Levels of trypsin inhibitors in food and feed products

Data on trypsin inhibitor contents of some commonly used food and feed products are reviewed in Table 1. Soyabeans are the most concentrated source of trypsin inhibitors among common food and feed products. Contents of tryspin inhibitors (mostly the Kunitz inhibitor) in soyabeans have been reported to vary from 8·6–48·2 mg/g sample or from 20·3–122·6 mg/g protein (Table 1). This large variation in contents of trypsin inhibitors could be due to differences in cultivars and, perhaps, to use of various methods of determination. Compared to raw soyabeans, peas, and other grain legumes, and properly processed soyabean products contain considerably lower levels of trypsin inhibitors.

Table 1 Trypsin inhibitor content of some common soya protein and other legume protein products

* Assuming a protein content of 50 % in soya flour.

Since the predominant trypsin inhibitors in soyabeans are located mostly with the main storage proteins in the cotyledons, they tend to fractionate with the storage proteins when soyabeans are processed into food ingredients⁽Reference Anderson and Wolf⁹⁾. Therefore, when soyabeans are defatted to produce raw flour, trypsin inhibitors become concentrated to levels of 28·0–65·8 mg/g sample or 57·8–131·6 mg/g protein.

Due to their proteic nature, trypsin inhibitors can be inactivated by heat processing including extrusion, infrared radiation, micronizing, boiling, autoclaving, steam processing or flaking⁽Reference Gatel¹⁴⁾ or they can be removed by fractionation⁽Reference Friedman and Brandon⁷⁾. The extent of heat inactivation of trypsin inhibitors depends upon a number of factors including the initial endogenous level, temperature, heating time, particle size, moisture and perhaps crop species and cultivar⁽Reference Friedman and Brandon^7, Reference Liener^8, Reference Gatel¹⁴⁾.

Most properly processed commercial soyabean products for human consumption including concentrated soyabean protein products such as concentrates (70 % protein) and protein isolates (90 % protein), soya-based infant formulas, soya milk and miso, have been subjected to sufficient heat treatment to inactivate up to 80 % of the trypsin inhibitor activity in raw soya flour⁽Reference Liener⁸⁾. Application of prolonged heating required to destroy all inhibitor activity would adversely affect protein digestibility and quality of the soyabean products. For example, heating at 100°C for 10 min reduced trypsin inhibitor activity by about 80 % and resulted in an optimal PER (Protein Efficiency Ratio) of about 3·1. However, prolonged heating to further reduce trypsin inhibitor activity resulted in a lower PER of about 2·9⁽Reference Rackis¹⁹⁾.

In the absence of regulatory upper safe limits of dietary trypsin inhibitors, there is no guarantee that each and every commercial product would be properly processed and, consequently, would contain minimal residual levels of trypsin inhibitors, which may vary greatly with the extent of heat and other processing conditions used in the preparation of soyabean products. For example, several commercial soya beverages have been reported to contain significant levels of residual trypsin inhibitor activity (up to 71 and 64 % of that of whole soybeans) against bovine and human trypsin, respectively (Fig. 1a and 1b; Xiao CW, Wood CM, Robertson P, Gilani GS, unpublished data). Similarly, some soya-based infant formulas have been reported to retain up to 28 % of the trypsin inhibitor activity⁽Reference Peace, Sarwar and Touchburn¹³⁾. An outbreak of gastrointestinal illness in individuals who had consumed an unprocessed soya protein extender in tuna fish salad documented the fact that inadequately processed soya products can find their way into the human food chain⁽Reference Liener⁸⁾.

Fig. 1 Inhibition of bovine (a) and human (b) trypsin by SBTI (soyabean trypsin inhibitors) in some commercial soyabean beverages (A, B, C, and D) sold in Canada (Xiao CW, Wood CM, Robertson P& Gilani GS, unpublished data).

Antinutritional effects of trypsin inhibitors

The feeding of raw soyabean protein preparations or extracted inhibitors from soyabeans caused an enlargement of the pancreas in susceptible animals⁽Reference Friedman and Brandon⁷⁾. Similarly, feeding of a soya protein isolate has been reported to significantly increase pancreatic weight of male and female rats but to significantly reduce their spleen weight (Fig. 2). Exposure to soyabean trypsin inhibitors results in increased synthesis and secretion of proteases (such as trypsin, chymotrypsin and elastase) and pancreatic hypertrophy and hyperplasia in animal models⁽Reference Friedman and Brandon⁷⁾. The increased secretion of proteases supported the suggestion that the growth depression caused by trypsin inhibitors was the consequence of an endogenous loss of amino acids in the form of enzymes being secreted by a hyperactive pancreas. Trypsin and chymotrypsin are particularly rich in sulphur-containing amino acids. Therefore, the effect of a hyperactive pancreas would be to divert these amino acids from the synthesis of body tissue proteins to the synthesis of enzymes, which are subsequently lost in the faeces⁽Reference Friedman and Brandon⁷⁾.

Fig. 2 Effects of feeding casein (control) and SPI (soya protein isolate) for 90 days on spleen and pancreas weights of female and male rats (Data were abstracted from Huang et al. ⁽Reference Huang, Wood and L'Abbe²⁰⁾). ^a,b Means for spleen or pancreas weights between the two dietary treatments (casein and SPI) in the same gender of rats with unlike superscripts differ significantly (P < 0·01).

Protein and amino acid digestibility and protein quality have been reported to be negatively affected in animal models by the presence of high levels of dietary trypsin inhibitors and other antinutritional factors from soyabeans, kidney beans and other grain legumes⁽Reference Gilani, Cockell and Sepehr⁵⁾. The effects of feeding a food-grade defatted soya flour (Nutrisoy) and autoclaved Nutrisoy on protein and amino acid digestibility were studied in growing pigs⁽Reference Li, Sauer and Huang²¹⁾. The trypsin inhibitor activities in the Nutrisoy and autoclaved Nutrisoy diets were 13·4 and 3·0 g/kg, respectively. The ileal protein and amino acid digestibility in the diet based on unheated Nutrisoy were about 50 % lower compared to those in the diet based on autoclaved Nutrisoy (Table 2).

Table 2 Effects of feeding raw soyabean flour (Nutrisoy) and autoclaved Nutrisoy on the ileal digestibility (%) of protein and selected amino acids in growing pigs*

^a,b Means in the same row between the two diets with unlike superscripts differ significantly (P < 0·01).

* Data were abstracted from Li et al. ⁽Reference Li, Sauer and Huang²¹⁾. Two maize starch-based diets were fed which contained 200 g/kg diet from either Nutrisoy (a food-grade defatted soya flour containing active trypsin inhibitors) or autoclaved Nutrisoy (containing reduced amounts of trypsin inhibitors).

† Digestibility after correction for dietary supplementation of methionine.

The influence of thermal processing (raw; home processing, boiling in hot water at 100°C for 10 min; and commercial canning) on amino acid digestibility in red kidney beans (Phaseolus vulgaris L.) has been investigated⁽Reference Wu, Woodie and Williams²²⁾. Digestibility was determined as true faecal digestibility in rats fed red kidney beans as the sole source of dietary protein in diets containing 10 % protein. The raw bean diet containing the highest amounts of antinutritional factors had the lowest amino acid digestibility values. The digestibility values for the first limiting amino acid (methionine + cystine) and valine in the raw bean diet were negative, whereas the values for other IAA, arginine and histidine ranged from 4–32 % (Table 3). Home processing and commercial canning substantially improved the digestibility of amino acids. The beneficial effect of home processing was more pronounced than that of commercial canning (Table 3). The lower amino acid digestibility values for the canned beans compared to those for the home processed beans would suggest an adverse effect of severe heat treatment used during canning on amino acid digestibility, especially that of the first limiting amino acid, methionine + cystine.

Table 3 Effects of processing on true rat faecal digestibility (%) of selected amino acids in red kidney beans*†

^a,b,c Digestibility values within a row with unlike superscript letters among the three diets were significantly different (P <  0·05).

* Data were abstracted from Wu et al. ⁽Reference Wu, Woodie and Williams²²⁾. Diets were formulated to contain 10 % protein. A protein-free diet was fed to estimate metabolic faecal amino acids; used in the calculations of true digestibility.

† Treatments: raw, uncooked dry beans; home-cooked beans (boiled in water, 100°C for 120 min); canned, commercially canned beans, Progresso; Casein, ANRC casein.

In lentils and beans, the true faecal digestibility of limiting amino acids such as methionine (41–60 %), cystine (0–75 %), tryptophan (47–76 %), and threonine (62–77 %) were substantially lower than the digestibility of protein (72–86 %) (Table 4). This would suggest that protein digestibility may not be a good approximation of the bioavailability of amino acids in grain legumes.

Table 4 Values (%) for the true digestibility of protein and limiting amino acids in lentils and beans, as determined by the rat balance method*

* Data were abstracted from Sarwar & Peace⁽Reference Sarwar and Peace²³⁾.

The presence of trypsin inhibitors in legumes such as soyabean not only has adverse effects on protein and amino acid digestibility, it also negatively impacts on protein quality. For example, three breeding lines of soyabean developed in Nigeria in raw form that contained 20·3–51·1 mg/g trypsin inhibitors had true faecal protein digestibilities of 47–58 % and had negative PER values ( − 0·46–0·88) compared to a PER value of 2·47 for a casein control (Table 5). Boiling (for 30 min) was more beneficial than autoclaving (for 20 min) in reducing levels of trypsin inhibitors (2·2–12·7 vs. 15·9–22·5 mg/g protein), and in improving true faecal protein digestibility (75–93 vs. 60–70 %) and PER values (1·35–2·30 vs. 0·91–1·33) (Table 5). An early study in humans showed that intake of raw soyabean meal supported 20 % lower nitrogen retention compared to that of heated soyabean meal⁽Reference Lewis and Taylor²⁴⁾.

Table 5 Effects of processing on contents of trypsin inhibitors, protein digestibility and protein quality of three soyabean breeding lines developed in Nigeria*

^a,b,c Means for trypsin inhibitor contents, true protein digestibility or PER within each soyabean line bearing unlike superscript letters were significantly (P < 0·05) different.

* Data were abstracted from Giami⁽Reference Giami¹¹⁾. The trypsin inhibitor contents, true protein digestibility and PER of the casein control diet were 0·00, 95 and 2·47, respectively.

Amounts of tannins in foods and feeds

The limited published data on tannin contents of various foods and feedstuffs are summarized in Table 6. Among important food and feed products, sorghum, millet, various types of beans and peas may contain considerable amounts of tannins (up to 72 g/kg) (Table 6). Some forage crops such as browse legumes may contain even higher amounts of tannins (up to 111 g/kg). Sorghum is an important food crop in many parts of Africa, Asia and the semi-arid tropics worldwide due to its drought resistance⁽Reference Duodu, Taylor and Belton³⁰⁾. Similarly, other cereal crops and beans and peas, which are high in tannins, form the main sources of nutrients in diets of people living in the economically disadvantaged countries of the world. Therefore, the total dietary intake of tannins in these countries could be considerable. The prevalence of protein malnutrition in these countries would further aggravate the antinutritional effects of dietary tannins. In general, tannins are resistant to heat⁽Reference Jansman and Longstaff²⁷⁾. Several technological treatments have been studied to reduce tannin contents of food and feed products including sorghum and fababean⁽Reference Jansman and Longstaff²⁷⁾. These included dehulling, soaking in water or alkaline solutions, addition of chemicals with a high affinity for tannins such as polyvinylpyrrolidone and polyethylene glycol or gelatin and germination. However, most of these processing treatments appeared to be rather laborious, expensive or ineffective⁽Reference Jansman and Longstaff²⁷⁾. More recently, genetic manipulation has been successful in producing fababean cultivars containing highly reduced levels of tannins⁽Reference Krépon, Marget and Peyronnet²⁸⁾.

Table 6 Contents of Tannins in some food and feeds products

Antinutritional effects of tannins

While tannins protect the grains against insects, birds and fungal attacks, this agronomic character is accompanied with reduced nutritional quality⁽Reference Duodu, Taylor and Belton³⁰⁾. It is well known that tannins are potential protein precipitants and they reduce protein and amino acid digestibility in animals fed tannin-containing cereals such as sorghum, and grain legumes such as field beans and fababean⁽Reference Jansman, Verstegen and Huisman³¹⁾. It is believed that under optimal conditions, sorghum tannin is capable of binding and precipitating at least 12 times its own weight of protein⁽Reference Jansman, Verstegen and Huisman³¹⁾.

The influence of feeding 20 sorghum cultivars varying in tannin (catechin equivalent) content of 0 to 3·88 to caecectomized cockerels on amino acid digestibility has been studied⁽Reference Elkin, Freed and Hamaker³²⁾. There were significant overall inverse relationships between tannin content and the mean digestibility of IAA or dispensable amino acids. It was also noted that sorghum varieties with similar tannin contents may vary greatly in their amino acid digestibility. The quantity of α-Kafirin (low digestibility principal storage protein) in various sorghum varieties was also inversely related to the amino acid digestibility, and may explain the somewhat inconsistent relationship between tannin content and amino acid digestibility in sorghum⁽Reference Elkin, Freed and Hamaker³²⁾.

Addition of increasing amounts of tannin-rich fababean hulls to a casein diet resulted in a linear decrease in the apparent rat faecal digestibility of total (r² = 0·97) and individual (r² = 0·27 to 0·99) amino acids⁽Reference Jansman, Frohlich and Marquardt²⁵⁾. The digestibility of most IAA was affected to a lesser degree than that of some of the dispensable amino acids, particularly proline, glycine and glutamic acid (Table 7). It was hypothesized that the marked reduction in digestibility of these dietary dispensable amino acids was primarily due to the interactions of tannins with the proline-rich proteins that were secreted by the parotid gland, as these three amino acids make up 73 % of the weight of isolated proline-rich proteins from parotid-glands of tannin-fed rats⁽Reference Jansman, Frohlich and Marquardt²⁵⁾.

Table 7 Influence of the addition of varying amounts of tannins extracted from fababean on apparent rat faecal digestibility values for amino acids in casein*

^a,b,c Means for amino acid digestibility among the three diets bearing unlike superscript letters were significantly (P <  0·05) different.

* Data were abstracted from Jansman et al. ⁽Reference Jansman, Frohlich and Marquardt²⁵⁾.

In a dose-response study, increasing the amount of dietary tannin-rich fababean hulls (providing 0·0 to 1·99 % tannins as catechin equivalents) caused a linear increase in both the relative size of the parotid glands in the rat (multiple regression correlation coefficient, r² = 0·90) and the quantity of proline-rich proteins in the glands (r² = 0·89)⁽Reference Jansman, Frohlich and Marquardt²⁵⁾. Similarly, the consumption of high tannin sorghum or purified condensed tannins was shown to increase specifically the size of the parotid gland and the synthesis and secretion of proline-rich proteins in rats⁽Reference Mehansho, Asquith and Butler³³⁾. It was suggested that proline-rich proteins are secreted in the saliva and are bound to dietary tannins in the oral cavity to protect dietary protein. Binding of tannins to both dietary and endogenous proteins (such as digestive enzymes and proteins located at the luminal side of the intestinal tract) has been used to explain reduced protein and amino acid digestibility in tannin-containing diets⁽Reference Jansman, Frohlich and Marquardt^25, Reference Mehansho, Asquith and Butler³³⁾.

Phytic acid contents of food and feed products

Phytate contents of a large number of foods have been previously summarized⁽Reference Harland and Oberleas^34, Reference Reddy³⁸⁾. The data on phytate contents of some commonly consumed food and feed products are summarized in Table 8. Phytate is present in seeds, grains and nuts at concentrations of up to several percent on a dry weight basis. Due to its uneven distribution within grains, most of the phytate can be found in the germ of corn, the bran of wheat and the pericarp of rice⁽Reference Reddy³⁸⁾. Processing treatments that remove or concentrate these portions can, therefore, result in dramatic changes in the phytate content of the finished product. For example, rice polishings, wheat bran and middlings and defatted meals of oilseeds (soyabean, cottonseed, sunflower and canola) contain substantial amounts of phytates (up to 5·5 per cent (Table 8). In pulses (peas, lentils and beans), the greatest proportion of the phytate is found in edible cotyledons. Therefore, mechanical processing does not result in much reduction in phytate content. Moreover, phytate is relatively heat-stable, with only a small fraction being destroyed during heat processing, while the phytase enzymes that might break down the phytate are heat labile⁽Reference Ryden and Selvendran⁴⁴⁾. However, extrusion (with a central temperature and moisture content of 150°C and 20 g/100 dry weight, respectively) of hard-to-cook common beans was reported to reduce phytate contents by about 20–30 % (Table 8).

Table 8 Phytic acid contents of some common food and feed products

Antinutritional effects of phytate on protein digestibility and quality

Phytic acid has been reported to interfere with the proteolytic action of pepsin in a number of vegetable and animal proteins as determined in vitro, possibly through the formation of phytate:protein interaction complexes at low pH⁽Reference Vaintraub and Bulmaga⁴⁵⁾. Phytate has also been reported to inhibit trypsin activity in some studies but not in all investigations. Addition of phytate to multienzyme proteolytic assay systems was shown to significantly (up to 25 %) inhibit in vitro digestion of casein⁽Reference Lothia, Hoch and Kievernagel⁴⁶⁾. Fermentation treatment of finger millet to decrease phytate content by 23–26 % caused a 14 to 26 % improvement in in vitro digestibility of the finger millet protein using pepsin and pancreatin⁽Reference Antony and Chnadra⁴⁷⁾.

The effects of phytate on protein and amino acid digestibility and protein quality have involved studies of phytase supplementation to production rations for animals such as poultry and swine. In such studies, microbial phytase is added to animal rations to degrade phytate, improve phosphorous bioavailability and reduce the environmental impact of high phosphate effluent from intensive pig and poultry units⁽Reference Selle, Ravindran and Caldwell³⁹⁾. This environmental problem prompted the development and acceptance of microbial phytase feed enzymes for monogastric animals.

While the primary function of phytase added to animal feeds was to improve the availability of phytate-bound phosphorous, it has provided new insights into the antinutritional properties of phytates. The interaction of phytate with proteins may have substantial practical and economical significance in animal nutrition⁽Reference Selle, Ravindran and Caldwell³⁹⁾.

Studies on the effects of phytase supplementation in improving protein and amino acid digestibility and protein utilization in pigs and poultry have been reviewed⁽Reference Selle, Ravindran and Caldwell³⁹⁾. Only a few studies have been reported on the influence of phytase supplementation on the ileal digestibility of amino acids in pigs. Differences in methodology make comparisons among these studies rather difficult, but an increase of 3–10 % in the ileal digestibility of at least some amino acids has been reported which appears to correlate with the magnitude of improved growth rates and protein retention reported by others⁽Reference Selle, Ravindran and Caldwell³⁹⁾.

Phytase supplementation has been reported to significantly improve the digestibility of protein and amino acids in 5-wk old broilers fed a number of feedstuffs including three cereals (corn, sorghum, and wheat) and four oilseed meals (soybean meal, cottonseed meal, canola meal and sunflower meal) and two cereal by-products (wheat middlings and rice polishings)⁽Reference Ravindran, Cabahug and Ravindran³⁶⁾. In the latter study, apparent ileal protein digestibility increased by 2–6 % in different diets due to phytase supplementation of 1200 FTU/g kg diet⁽Reference Ravindran, Cabahug and Ravindran³⁶⁾. One FTU (unit of phytase) is defined as the quantity of enzyme that releases 1 μmol of inorganic phosphorus/min from 0·00 015 mol/L sodium phytate at pH 5·5 at 37°C. The mean apparent ileal digestibility of 15 amino acids increased by 2–9 % with phytase addition to various diets. For individual IAA, average improvements across various feedstuffs tested were 4–8 % with the greatest improvement being noted for threonine and valine⁽Reference Ravindran, Cabahug and Ravindran³⁶⁾. Dietary phytate concentration was negatively correlated with the inherent protein digestibility (r = − 0·81, P < 0·001) and amino acid digestibilities (r = − 0·85, P < 0·001) of the feedstuffs tested.

Supplementation of a marginally (91 % of the recommended level) lysine-deficient diet (wheat-soyabean meal-sorghum) for broiler chicks with increasing levels of microbial phytase significantly improved (3–5 %) the apparent ileal digestibility of protein and amino acids⁽Reference Ravindran, Selle and Ravindran⁴⁸⁾. Statistically significant linear dose responses were noted for the digestibility of protein and all amino acids using phytase supplementation levels up to 1000 FTU/kg diet. Phytase supplementation also increased apparent metabolizable energy in that study, attaining a plateau effect at 750 FTU/kg diet⁽Reference Ravindran, Selle and Ravindran⁴⁸⁾.

The beneficial effects of phytase supplementation on the protein and amino acid digestibility of various feedstuffs for poultry and swine could be due to the release of protein from protein-phytate complexes occurring naturally in feedstuffs, the prevention of formation of binary and tertiary protein-phytate complexes within the gut, the alleviation of the negative impact of phytate on digestive enzymes, and the reduction in endogenous amino acid losses⁽Reference Selle, Ravindran and Caldwell³⁹⁾.

D-amino acid contents of some common food products

Data on D-amino contents of foods have been recently reviewed⁽Reference Csapó, Albert and Csapóo-Kiss⁶²⁾. Although some insects, worms and marine invertebrates contain substantial quantities of D-amino acids, such organisms are not major components of the human diet. However, in communities where marine shellfish is an important source of food, the antinutritional implications of D-amino acids must be taken into account⁽Reference Csapó, Albert and Csapóo-Kiss⁶²⁾. Milk, meat and various grains do not contain substantial quantities of D-amino acids. However, during the course of preparation for consumption, processing treatments are applied which may give rise to racemization. The influence of processing treatments on racemization of amino acids was investigated by determining D-aspartic acid concentrations⁽Reference Payan, Cadilla-Perezrios and Fisher⁶³⁾. In this study, raw milk contained the lowest level of D-aspartic acid (1·48 %) but quantities of D-aspartic acid increased with the extent of processing. For example, fat-free milk powder, kefir, evaporated milk, yoghurt and milk-based infant formulas contained 2·15, 2·44, 2·49, 3·12 and 4·95 % of D-aspartic acid, respectively⁽Reference Payan, Cadilla-Perezrios and Fisher⁶³⁾. A D-aspartic acid content of 31 % was found in casein heated at 230°C for 20 min⁽Reference Csapó, Albert and Csapóo-Kiss⁶²⁾. Similarly, high ratios of D-aspartic acid have been reported in textured soya protein and bacon (up to 13 %), alkaline/heat treated soya protein (up to 27·7 %), and alkaline/heat treated zein (up to 40·2 %)⁽Reference Csapó, Albert and Csapóo-Kiss^62, Reference Masters and Friedman⁶⁴⁾. Among common food products, substantial quantities of D-aspartic acid were reported in crackers made from wheat flour (9·5 %), Mexican pancake (11·6 %) and corn cake (15·4 %)⁽Reference Finley⁶⁵⁾. In mature cheeses, D-aspartic acid, D-alanine and D-glutamic acid were reported to be up to 68 %, suggesting that foods which have undergone microbial fermentation would contain substantial quantities of D-amino acids⁽Reference Palla, Marchelli and Dossena⁶⁶⁾. In general, the IAA do not racemize rapidly unless exposed to high temperatures⁽Reference Csapó, Albert and Csapóo-Kiss⁶²⁾.

LAL-amino acid contents of some common food and feed products

LAL (an unnatural amino acid derivative) is formed when proteins are subjected to an alkaline treatment. It is formed mainly by the addition of an ε-amino group of lysine residue to the double bond of a dehydroalanine residue that has been generated by the β-elimination reaction of cystine, phosphoserine, or glycoserine residue⁽Reference Masters and Friedman⁶⁴⁾. The amount of LAL formed in processed protein products is dependent upon temperature, concentration of alkali, time of exposure to alkali, type of protein, and type of cations in the solution⁽Reference Struthers, Dahlgren and Hopkins⁶⁷⁾. Heat treatment under non-alkaline conditions was also reported to form LAL in a variety of proteins⁽Reference Struthers, Dahlgren and Hopkins⁶⁷⁾.

Although protein-containing food and feed products are commonly subjected to alkaline/heat treatments, information on the amounts of LAL found in food products that are part of the every day diet are limited. The published data vary widely according to the treatments applied. The most representative results are summarized in Table 12. Domestic cooking may produce up to 1820 ppm LAL in foods (such as sausage, chicken meat and eggs) initially free of LAL. Commercial food products that have undergone processing treatments may contain LAL in variable amounts according to the type of product and certainly the conditions of preparation including alkaline/heat treatment. Sterilized milk and milk powders have been reported to contain up to 1160 ppm LAL. Special attention has been paid to the high LAL contents of sterilized liquid concentrate infant formulas which may contain as much as 2120 ppm LAL. Since such formulas are often the sole source of protein for infants over a long time period, it has been recommended that the LAL content of infant formulas be kept under < 200 ppm⁽Reference Friedman⁶⁹⁾. Protein-rich ingredients such as dried egg white, soya protein isolate and sodium caseinates have been reported to contain significant amounts (up to 6900 ppm LAL) (Table 12). Among the common food products, whipping agents are known to contain the highest levels of LAL, i.e. up to 53 150 ppm LAL.

Table 12 Levels of lysinoalanine (LAL) in some common food products

Antinutritional effects of protein-bound D-amino acids and LAL

Protein-bound D-amino acids formed during processing, especially at alkaline pH, may have adverse effects on protein digestibility and the quality and safety of processed foods. When absorbed, D-amino acids may be made utilizable by the action of racemases or epimerases or D-amino acid oxidases⁽Reference Friedman⁶⁰⁾. The amino acid oxidase system (which varies in amount and specificity of oxidases in different animal species) may become saturated when foods containing high concentrations of D-amino acids are consumed. Proteins containing D-amino acids can be hydrolyzed at peptide bonds containing L-amino acids. However, the hydrolysis rates may be slower than those for corresponding unprocessed proteins. These changes adversely affect the nutritional quality and safety of foods by generating biologically nonutilizable forms of amino acids, creating D-D, D-L, and L-D peptide bonds partly or fully inaccessible to proteolytic enzymes. Moreover, these racemized proteins may compete with proteins that do not have racemized amino acids for the active site of digestive proteinases in the gut and thus adversely affect the biological utilization of the unracemized proteins. The slower absorption of free and peptide-bound D- compared with L-amino acids may result in decreased protein digestibility⁽Reference Pappenheimer, Karnovsky and Maggio⁷⁰⁾. It is not known whether D-amino acid containing oligopeptides can change the microflora of the digestive tract. Wide variations in the biological utilization of free D-methionine by various animal species have been reported⁽Reference Friedman⁶⁰⁾. Free D-methionine was well utilized by mice and rats but was poorly utilized by humans when consumed either orally or during total parenteral nutrition.

The formation of D-amino acids and LAL and their influence on protein digestibility has also been studied⁽Reference de Vrese, Frik and Roos⁷¹⁾. Three protein sources (casein, β-Lactoglobulin and wheat protein) were subjected to heat and alkaline treatments (heating for 6 or 24 h at 65°C, pH 10·5–11·5). Treatment of these proteins for 24 h increased levels of D-amino acid residues. For example, about 11–15 % of L-asparagine and aspartic acid, the most susceptible amino acids, were racemized in the three protein sources⁽Reference de Vrese, Frik and Roos⁷¹⁾. Moreover, the alkaline/heat treatment resulted in increased amounts of LAL, and about 10–12 % of the total lysine was converted to LAL in these protein sources. True ileal protein digestibility of the treated casein, β-lactoglobulin and wheat protein, decreased by 13, 14 and 17 %, respectively, when fed to minipigs⁽Reference de Vrese, Frik and Roos⁷¹⁾. Similarly, the heat/alkaline treatment of casein caused significant reductions (up to 17 %) in digestibility of aspartate, serine and glycine. However, digestibility of other amino acids was not affected. This study provides evidence that even small amounts of D-amino acids and LAL within a protein can impair protein digestibility. This significant impairment was already present at a level of 8 % D-asparagine + aspartic acid as found in treated β-lactoglobulin (24 h at 65°C, pH 10·5)⁽Reference de Vrese, Frik and Roos⁷¹⁾.

The health concerns of LAL are twofold: LAL formation in foods results in a loss of the IAA, lysine, cysteine and threonine, and reduced protein digestibility; and secondly in kidney damage^(72, Reference Sternberg, Kim and Schwende⁷³⁾. Nephrocytomegaly (i.e. enlarged kidney cells) and nephrokaryomegaly (enlarged nuclei) are unique lesions induced in rats by LAL. The amount of dietary LAL needed to produce nephrocytomegaly depends on whether LAL is fed as the free amino acid or bound in a protein. Enlarged nuclei have been reported in rats by feeding as little as 1200–1400 ppm protein- bound LAL⁽Reference Gould and MacGregor^74, Reference Karayiannis, MacGregor and Bjeldanes⁷⁵⁾. In comparison, feeding of free LAL was found to produce nephrocytomegaly at much lower levels (100–250 ppm)⁽Reference DeGroot, Slump and Feron^76, Reference Woodard⁷⁷⁾. L-lysyl-D-alanyl-LAL, the most potent isomer of the four optical isomers of LAL, induced nephrocytomegaly when fed at levels as low as 30 ppm, whereas D-lysyl-D-alanyl-lysinoalanine did not produce lesions at less than 1000 ppm⁽Reference Slump⁷⁸⁾. LAL, which is a strong chelator of mineral nutrients such as calcium, iron, copper and zinc, may exert its toxic effect by metal binding in renal tubule cells. The human kidney is more susceptible to damage by LAL than that of several animal species⁽Reference Friedman⁶⁹⁾. The altered mineral (iron and copper) status due to consumption of LAL from processed foods was demonstrated in one of the first in vivo studies in rats⁽Reference Sarwar, L'Abbé and Trick⁷⁹⁾. Liver and kidney iron levels were greatly reduced which is of particular concern, as this is the mineral in which status is often already reduced in the elderly and infants, persons likely to consume sole-source foods. The susceptibility of humans to the nephrotoxic effect of LAL is unknown. Because nephrotoxic effects were not observed after long-term feeding of alkali-treated soya protein to baboons, consumption of low amounts of dietary LAL is probably safe for human consumption⁽Reference Friedman⁶⁹⁾ However, further analytical data are needed for assessment of the actual quantity of LAL ingested⁽⁷²⁾. In addition, the influence of chronic consumption of alkaline-treated foods high in LAL on the balance of copper and other minerals should be examined.

It has been recommended that the LAL content of infant formulas, which are often the sole source of protein for some infants, should be kept at < 200 ppm⁽Reference Friedman⁶⁹⁾. Significant amounts of LAL were found in infant formulas and formulated liquid diets sold in Canada. The liquid concentrate forms of milk-based infant formulas and commercially available formulated liquid diets were found to contain up to 1200 and 2400 ppm of LAL in the formula/diet protein, respectively (Gilani GS, unpublished data). It is not known whether growing children and infants are more sensitive to the adverse effects of LAL than are adult humans.

To establish an NOAEL (no observed adverse effect level) for dietary LAL, a 16-wk rat study investigating the effect of alkaline-treated soya protein isolate (to provide 0·05, 0·10, 0·20 and 0.30 % of dietary LAL) on growth, protein and mineral status were investigated (Gilani GS, L'Abbé MR, unpublished data). Body weights of rats fed a casein control, and 0·05, 0·10, and 0·20 % LAL diets were 522 ± 25^a, 500 ± 22^a, 370 ± 20^b and 280 ± 16^c g, respectively. Growth data suggested that the NOAEL for protein-bound LAL would probably be between 0·05 and 0·10 % of diet. Alkali-treated proteins containing LAL have lower digestibility compared to untreated proteins. For example, an inverse relationship between the LAL content of casein and the extent of in vitro proteolysis by trypsin has been demonstrated⁽Reference Friedman, Zahnley and Masters⁸⁰⁾. The biological utilization of LAL as a source of lysine in a mouse growth assay was only 3·8 % that of crystalline lysine⁽Reference Friedman, Gumbmann and Savoie⁸¹⁾. Similarly, LAL was completely unavailable as a source of lysine to the rat but it was 37 % bioavailable to the chick⁽Reference Robbins, Baker and Finley⁸²⁾. The alkaline treatment of soya protein reduced protein digestibility from 97 to 83 % in rats, and lowered body weight gain in baboons⁽Reference Friedman⁶⁹⁾. Similarly, alkaline/heat treatment had significant negative effects on the true faecal protein digestibility of lactalbumin (99 vs. 73 %) and soya protein isolate (96 vs. 68 %) in rats⁽Reference Sarwar, L'Abbé and Trick⁷⁹⁾. The protein quality of these protein sources as predicted by rat growth was also significantly reduced (Table 13). The treated proteins contained considerably higher levels of LAL compared to the untreated proteins. The amount of LAL in the treated lactalbumin was more than two-fold higher than in the treated SPI (soya protein isolate). As expected, the formation of LAL in the treated proteins was associated with a loss of lysine (19–20 %), cystine (73–77 %), and serine (18–30 %). There was also a loss of threonine (35–45 %) in the treated proteins (Table 13). Most other amino acids were not greatly affected by the alkaline treatment of the two proteins. Since the methodology used did not distinguish between D- and L-isomers of amino acids, the influence of the processing conditions on the formation of D-amino acids could not be determined in this study⁽Reference Sarwar, L'Abbé and Trick⁷⁹⁾. Exposing soyabean protein isolate to alkaline conditions (pH 8–14) for various time periods (10–480 min), and temperatures (25–95 at 19°C intervals) has been reported to destroy all of the cystine and part of the arginine, lysine, serine and threonine residues⁽Reference Friedman⁶⁹⁾. These losses were accompanied by the appearance of LAL and unidentified ninhydrin-positive compounds, and the formation of D-amino acids. Therefore, possible causes for the reduction in protein digestibility and quality following heat/alkaline treatment may include destruction of lysine, threonine, cystine and arginine, isomerization of L-amino acids to less digestible D-isomers, formation of inter- and intra-molecular cross-links, and inhibition of proteolytic enzymes⁽Reference Friedman^69, Reference Sarwar, L'Abbé and Trick⁷⁹⁾.

Table 13 Selected amino acids, true faecal protein digestibility (rat) and protein quality of untreated and alkaline/heat-treated lactalbumin and soya protein isolate (SPI)*

* Data were abstracted from Sarwar et al. ⁽Reference Sarwar, L'Abbé and Trick⁷⁹⁾.

† Protein sources were subjected to alkaline treatment with 0·1 N NaOH at room temperature for 1 h, followed by heat treatment at 75°C for 3 h, neutralization with 10 N HCl to pH 7·5, with ultrafiltration to remove salts and spray drying of the ultrafiltrate retentate.

§ Determined in rats; RPER (relative protein efficiency ratio) values were calculated using the following equations: RPER = [PER of test diet/PER of control diet] × 100; where PER (protein efficiency ratio) = weight gain of test rat/protein consumed by test rat.

Protein digestibility and quality of some enteral products based on caseinates and SPI were inferior to casein⁽Reference Sarwar and Peace⁸³⁾. For example, the true faecal protein digestibility of five commercial enteral products, as determined in rats, was significantly lower than that of casein (89–92 vs. 95 %). The enteral products also contained higher levels of LAL than casein (998–2333 vs. 0 μg/g protein). Since the formation of LAL was also reported to occur in a variety of proteins under nonalkaline conditions⁽Reference Friedman⁶⁹⁾, the lower protein digestibility of the enteral products could be explained by the presence of LAL in these products⁽Reference Sarwar and Peace⁸³⁾. Similarly, heat treatment at pH 12·2 was reported to cause a significant reduction in protein digestibility and NPU (net protein utilization) of casein and soyabean⁽Reference Friedman⁶⁹⁾.

In comparing the protein nutritional values of milk-based infant formulas sold in Europe, the formation of LAL was found to be one of the most sensitive predictors of protein damage in infant formulas caused by heat processing⁽Reference Pompei, Rossi and Mare⁸⁴⁾. It was found that liquid forms of milk-based formulas contained up to ten times more LAL than powder forms; suggesting more severe heat treatment in the preparation of liquid forms compared to powders⁽Reference Pompei, Rossi and Mare⁸⁴⁾. The question as to whether either D-amino acids and D-amino acid-containing peptides or cross-links such as LAL alone are responsible for lower protein digestibility, or whether these effects are additive remains unresolved. Both racemization and cross-links were shown to inhibit proteolysis and to decrease protein and peptide digestibility with in situ experiments using isolated loops of rat intestine⁽Reference Lister, Sykes and Baily⁸⁵⁾. The adverse effects of D-amino acids and LAL formed during heat processing of proteins may have considerable implications in animal nutrition. For example, feeding roller-dried milk powder to calves may impair their growth. Some NaOH and/or heat treated feedstuffs may contain considerable amounts of D-amino acids and LAL, for example, as a consequence of alkaline detoxification of aflotoxins. The diminished nutritive value for a racemized, heat and alkali-treated dietary protein may also have relevance for human nutrition. This reduction in protein nutritional value may not only be due to a reduction in L-amino acid content but also of a diminished overall digestibility. Even if many IAA amino acids are not racemized to D-isomers and would, after breakdown of the protein, be available for absorption, adjacent D-amino acids might nevertheless interfere with their absorption⁽Reference Paquet, Thresher and Swaisgood⁸⁶⁾. The effect of adjacent amino acids on the bioavailability of methionine in some tripeptides found in casein and soya protein has been examined⁽Reference Sarwar, Paquet and Peace⁸⁷⁾. The relative bioavailability of methionine (L-methionine = 100) in Ala-Met-Ala; Ala-D-Met-Ala; Valine-Met-Phe; Val-D-Met-Phe; Thr-Met-Arg; Thr-D-Met-Arg; Thr-Met-Lys was determined using a rat growth method (NPR, Net Protein Ratio) as the performance index. Crystalline D-met was completely bioavailable to the growing rat, but in the tripeptide form, D-Met (0–45 %) was markedly less bioavailable than L-Met (38–71 %) (Table 14). The bioavailability of Met was also influenced by the side chain of the adjacent amino acids. Met in the tripeptides with the bulky amino acids (Val-Met-Phe, Thr-Met-Arg, Thr-Met-Lys) was less bioavailable than in those with lighter amino acids (Ala-Met-Ala). D-Met in the tripeptides with bulky amino acids (Val-D-Met-Phe, Thr-D-Met-Arg) was completely unavailable for rat growth. The lower bioavailability of D-met when compared to L-Met in Ala-Met-Ala or Val-Met-Phe (Table 14) may be due to lower hydrolysis (by peptidases of the brush border membrane of the intestinal mucosa) of the respective peptide containing D-Met than that containing L-Met⁽Reference Paquet, Thresher and Swaisgood⁸⁶⁾. These findings suggested that protein-bound D-Met (formed during processing) may be considerably lower in bioavailability than protein-bound L-Met. Moreover, bioavailability of protein-bound Met may be influenced by the amino acids adjacent to Met in the polypeptide chain⁽Reference Sarwar, Paquet and Peace⁸⁷⁾.

Table 14 Net protein ratio (NPR) and methionine bioavailability values for some Synthetic tripeptides containing L- and D-methionine (Met)*

† Means (n = 8); the NPR of the casein + Met diet was 5·60, suggesting that the experimental conditions of the feeding study were properly controlled.

^a-d Means within the same column bearing unlike superscript letters differ significantly (P < 0·05).

* Data were abstracted from Sarwar et al.(1985).