Laurent Savoie

Relationship between dietary proteins, their in vitro digestion products, and serum cholesterol in rats☆

A relationship was assessed between the amino acid composition of 9 protein sources or of their in vitro digestion products and total serum cholesterol in rats. Three animal proteins (casein, beef, fish) and 6 vegetable proteins (soy, pea, peanut meal, rapeseed, oatmeal, wheat gluten) were tested. The intact protein sources were submitted to an enzymatic proteolysis according to a new in vitro digestion method. Each protein source was hydrolyzed for 30 min with pepsin at pH 1.9, then with 10 mg pancreatin at basic pH in a dialysis cell. The digestion products diffused through the dialysis membrane of the cell and were collected by a circulating sodium phosphate buffer over a 6-h period. They were likely to correspond to end products luminal in vivo digestion. The aromatic and the basic amino acids were present in higher proportions in the digestion products than in the intact protein sources, reflecting the specificity of the proteolytic enzymes. Total serum cholesterol was measured on male Sprague-Dawley rats fed cholesterol-free or cholesterol-enriched (1% cholesterol, 0.5% cholic acid) semipurified diets containing protein sources. Total serum cholesterol ranged from 70 mg/dl with the pea diet to 98 mg/dl with the peanut meal diet in rats fed cholesterol-free diets and from 163 mg/dl with the wheat gluten diet to 313 mg/dl with the casein diet in rats fed the cholesterol-enriched diets. These results suggested no specific effect of protein from animal or vegetable origin on total serum cholesterol in rats. In rats fed cholesterol-enriched diets, significant correlations were observed between total serum cholesterol and tyrosine content or leucine/isoleucine ratio of digestion products. These correlations were stronger than those observed with intact protein sources.

Circadian variation of food intake and digestive tract contents in the rat

The variation of food intake and digestive tract contents over a period of 24 hours was measured in the free feeding rat kept under a 12:12 hr light:dark cycle (lights on at 0700 hr). After a cessation between 0700 and 1000 hr, the rate of food intake increased progressively during daytime followed by a marked increase and a plateau after lights went off. In the dark, dry matter in the stomach was almost ten times the levels of the light phase. In the major part of the high feeding period, the rates of food intake and gastric emptying equilibrated. Dry matter content of the intestine also followed circadian variations that were specific for each segment. The time-to-time content of the mid-gut segment was closely related to gastric emptying. The extent of dry matter absorption in the proximal intestine decreased continuously from the time lights went on and reached a minimum at 22 hr. Absorption in the distal segments was much less subjected to variation over 24 hours. In the free feeding situation, the changes induced by the light cycle on the rhythm of food intake were accompanied by marked modifications of digestive tract contents including those of the intestine. Data indicate a close relationship between gastric function and the middle portion of the intestine.

Comparison of in vitro systems of protein digestion using either mammal or fish proteolytic enzymes

Hydrolysis of three different proteins by either crude fish digestive extracts or purified mammal proteases was assayed using two different in vitro systems. The closed system was a modification of the pH-stat method including a previous acid digestion. The open system used a digestion cell containing a semi-permeable membrane which allowed continuous separation of the final products of hydrolysis with a molecular cut-off of 1000 Da. Assays in both systems resulted a similar arrangement of the tested proteins in relation to their ability to be hydrolyzed, with casein>fish meal> or =soybean meal. With the exception of casein, no significant differences were found between results produced by any of the enzyme sources using the closed system. In constrast, significantly higher hydrolysis of all proteins was produced by mammal enzymes under conditions operating in the open system. Differences in the rate of release of amino acids measured in this latter system were related both to the type of protein and the origin of the enzymes. When using purified mammal enzymes, release of lysine or phenylalanine from casein and soybean was high, but low from fishmeal. Isoleucine and valine present in fishmeal were preferentially hydrolyzed by commercial enzymes, but glycine and proline by fish enzymes.

In vitro amino acid digestibility of food proteins as measured by the digestion cell technique

The digestibility of proteins and individual amino acids of nineteen selected foods was determined by anin vitro assay. Samples were hydrolysed with pepsin for 30 minutes in an acidic medium; the pH was then raised to 7.5 and the mixture poured into the dialysis bag (molecular weight cut-off 1000) of a digestion cell with pancreatin. Digestion products, mixtures of free amino acids and low molecular weight peptides which pass through the dialysis membrane, were collected for 6 hours by sodium phosphate buffer circulation. All proteins from animal sources displayed a digestibility similar to casein, except for breakfast sausage. Vegetable proteins showed intermediate digestibility, except for cereals (lower) or peanut butter (higher). Target amino acids of enzymes were generally more readily hydrolysed. However, compared to other animal proteins, glycine in milk products, valine, isoleucine, methionine and lysine in breakfast sausage and hot dog, and histidine in tuna were more easily released. Overheating of non-fat dried milk not only reduced the lysine digestibility, but also that of methionine, phenylalanine, histidine and cystine. Among vegetable proteins, wheat products were characterized by a relatively greater release of threonine, isoleucine and histidine, and peas by a lower digestibility of methionine and lysine. Proline of soy isolate and isoleucine of pinto bean were resistant to hydrolysis while arginine of pinto beans and of rice-wheat-gluten was easily released.

Sequential release of amino acids and peptides during in vitro digestion of casein and rapeseed proteins

Abstract An in vitro proteolysis method was used to study the kinetics of digestion of casein and rapeseed proteins. After a pepsin predigestion, the protein sources were submitted to a 24-hour pancreatin hydrolysis, with the continuous removal of digestion products by dialysis. Dialysates were collected at 3-hour intervals, and analysed for content of nitrogen, amino acids and low molecular weight peptides. At the end of the experimental period, casein had a higher overall digestibility (97%) than rapeseed (83%). The difference was mostly due to a high rate of digestion during the first 3 hours for casein, as nitrogen release was similar for both proteins at subsequent intervals. Much more variation was observed in individual amino acid digestibility. In casein, arginine, tyrosine and methionine were the most rapidly released, being more than 95% digested after 12 hours. In rapeseed, the amino acids were generally hydrolysed more gradually. Only tyrosine and methionine were more than 95% released by 24 hours. In general, highly digestible amino acids were mostly released as free amino acids. But differences were also noted between proteins for the distribution of individual amino acids in low molecular weight peptides. With casein, isoleucine, valine and threonine were found in large proportion in peptides, and arginine was mostly released as a free amino acid. With rapeseed, histidine was preferentially released in peptides, while methionine appeared mostly in the free form from 6 to 12 hours. These findings give new insight into the effect of protein nature upon the kinetics and form of release of digestion products. Differences in sequential hydrolysis also suggest that subsequent absorption and utilisation of dietary amino acids in the living organism may be largely dependent on the luminal digestion process.

Improvement of the Nutritional Quality of a Traditional Complementary Porridge Made of Fermented Yellow Maize (Zea Mays): Effect of Maize–Legume Combinations and Traditional Processing Methods

Background Blends with a cereal–legume ratio of 70:30 have been introduced in many communities for use in the preparation of complementary foods with augmented protein quality. These foods should meet World Health Organization estimated energy and nutrient needs from complementary foods. Objective To increase energy and nutrient densities and nutrient availability in a traditional complementary porridge. Methods Yellow maize was processed by lactic acid fermentation. Peanuts (Arachis hypogea) and beans (Phaseolus vulgaris) were processed by germination, roasting, dehulling, and a combination of germination and roasting. Blends were prepared from processed peanuts and beans and cooked into porridges with viscosities less than 3,000 cp. Traditional porridge was the control and consisted of fermented yellow maize only. The porridges were analyzed for their physicochemical and nutritional properties. Results Blends increased energy and nutrient densities in porridges compared with the control (p < .05). The maize–peanuts combination yielded porridges with higher energy densities and improved nutritional quality compared with the maize–beans combinations. In vitro availability of iron did not change (p > .05) with formulation of the blends except for porridges made from maize and germinated peanuts, but there was a significant increase in zinc in vitro availability, whereas a decrease was observed for calcium in vitro availability. The energy densities of maize–peanuts porridges were sufficient to cover energy required from complementary foods for infants aged 6 to 11 months receiving four meals of complementary foods per day and an average amount of energy from breastmilk. Conclusions Maize-legume blends can efficiently improve the nutritional quality of traditional porridge. Peanuts are the best legume complements.

Analysis of bound amino acids in the plasma of fed rats : a new preparation procedure

Abstract Oligopeptides have been proposed as an important form of transport of amino acids in circulation, but current preparation and analysis methods do not allow detection of peptides in plasma samples. In order to measure the concentration and composition of bound amino acids in comparison with those under free form in plasma, blood samples were collected in portal vein and aorta of rats fed a 20% rapeseed protein diet. Samples were deproteinized with sulfosalicylic acid, and centrifuged in 3000 molecular weight cut-off filters. Analyses were performed by ion-exchange chromatography before and after hydrolysis of filtrates in 6N HCl, to assess by difference the amount of bound amino acids in plasma. Hydrolyzed samples from portal vein and aorta contained, respectively, 8.2% and 10.3% more total amino acids than nonhydrolyzed samples ( P

Enzymatic digestion method with dialysis to assess proteinodamage; Application to alkali-treated proteins containing lysinoalennane

Abstract An in vitro method is proposed to study protein digestion and amino acid availability. It uses a pepsin-pancreatin proteolysis and continuous removal of digestion products by dialysis through a membrane to simulate absorption in the intestine and to prevent proteolysis inhibition. Digestion was evaluated by measuring the amount of dialyzable products consisting of a mixture of amino acids and small peptides. High extent of digestion of casein, soybean and rapeseed proteins was achieved in a relatively short period with this method; some amino acids were released rapidly, mainly basic and aromatic amino acids, while proline and dicarboxylic amino acids were released at slow rates. The proteins were submitted to heat and alkali treatment that induces the formation of lysinoalanine (LAL) at the expense of lysine. The in vitro method was used to assess the effect of this treatment on protein digestion. Heat and alkali treatment at 60° and 80°C reduced in vitro protein digestibility in the following decreasing order: casein > soybean protein > rapeseed protein. A maximum effect on protein digestion did not correspond to maximum change in the digestibility of specific amino acids. Lysinoalanine was poorly released by digestive enzymes except in severely treated casein (pH 12, 60° or 80°C for 4 h). This illustrates the nutritional consequences of processing and the possibility of using the in vitro method for protein quality evaluation.

Extent of damage to amino acid availability of whey protein heated with sugar

The effect of heat treatments, at various water activities (aW), on digestibility and on the availabilities of amino acids of whey protein samples in the presence of lactose was estimated by an in vitro digestion method with continuous dialysis. Four aW (0.3, 0.5, 0.7 and 0.97), three temperatures (75, 100 and 121 degrees C) and three heating periods (50, 500 and 5000 s) were selected. The initial lysine:lactose molar ratio was 1:1. Amino acid profiles showed that excessive heating of whey (121 degrees C, 5000 s) destroyed a significant proportion of cystine at all aW, lysine at aW 0.3, 0.5 and 0.7, and arginine at aW 0.5 and 0.7. At aW 0.3, 0.5 and 0.7, protein digestibility decreased (P less than 0.05) as the temperature increased from 75 to 121 degrees C for a heating period of 5000 s, and as the heating time was prolonged, from 500 to 5000 s at 121 degrees C. Excessive heating also decreased (P less than 0.05) the availabilities of all amino acids at aW 0.3, 0.5 and 0.7. The availabilities of lysine, proline, aspartic acid, glutamic acid, threonine, alanine, glycine and serine were particularly affected. Severe heating at aW 0.97 did not seem to favour the Maillard reaction, but the availabilities of cystine, tyrosine and arginine were decreased, probably as a result of structural modifications of the protein upon heating. Heating whey protein concentrates in the presence of lactose not only affected lysine, but also impaired enzymic liberation of other amino acids, according to the severity of heat treatments and aW.

Relationship between amino acid intestinal effluent in rat and in vitro protein digestion products

Abstract Amino acid absorption as measured by proto-aortic differences was studied in rats given 10% protein diets ad libitum. Free plasma amino acid (PAA) levels were determined in portal and aortic blood sampled at 2200 h (low intestinal absorption time), 0400 h were beef, casein, rapeseed, soybean and gluten. These proteins were also submitted to an in vitro digestion process by which free amino acids and low molecular weight peptides (digestion products) were collected as they were released after pepsin and pancreat in hydrolysis. Even though most essential amino acids (EAA) were preferentially hydrolysed during the 6-h digestion, the pattern of release was different for each protein. Essential PAA of intestinal effluent (porto-aortic difference) were compared with both in vitro digestion products and protein EAA. In most instances, correspondence between EAA patterns and digestion products was higher. Correlations were rather low for beef and casein (r=0.70), but higher with rapeseed and particularly with soybean and gluten (r=0.92 between PAA of 2200 h intestinal effluent and peptide fraction of in vitro digestion products for soybean, and r=0.93 between 0700 h intestinal effluent and peptide fraction for gluten). It can thus be stated that compared to protein amino acid composition, in vitro digestion products appear to be better predictors of amino acid bioavailability.