Comparison of interlaboratory variation in amino acid analysis and rat growth assays for evaluating protein quality

Estimates of inter- and intralaboratory variation of protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR), relative NPR (RNPR), and nitrogen utilization (NU) were compared with those of amino acid analysis in the same batches of 7 protein sources (ANRC casein, egg white solids, minced beef, soy assay protein, rapeseed protein concentrate, pea flour, and whole wheat flour). Interlaboratory variation (estimated as between-laboratories coefficients of variation, CV) of NPR and RNPR (up to 6.0%) was lower than that of PER (up to 20.2%) and RPER (up to 18.5%). The interlaboratory determination of NPR and RNPR was also more reproducible than that of most essential amino acids (CV up to 10.0%), especially tryptophan (CV up to 23.7%), cystine (CV up to 17.6%), and methionine (CV up to 16.1%). Intralaboratory variation (estimated as within-laboratories CV) of amino acid analysis (up to 4.7%), however, was comparable to that of protein quality indices in most protein sources (up to 6.0%). The significant (P less than 0.01) positive correlations (r = 0.68-0.74) between amino acid scores and protein quality indices based on rat growth were further improved when amino acid scores were corrected for digestibility of protein (r = 0.73-0.78) or individual amino acids (r = 0.79-0.82).     

Assessment of protein quality methodology for infant formulas

Rat bioassay was used to assess the protein quality of powdered infant formulas and to evaluate the feasibility of using modified casein diets (containing the same source and level of fat and carbohydrate contributed by the infant formulas) as reference standards. Modification of the casein diet to match the milk-based formulas caused a significant reduction in weekly protein efficiency ratios (PER) and net protein ratios (NPR) at the third and fourth weeks. Modification of the casein diet to stimulate the soy-based formulas had no significant effect on NPR values; PER values were more varied. When PER and NPR values of the powdered milk-based formulas were expressed relative to the unmodified reference standard, the relative values were lower than when each matched reference was used. With few exceptions, the relative weekly PER values of the soy-based formulas were similar regardless of the standard used. The relative NPR values of the formulas had a pattern similar to the relative PER values. The data indicate that protein quality evaluation of infant formulas using rat bioassay warrants the use of matched casein reference diets for each type of formula.     

In vivo rat assay for true protein digestibility: collaborative study

Eight laboratories participated in a collaborative study to estimate precision of a standardized rat assay for determining true protein digestibility in selected animal, fish, and cereal products. Each of 7 test protein sources (casein, tuna fish, macaroni/cheese, pea protein concentrate, rolled oats, pinto beans, and nonfat dried milk) was fed as the sole source of protein at a 10% protein level in mixed diets. Each diet was fed to 2 replicate groups of 4 rats each for a 4-day acclimation period and a 5-day balance period. Mean digestibilities ranged from 98.6% for casein to 72.6% for pinto beans. Repeatability standard deviations ranged from 0.5 to 2.0%; the mean relative standard deviation for repeatability was 0.9% (range 0.5-2.8%). Reproducibility standard deviations ranged from 1.2 to 3.2%, and the mean relative standard deviation for reproducibility was 2.4% (range 1.3-4.4%). The method has been approved interim official first action for determining true protein digestibility in foods and ingredients.     

Dietary supplement of G-rutin reduces oxidative damage in the rodent model.

Dietary supplement of bioflavonoid rutin and calorie restriction were examined to investigate possible reduction in oxidative DNA damage, protein oxidation, and lipid peroxidation in animals. Rats were fed ad libitum 20% casein semipurified diet or a diet that was supplemented by 2.5% water soluble rutin derivative, 4(G)-alpha-glucopyranosylrutin (G-rutin) for 18 days. Two other groups of rats were fed the respective diets at 60% of the mean food intake of the ad libitum fed animals for the same period. Urinary excretion of thymine glycol and thymidine glycol, indices of DNA base damage in the whole body, were significantly low in the G-rutin-supplemented groups. The protein carbonyl contents, a measure of protein oxidation, were significantly low in the liver of G-rutin-supplemented groups and calorie-restricted groups. The data indicate that G-rutin provides an antioxidant defense in rodents against free radical-caused oxidative damage of DNA and proteins.     

Isoflavone glycitein diminished plasma cholesterol in female golden Syrian hamsters

The soybean isoflavones, daidzein, genistein, and glycitein, were hypothesized to act as cholesterol-lowering components, separate from soy protein. Pure synthetic daidzein, genistein, or glycitein (0.9 mmol/kg diet) or a casein-based control diet was fed to groups of 10 female Golden Syrian hamsters for 4 weeks. Hamsters fed glycitein had significantly lower plasma total (by 15%) and non-HDL (by 24%) cholesterol compared with those fed casein (P<0.05). Daidzein and genistein's effects on these lipids did not differ from the effects of either casein or glycitein. Plasma HDL cholesterol and triglyceride concentrations were not significantly affected by dietary treatments. The percentage of urinary recovery of the ingested dose of each isoflavone was glycitein>daidzein>genistein (33.2%, 4.6%, 2.2%, respectively), with the apparent absorption of glycitein significantly greater than that of the other isoflavones. These data suggest that glycitein's greater cholesterol-lowering effect was due to its greater bioavailability, as reflected in its urinary recovery compared with that of the other isoflavones.     

Nutritional quality of sesame seed protein fraction extracted with isopropanol

The nutritional effect of diet containing decorticated sesame seed extracted with isopropanol (DSS-Iso) was evaluated on growth performance, food efficiency ratio, plasma and tissue lipid profile, plasma protein content, and erythrocyte membrane lipid profile of rats on a comparative basis with diets containing casein (control), soybean meal, and decorticated sesame seed extracted with hexane (DSS-Hex). Rats fed a DSS-Iso-based diet showed body weight gain and food efficiency ratio similar to those of the control groups fed diets prepared with casein, soybean meal, and DSS-Hex. However, dietary proteins exerted a separate effect on plasma lipid concentrations of the rats. Rats fed a DSS-Iso-based diet showed significant decreases in plasma total cholesterol (p < 0.01), triglyceride (p < 0.01), and VLDL+LDL cholesterol (p < 0.01) concentrations in comparison to the rats fed diet containing casein. No significant differences in plasma lipid concentrations were observed for the rats fed diets prepared with soybean meal, DSS-Hex, and DSS-Iso. Rats fed the different dietary proteins did not show much variation in plasma proteins, liver lipids, and erythrocyte membrane lipid concentrations, which suggests that DSS-Iso could be a suitable edible protein like casein or soybean meal.     

Effect of particle size on the biological availability of reduced iron.

A sample of reduced iron was separated by air elutriation into several fractions based on particle size; and the relative biological values (RBVs) of 3 fractions containing fine, medium, and coarse particles were determined with rats and chicks, using diets containing either skim milk powder or casein as the major source of protein. The RBV of reduced iron for rats was shown to be dependent on particle size by curative and prophylactic assay procedures, with the source of protein having no marked effect. In contrast to rats, chicks utilized reduced iron to a higher degree, especially the iron from coarse particles. Prophylactic assays with rats indicated that a 3-4 week feeding period from weaning at 21 days of age gave RBVs which were not significantly different from those obtained by repletion of hemoglobin of anemic animals. After 5 weeks on the test diets, however, the rats lost their ability to distinguish between coarse and medium particles of reduced iron when fed with a diet containing casein as the source of protein.     

Intestinal Contents Supernatant Viscosity of Rats Fed Oat-Based Muffins and Cereal Products

The intestinal contents viscosities of oat-based breakfast cereals and muffins were examined. Male Sprague-Dawley rats were adapted for four days to a semipurified diet (AIN-76A). Following an overnight fast, the animals were meal-fed 5 g of either the AIN-76A diet (containing 5% cellulose), the AIN-76A diet containing 2% guar gum, whole-grain oat flour, one of five cereals (corn flakes, cooked oatmeal, uncooked oatmeal, cooked oat bran, or Cheerios), or one of two types of muffins (containing whole-grain oat flour or oatmeal). Two hours after presentation of the meal, the animals were killed, the small intestines removed, and the contents collected. The contents were centrifuged, and the viscosity values of the undiluted supernatants were determined. The supernatant viscosity from rats fed the AIN-76A diet was negligible (<5 mPa·sec), whereas that from rats fed guar gum was high (396 ± 117 mPa·sec). Of the cereals fed, corn flakes resulted in the lowest viscosity (<5 mPa·sec). However, all oat-based cereals resulted in high intestinal contents supernatant viscosity levels (cooked oatmeal 368 ± 128, uncooked oatmeal 307 ± 107, cooked oat bran 301 ± 85, Cheerios 199 ± 58, mPa·sec) with no statistically significant differences. The intestinal contents viscosity values for the whole-grain oat flour muffin and oatmeal muffin were 233 ± 52 and 111 ± 26 mPa·sec, respectively, a statistically significant difference (P < 0.05). This suggests that the form of the oat within a food may influence the degree of viscosity produced within the small intestine after that food is consumed.     

Use of a rat model to evaluate tall fescue seed infected with introduced strains of Neotyphodium coenophialum

Experimental cultivars of the pasture grass tall fescue are infected with unique strains of the fungal endophyte Neotyphodium coenophialum, which produce low concentrations of ergot alkaloids. A rat model was evaluated as a tool for rapid, initial screening of experimental cultivars considered to be nontoxic. Rats were fed diets that included seed from experimental cultivars of tall fescue with introduced strains of N. coenophialum and a toxic control diet containing seed of the cultivar Kentucky 31 (KY31), with its endemic strain of N. coenophialum. Rats were preconditioned to a nontoxic diet and then fed treatment diets for 13 days with 5 days at thermoneutrality (21 degrees C) followed by 8 days under heat stress (31 degrees C). For most of the 13-day treatment period, rats fed KY31 exhibited depressed daily intake compared to those fed diets of cultivars with introduced endophytes (P < 0.05). In addition, rats fed KY31 exhibited significantly less weight than rats on other diets after heat treatment was imposed. For all initial trials and repeated trials, total intake and total gain calculated at the end of each trial were the most consistent indicators of toxicity.     

Soybean meal fermented by Aspergillus awamori increases the cytochrome P-450 content of the liver microsomes of mice

The effect of soybean meal fermented by Aspergillus awamori on the acute lethality of acetaldehyde, pentobarbital sleeping time, and cytochrome P-450 content of the hepatic microsomes was studied in mice. Most of the daidzin and genistin in soybean meal (SBM) were converted into the respective aglycones, daidzein and genistein, by fermentation. In experiment 1, mice were fed isonitrogenic test diets with one of the following five protein sources for 28 d: casein, SBM, fermented and hot-air-dried SBM (FSBM-HD), fermented and freeze-dried SBM (FSBM-FD), or methanol-extracted FSBM-FD (FSMB-FD-R). The acute lethality of acetaldehyde in mice fed the FSBM-FD diet was significantly lower than that in mice fed the SBM, FSBM-HD, or FSBM-FD-R diet. In experiments 2 and 3, mice were fed isonitrogenic test diets with one of the following four protein sources for 28 d: casein, SBM, FSBM-FD, and FSBM-FD-R. The pentobarbital sleeping time was significantly shorter and the cytochrome P-450 content was significantly higher in the mice fed the FSBM-FD diet than the respective value in mice fed the other test diets. In experiment 4, mice were fed one of eight diets which contained different levels of aglycone obtained by varying the proportion of FSBM-FD and FSBM-FD-R, for 28 d. The cytochrome P-450 content in hepatic microsomes increased as the dietary level of isoflavonoid aglycones increased, but there was a saturation phenomenon. These results suggest that soy isoflavonoid aglycones are more potent inducers of cytochrome P-450 than isoflavonoid glycosides.     

Lower weight gain and hepatic lipid content in hamsters fed high fat diets supplemented with white rice protein, brown rice protein, soy protein, and their hydrolysates

The physiological effects of the hydrolysates of white rice protein (WRP), brown rice protein (BRP), and soy protein (SP) hydrolyzed by the food grade enzyme, alcalase2.4 L, were compared to the original protein source. Male Syrian Golden hamsters were fed high-fat diets containing either 20% casein (control) or 20% extracted proteins or their hydrolysates as the protein source for 3 weeks. The brown rice protein hydrolysate (BRPH) diet group reduced weight gain 76% compared with the control. Animals fed the BRPH supplemented diet also had lower final body weight, liver weight, very low density lipoprotein cholesterol (VLDL-C), and liver cholesterol, and higher fecal fat and bile acid excretion than the control. Expression levels of hepatic genes for lipid oxidation, PPARα, ACOX1, and CPT1, were highest for hamsters fed the BRPH supplemented diet. Expression of CYP7A1, the gene regulating bile acid synthesis, was higher in all test groups. Expression of CYP51, a gene coding for an enzyme involved in cholesterol synthesis, was highest in the BRPH diet group. The results suggest that BRPH includes unique peptides that reduce weight gain and hepatic cholesterol synthesis.     

Nutritional responses of rats to diets based on chickpea ( Cicer arietinum L.) seed meal or its protein fractions

The aim of this study was to isolate the protein fractions from chickpea, var. IAC-Marrocos, as well as to evaluate its in vivo nutritional protein quality. Among the proteins, albumins showed better nutritional value in the in vivo assays and amino acid contents, despite their higher trypsin inhibitor contents. Trypsin inhibitors were found to be heat labile in all samples, but the digestibility results for unheated and heated flour and albumins suggest that their contents are not very decisive. The PER values for casein (not supplemented) were very similar to those of heated flour and unheated or heated albumin and total globulins. The albumin and glutelin fractions showed the best results for PDCAAS, however, lower than those of casein. Despite the high digestibility of the globulin the very low essential amino acid content lowered its PDCAAS, and it had the lowest values.     

Evaluation of infant formula protein quality: comparison of in vitro with in vivo methods

Two-week protein efficiency ratio (2-wk PER), net protein ratio (NPR), calculated-protein efficiency ratio (C-PER), and discriminant computed-protein efficiency ratio (DC-PER) of milk- and soy-based infant formulas were compared to a 4-week protein efficiency ratio (PER). Expressed relative to ANRC casein, 2-week PER and NPR correlated significantly (P less than 0.01, r = 0.90) with PER. Although C-PER and DC-PER also correlated significantly (P less than 0.01) with PER, r = 0.71 and r = 0.87, respectively, these in vitro methods did not distinguish differences in protein quality among soy-based infant formulas. C-PER and DC-PER, as currently designed, are not applicable to the measurement of protein quality for all types of infant formulas.     

Interlaboratory study of blood selenium determinations

Fifty-one laboratories from 14 countries participated in a survey on the determination of selenium (Se) in 8 bovine blood samples with Se concentrations ranging from 0.2 mumol/L (0.016 microgram/mL) to 14 mumol/L (1.1 micrograms/mL). The methods used (and the percentage of participants using each method) were fluorometry (61), hydride-generation atomic absorption spectrophotometry (AAS) (23), graphite-furnace AAS (6), gas chromatography (4), neutron activation analysis (4), and X-ray fluorometry (2). There was little difference in the mean Se results obtained by fluorometry or hydride-generation AAS (P greater than 0.05). Mean intralaboratory coefficients of variation (CVs) from known replicates ranged from 4 to 14% for all samples. Interlaboratory CVs were related to blood Se concentration and increased to 55% at Se levels below 0.4 mumol/L (0.032 microgram/mL). Laboratories that used quality control (QC) schemes had lower interlaboratory CVs than those that did not, but the advantage began to diminish at blood Se concentration below 0.4 mumol/L (0.032 microgram/mL). The high interlaboratory CVs, coupled with the false assurance from the low intralaboratory CVs and the ineffectiveness of the QC schemes at blood Se concentrations below 0.4 mumol/L (0.032 microgram/mL), are of concern in diagnosis of marginal Se deficiency in livestock where the concentrations of interest are in the range 0.15-0.5 mumol/L (0.012-0.039 microgram/mL).     

Improving digestibility of soy flour by reducing disulfide bonds with thioredoxin

The Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI) of trypsin and chymotrypsin contain disulfide bonds. Glycinin, the major storage protein in soybeans also contains disulfide bonds. Treatment of soy white flour with a NADP-thioredoxin system (NTS) effectively reduced disulfide bonds in soy flour and increased protein digestibility by trypsin and pancreatin as measured by the pH stat method. Treatment of soy flour with NTS increased the digestibility compared to soy white flour by 29.3 and 60.6% for trypsin and pancreatin, respectively. NTS-treated soy flour had similar digestibility by trypsin to autoclaved soy flour and casein, but digestibility by pancreatin was less than autoclaved soy flour and casein. The degree of reduction by NTS was highly correlated to the degree of hydrolysis (DH) by trypsin (R(2) = 0.93) and pancreatin (R(2) = 0.99). The DH of NTS-treated soy flour by trypsin is reflective of both inactivation of trypsin inhibitors and overall protein digestibility while pancreatin hydrolysis is reflective of only overall protein digestibility.     

Effects of soy protein isolate feeding on severe kidney damage in DOCA salt-treated obese Zucker rats

This study assessed the effects of soy protein isolate (SPI) on severe kidney damage in deoxycorticosterone acetate (DOCA) salt-treated obese Zucker rats. These rats underwent heminephrectomy and were fed either casein or SPI diet for 12 weeks. From weeks 8 to 10 of the experiment, kidney damage was induced by biweekly injection of 25 mg/kg DOCA and administration of 0.5% NaCl (w/v) ad libitum. Urinary protein and N-acetyl-β-d-glucosaminidase excretions of SPI rats were much lower than those of casein rats at weeks 1 (p < 0.01) and 2 (p < 0.05) after DOCA treatment. Abnormal mineral excretions induced by DOCA treatment in casein rats were hardly detected in SPI rats. Severe atrophy of tubular epithelium and some flattened/detached renal tubules were also observed in casein rats, but not in SPI rats. These results indicate that consecutive treatment of SPI protects against renal dysfunction, particularly tubulointerstitial nephritis, in DOCA salt-treated obese Zucker rats.     

Physicochemical and functional properties of buckwheat protein product

This study was conducted to compare the physicochemical and functional properties of buckwheat protein product (BWP), soy protein isolate (SPI), and casein. BWP was prepared from buckwheat flour by the method including alkaline extraction and isoelectric precipitation. The amino acid composition of BWP was very similar to that of buckwheat flour. The protein solubility (PS) of BWP was much greater than that of SPI at all pH levels (pH 2-10) but lower than that of casein at pH 7-10. The isoelectric point of BWP was around pH 4. The higher aromatic hydrophobicities (ARH) of BWP, SPI, and casein were obtained at lower pH levels (pH 2-3). The emulsifying stability (ES) of BWP was lower than those of SPI and casein at high pH levels (pH 7-10). At all pH levels, BWP formed a thin emulsion. Regression analysis showed that the ARH of BWP was significantly associated with the ES. Although the water holding capacity of BWP was quite lower than that of SPI, its fat absorption capacity was slightly higher than those of SPI and casein. These results indicated that the physicochemical properties of BWP were different from those of SPI or casein. Thus, BWP is a potential source of functional protein for possible food application.     

Technological and Nutritional Properties of Flours and Tortillas from Nixtamalized and Extruded Quality Protein Maize (Zea mays L.)

Nixtamalized and extruded flours from quality protein maize (QPM, V‐537C) and tortillas made from them were evaluated for some technological and nutritional properties and compared with the commercial brand MASECA. Both QPM flours showed higher (P < 0.05) protein content, total color difference, pH, available lysine, and lower (P < 0.05) total starch content, Hunter L value, water absorption index, gelatinization enthalpy, resistant starch, and retrograded resistant starch than nixtamalized MASECA flour. Tortillas from nixtamalized and extruded QPM flours had higher contents of essential amino acids than tortillas from MASECA flour, except for leucine. Tortillas from processed QPM flours also showed higher (P < 0.05) values of the nutritional indicators calculated protein efficiency ratio (C‐PER 1.80–1.85 vs. 1.04), apparent and true in vivo protein digestibility (78.4‐79.1 vs. 75.6% and 76.4–77.4 vs. 74.2%, respectively), PER (2.30–2.43 vs. 1.31), net protein retention (NPR; 2.88–2.89 vs. 2.11), and protein digestibility corrected amino acid score (PDCAAS; 54–55 vs. 29% based on preschool children and 100 vs. 85% based on adults) than MASECA flour. The use of QPM for flour and tortilla preparation may have a positive effect on the nutritional status of people from countries where these products are widely consumed.     

Chlorogenic acid moderately decreases the quality of whey proteins in rats

During processing and storage, phenolic compounds (PCs) may react with food protein bound amino acids (AAs). Such reactions have been reported to change physicochemical and to decrease in vitro digestion properties of proteins. A rat growth and nitrogen (N) balance study was conducted to prove whether derivatization with chlorogenic acid (CA) affects the nutritional quality of beta-lactoglobulin (beta-LG). Test diets (10% protein level) contained nonderivatized beta-LG (LG, treated under omission of CA), low derivatization level beta-LG (LGL), high derivatization level beta-LG (LGH), or casein supplemented with l-methionine (0.3% of diet; C+met) as an internal standard. An additional group received untreated beta-LG supplemented with pure CA (1.03% of diet; LG+CA). The AA composition of test proteins, plasma AAs, and liver glutathione (GSH) concentrations were determined. Protein digestibility-corrected amino acid score (PDCAAS) was calculated using human or rat AA requirement patterns and rat fecal digestibility values. N excretion was significantly higher in feces and lower in urine of rats fed with LGH as compared to LG and LGL. Consequently, true N digestibility (TND) was significantly lower with LGH as compared to LG and LGL. The lower content of methionine, cysteine, lysine, and tryptophan in LGH corresponded to a reduced TND. Net protein utilization (NPU) was not different between treated beta-LG fed diet groups but was lower than in LG+CA and C+met fed groups. Only at a relatively high level of derivatization with CA, the otherwise good nutritional quality of beta-LG is affected so that TND is reduced, while NPU still remains unaffected. Derivatization of beta-LG with CA does not seem to lead to an additional deficiency in a specific indispensable AA in growing rats fed with 10% protein.     

Effects of Fortification and Enrichment of Maize Tortillas on Growth and Brain Development of Rats Throughout Two Generations

The growth and brain development of laboratory rats fed typical indigenous tortilla‐based diets were determined throughout two generations. The experiment compared three different types of tortilla‐based diets: regular tortillas produced from dry masa flour (RDMF), tortillas obtained from fresh masa (FM), and tortillas produced from dry masa flour fortified with 6% defatted soybean and enriched with vitamins B₁, B₂, niacin, and folic acid and microminerals iron and zinc (FEDMF). Female rats were mated 58 days postweaning with males belonging to the same treatment with the objective of obtaining second generation pups that were further subjected to regular lactation and 28 day postweaned growth. A comparison between growth of first and second generation rats was determined. In addition, representative animals of each physiological stage were first exsanguineted for hematocrit determination and then slaughtered with the aim of obtaining femur and brain tissues. Cerebral DNA and number of neurons were determined in each of the brains sampled. Growth of rats fed FEDMF was significantly higher (P < 0.05) in both generations than counterparts fed RDMF or FM. The difference among treatments was more evident in second generation rats. Pregnancy rate, number of newborns per litter, litter weight, and pup's survival rate was higher for the control and FEDMF treatments. Femur growth was also higher (P < 0.05) for first‐generation male adult rats fed control and FEDMF. The concentration and total content of cerebral DNA and number of neurons in males and females belonging to the first generation was similar. However, for second generation rats, these values were lower for animals fed regular tortilla diets. This data clearly demonstrates that the negative effects of malnutrition on brain development of pups occurred during gestation and lactation.