Protease Inhibitors in Processed Plant Foods
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161 Journal of Food Protection, Vol. 50, No. 2, Pages 161-166 (February 1987) Copyright International Association of Milk, Food and Environmental Sanitarians Protease Inhibitors in Processed Plant Foods ROBERT A. BURNS Department of Nutritional Science, Mead Johnson Nutritional Group, Evansville, Indiana 47721 (Received for publication January 16, 1986) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/2/161/1651248/0362-028x-50_2_161.pdf by guest on 24 September 2021 ABSTRACT infestations or damage. Hence, the protease inhibitor ac tivity found in seeds or tubers will vary with variety, tim Plants contain a wide variety of protein protease inhibitors. ing and conditions of harvest, and duration and condi However, most is known about the serine protease (trypsin and chymotrypsin) inhibitors found in legumes, particularly soy tions of storage (43). Plant inhibitors have been identified beans. These inhibitors in unheated legume protein (a) impair for enzymes from four major protease classes. The en the protein's nutritional quality, (b) induce pancreatic hyper zymes of importance regarding mammalian protein diges trophy in some but not all experimental animals, (c) enhance tion and nutrition which are susceptible to plant protease the action of chemical pancreatic carcinogens in Wistar rats but inhibitors include: the carboxyl protease, pepsin; the not hamsters or mice, (d) are reported to be carcinogenic to serine proteases, trypsin, chymotrypsin and elastase; and the pancreas of Wistar rats and (e) inhibit certain experimental the metallo-proteases, carboxypeptidases A and B. There tumors in rats, mice and hamsters. The physiological signifi are also plant protease inhibitors which inhibit the mam cance of the low residual protease inhibitor levels in commer malian plasma serine proteases, kallikrein and plasmin, cially processed plant proteins and human foods prepared from and the plant sulfhydryl proteases, bromelain, ficin and such proteins remains to be resolved. Plant proteins prepared papain (43). for human consumption, however, contain low levels of pro tease inhibitor activity which are of no nutritional concern in The protein protease inhibitors found in plants gener animals or humans. ally contain little or no carbohydrate and have a molecu lar weight ranging from 4,000 to 80,000. The widely studied Bowman-Birk protease inhibitor from soybeans Protease inhibitors are widely distributed throughout contains 71 amino acids with a molecular weight of nature and are found in plants, animals and microor 8,000, and the Kunitz inhibitor from soybeans contains ganisms. The scope of this overview will be restricted 198 amino acids with a molecular weight of 23,000. to protein protease inhibitors occurring in plants, par Many of the larger protease inhibitors are polymeric con ticularly plants contributing to the human diet. The taining up to four subunits. The amino acid sequences overview will include a brief description of protein pro of many protease inhibitors have been determined, and tease inhibitors, a discussion of their nutritional, phys a characteristic feature is a large degree of sequence iological, toxicological, and therapeutic significance, homology both within the same inhibitor and between in and a review of the effects of food processing and food hibitors from different plants (43). All protease inhibitors preparation procedures on protease inhibitor activity. have one or more peptide bond (reactive site) which in teracts with the corresponding enzyme active site. The OCCURRENCE AND PROPERTIES OF PROTEIN PRO reactive sites are normally found closely associated with TEASE INHIBITORS IN PLANTS half cystine residues linked by a S-S bridge (24,25). Often two or more enzymes can be inhibited by the same Protease inhibitors have been found in a great variety protease inhibitor. In these instances, the different en of plants, including most legumes and cereals and certain zymes can compete for the same site on the inhibitor or fruits (apples, bananas, pineapples and raisins) and veget can be inhibited by separate sites on the inhibitor ables (cabbage, cucumbers, potatoes, spinach and to molecule. The reactive sites of inhibitors react with active matoes) (4,43). It has been estimated that between 5 to sites on the corresponding enzyme in a similar manner 10% of the soluble proteins in barley, wheat and rye to the interaction between substrates and enzyme. Unlike grains are protease inhibitors (35). These levels of in substrates, however, the inhibitors form an extremely hibitors approximate the lower estimates of the levels stable complex with the enzyme which dissociates found in mammalian pancreas and plasma (4,49). The slowly. It has still not been entirely resolved why these quantity of protease inhibitors depends on variety and proteins are inhibitors, not just substrates for their respec physiological status of the plant and on levels of insect tive enzymes (24). JOURNAL OF FOOD PROTECTION, VOL. 50, FEBRUARY 1987 162 BURNS NUTRITIONAL AND PHYSIOLOGICAL SIGNIFICANCE (21), varying heat treatments (41) and various soy protein OF PROTEASE INHIBITORS fractions (37). Pancreatic hypertrophy and the over-secre tion of pancreatic enzymes can cause part of the growth It has been known for many years that the nutritional depression in rats fed unheated soy protein because sulfur quality of the protein of legumes can be improved by amino acids are first-limiting in soy protein and pancrea heat treatment (17). The main heat labile factors in tic enzymes are rich in sulfur amino acids (29). legumes are protease inhibitors and lectins and have been The following mechanism has been proposed to explain extensively reviewed by Liener (29,30). Although pro the pancreatic hypertrophy caused by trypsin inhibitors in tease inhibitors are present in almost all legumes, the rats. The level of pancreatic secretions and pancreas size heat-labile trypsin inhibitors in soybeans have been most are normally regulated through cholecystokinin by the extensively examined due to the importance of soy pro level of free trypsin in the intestine. After ingestion of tein as a component in animal and human nutrition. In trypsin inhibitors or protein, free trypsin levels fall and the 1940's, it was first realized that the improved nutri the pancreas is stimulated to secrete more enzymes, in Downloaded from http://meridian.allenpress.com/jfp/article-pdf/50/2/161/1651248/0362-028x-50_2_161.pdf by guest on 24 September 2021 tional quality of soybeans for rats, chickens and mice due cluding trypsin (10,31). The persistent stimulation of pan to heating could be attributed to destruction of these heat- creatic secretion elicited by dietary trypsin inhibitors in labile trypsin inhibitors (29). Rackis et al. (41) have the rat results in pancreatic hypertrophy. Protease or tryp clearly demonstrated that as soy flakes are treated with sin inhibitors from other plants have not been as exten moist heat for increasing periods of time, the trypsin in sively examined as those from soybeans, but available hibitor activity decreases and protein quality as measured data indicate they exert their effects on physiology and by protein efficiency ratio (PER) or apparent nitrogen di nutritional quality of the protein by similar mechanisms gestibility increases progressively. (29). Trypsin inhibitor activity, however, is generally not a good predictor of the nutritional quality of soy protein TOXICOLOGICAL ASPECTS OF PROTEASE IN preparations. Kakade et al. (21) measured trypsin in HIBITORS hibitor activity and PER of 25 different varieties of soy bean and found no correlation between PER and trypsin Rapidly growing tissue has an increased susceptibility inhibitor levels. In a further experiment, Kakade et al. to chemical carcinogens. Therefore, it is not unreasonable (18) demonstrated that, for soy flakes, removal of trypsin that feeding raw soy flour to rats would cause pancreatic inhibitor by affinity chromatography increased the PER hypertrophy and increase the susceptibility of the organ from 1.4 to 1.9, but heat treatment causing a similar de to chemical carcinogens. This phenomenon was first re crease in trypsin inhibitor activity effected a greater in ported by Morgan et al. (36) in 1977. They demonstrated crease in PER from 1.4 to 2.7. The lower PER of trypsin that after male Wistar rats were fed raw soy flour and inhibitor-depleted soy protein compared to heated soy given weekly intraperitoneal injections of a pancreatic protein may be partially due to removal of the sulfur carcinogen, azaserine, for 19 wk, all pancreatic sections amino acid-rich component (trypsin inhibitor) of a protein examined had nodules in the acinar tissue. Control rats limiting in sulfur amino acids. However, measurements fed heated soy flour and injected with saline had no of in vitro digestibility and pancreatic hypertrophy sup nodules and other rats receiving either raw soy flour and port the author's conclusion that not all growth-inhibiting saline injections or heated soy flour with azaserine injec activity can be attributed to trypsin inhibitors, but that tions had fewer pancreatic nodules (36). The same inves native soy protein can be resistant to digestion by pro tigators have extended and verified these preliminary ob teolytic enzymes (18). Therefore, trypsin inhibitor activ servations in Wistar rats and in all instances the car ity alone cannot be used to assess soy protein quality as cinogenicity of azaserine