptides once they enter the circulation system and cross cell membranes. The majority of bioactive peptides vary essentially from di- to heptapeptides. They need to be resistant to the digestion process throughout the GIT and be able to be absorbed intact into the blood system to reach the target organs and tissues. Dairy and marine-derived products seem to be the major sources of food-derived bioactive proteins and peptides but other sources, such as eggs and plant-derived products including legumes, nuts and cereals (e.g. wheat, rice, maize), are of importance for providing bioactive peptides (Kim et al., 2012). Synthetic peptides are also being produced especially for pharmaceutical purposes since they can exhibit therapeutic roles in the treatment of several diseases (Freitas et al., 2013). Furthermore, they conclude that it is well-established that in vitro antioxidant activity cannot be directly correlated to health-promoting activity. Application of the full potential anti-oxidative capacity in food and feed ingredients or as pharmaceutical agent, however, seems to be rather far away, related to issues such as intestinal stability and absorbability and physiological efficacy (Freitas et al., 2013).
Protein sources and gut health in pigs
In pigs, health of the GIT can be defined as its capacity to exert its different functions allowing the animal to achieve its potential productive performance under a variety of environmental conditions (Jansman, 2016). Diet composition and protein sources included, can have major effects on development and functionality of the GIT. The effects of protein sources on gut health relate to the presence of specific constituents, release of bioactive peptides during protein digestion and to protein sources, its proteinaceous part as well as its other constituents, e.g. in plant sources, structural carbohydrates and oligosaccharides, interfering with the intestinal microbiota composition and colonization of pathogenic species.
One of the prime areas of research in relation to the application of bioactive constituents, either naturally present in feed ingredients of released during in vivo digestion of dietary proteins concerns gut heath. Improvement of gut function and health in pigs and poultry is a major challenge in animal production, under the constraint that pressure is high to reduce antibiotic use in animal production in many parts of the world. The function of the GIT is not only to digest and absorb nutrients, it also has an important barrier function. The barrier consist of various compartments, being a physical, an immunological barrier and a microbial barrier. The latter consist of an extensive and complex microbiota present in both the lumen and attached to the intestinal mucosa. The microbiota changes and increases in density from the proximal to the distal compartments of the intestinal tract (the small and large intestine). Dietary constituents and proteins and peptides released during the digestive process can interfere with each of these functions in a complex manner (Aumiller et al., 2015).
Rist et al. (2013) reviewed the effects of dietary protein sources on intestinal protein fermentation in pigs. The degradation of protein results in metabolites such as branched chain fatty acids (BCFA), but also in potentially toxic products including ammonia, amines, phenols and indoles. As increased intestinal protein fermentation suggests proliferation of potential pathogens (Hughes et al., 2000), several studies examined the effect of dietary protein source on composition of the intestinal microbiota. They concluded that available studies on the effect of dietary protein source on the composition of the intestinal microbiota are inconsistent and do not allow for recommending ultimate feeding strategies from this perspective. However, under conditions of increased stress, such as suboptimal hygienic and housing conditions or exposure to infections, they suggest that plant protein sources with a lower protein digestibility might increase the risk for the development of post weaning diarrhea, related to increased intestinal fermentation of protein and increased risk of proliferation of pathogenic bacteria.
In relation to gut health, anti-adhesive peptides could also be of relevance. It has been shown that anti-adhesive peptides against H. pylori, causing inflammation and gastric ulcers, are present in peas hydrolyzed with trypsin (Niehues et al., 2010). According to Becker et al. (2009), grain legumes may offer alternative adhesion sites other than the epithelium for pathogens such as E. coli, thereby reducing the numbers of pathogens attaching to the intestinal mucosa. For example, pea hulls exert a stronger in vitro binding capacity than faba bean hulls for ETEC K88 adhesins (Becker et al., 2009). It could be shown that faba bean hulls interfere more efficiently than pea hulls with the heat-labile entero-toxin LTp-I from ETEC which is binding to the intestinal epithelial GM1 receptor (Becker et al., 2012).
Specific feed ingredients such as milk products, plasma proteins, seaweed, algae are known for their functional properties in relation to gut health (Jansman et al, 2016). These relate to their specific effects on the intestinal microbiota (e.g. antimicrobial properties in case of milk and plasma proteins) to stimulating effects on the developing local immune system in young animals (e.g. seaweed and algae). For example dietary provision of a Laminaria spp. derived seaweed extracts, containing laminarin (a low-molecular-weight polysaccharide containing β-glucans) and fucoidan (a high-molecular-weight sulfated polysaccharide with anti-inflammatory, antiviral, antitumor properties) modulate the GIT environment, possess potent antibacterial and immunomodulatory properties, influence intestinal gene expression and enhance growth performance in pigs (Deville et al., 2007, Walsh et al., 2013, Heim et al. 2014a and 2014b). The former is suggested to be in part related to improvement of the digestive and absorptive function of the intestine.
The further increase in the worlds’ animal production, and strive for improving its sustainability requires identification of novel protein sources and efficient use of traditional sources. The former requires at least a proper understanding of their nutritional value. Improved knowledge on “other” functional effects of the use protein various sources, however, on the animal’s productivity, resilience and its gut and general health could contribute to maximize the value of protein sources in animal feeds. Use of novel omics based research tools and approaches and development and application of food based innovations to support human health will provide also new options to develop and apply functional properties of ingredients in diets for pigs and other production animals.