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).

Inter- and intra-laboratory variability in rat growth assays for estimating protein quality of foods.

Protein efficiency ratio (PER), relative PER (RPER), net protein ratio (NPR), relative NPR (RNPR), and nitrogen utilization (NU) methods were investigated in an interlaboratory rat growth study. Six collaborating laboratories studied 7 protein sources (ANRC casein; minced beef; soya assay protein, SAP; pea flour; whole wheat flour, WW; rapeseed protein concentrate, RPC; and egg white solids, EW), and their 10 supplementary or complementary mixtures (casein + Met, SAP + Met, pea flour + Met, WW + Lys, WW + casein, WW + beef, WW + SAP, WW + pea flour, WW + RPC, WW + EW). Test protein(s) were added at the 8% level (N X 6.25). Casein + Met was used as the reference protein. Interlaboratory variation (estimated as between-laboratories coefficients of variation) of PER (up to 17.2%) was greater than that of RPER (up to 14.9%), NU (up to 9.5%), NPR (up to 7.0%), and RNPR, which had the lowest variability (up to 4.7%). In most cases, intralaboratory variation (estimated as within-laboratories coefficients of variation) for all the methods was less than 5%.

Apparent nitrogen digestibility data: AACC-ASTM collaborative study.

Apparent nitrogen digestibility data were obtained from 4 laboratories for 6 protein sources and 2 diet levels, 6 and 10% protein, after a 2-day adaptation period during the AACC-ASTM protein efficiency ratio (PER) and net protein ratio (NPR) collaborative studies. For 5 protein sources fed as 10% of the diet, the interlaboratory variation as measured by coefficient of variation (CV) values was low (1.5-3.5%), indicating high precision of the method. Wheat flour (6% protein diet) had the highest variation and, therefore, the lowest precision (CV of 7.10%). The interlaboratory variation (CV value) for 3 of the 4 laboratories was considerably lower, less than half that for the 4 laboratories. An analysis of variance of apparent nitrogen digestibility data indicated significant (P less than 0.05) effects for the 4-laboratory group due to laboratories and protein diets at both 10 and 6% protein levels, and for the 3-laboratory group at the 10% protein level. The 3-laboratory ANOVA for the 6% diets indicated a significant effect (P less than 0.05) due to diet only.

Net protein ratio data: AACC-ASTM collaborative study.

Seven- and 14-day net protein ratio (NPR) data were obtained from 7 laboratories for 6 protein sources: ANRC casein, lean beef, lactalbumin, textured vegetable protein, and peanut flour were fed as 10% protein (N X 6.25) in the test diet. Wheat flour, casein, and textured vegetable protein were fed as 6% protein (N X 6.25) in the test diet. Weighed dry ingredients for each diet were sent to each collaborator , who mixed the dry ingredients, then added specified amounts of corn oil and water and mixed each complete diet thoroughly. Rats were adapted for 0, 2, or 4 days, and then were fed the test diets for 28 days for protein efficiency ratio (PER) diets. The animal weight gain and feed consumption data obtained after 7 or 14 days of feeding were used to calculate NPR values. Analyses of data were done before [net protein ratio (NPR)] and after (R-NPR [relative-NPR]) adjustment of the data from each laboratory by its results for the reference protein casein. From the analysis of variance for NPR, significant (P less than 0.05) interactions were observed among laboratories, protein sources, and adaptation times of the animals (0, 2, or 4 days). Inter- and intralaboratory variability were decreased by use of 14-day values compared with 7-day values. Adjustment of the NPR data to R-NPR did not lower the intralaboratory variability but did lower the interlaboratory variability of the data. Increasing adaptation time did not consistently decrease interlaboratory or intralaboratory variability or decrease coefficients of variation (CV) of R-NPR values.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein efficiency ratio: AACC/ASTM collaborative study.

Eight laboratories (7 of the laboratories conducted animal experiments) participated in a collaborative study to standardize some of the methodology associated with animal bioassays for determining protein efficiency ratios and to suggest improvements which would reduce the variation among laboratories. One-, 2-, 3-, and 4-week protein efficiency ratios (PER) with 0-, 2-, or 4-day adaptation periods were obtained from each laboratory, respectively, for 6 protein sources: casein, lean beef, lactalbumin, textured vegetable protein, peanut flour, and wheat flour. Analyses were computed for PER and adjusted PER (APER). From the analysis of variance for PER and APER, significant (P less than 0.05) effects were observed due to laboratories, adaptation length, protein sources, and/or interactions among these variables. In general, APER values show much less variation among laboratories than PER values. The reproducibility and repeatability variances were significantly (P less than 0.05) greater for an assay length of 2 weeks than they were for 3- or 4-week assays. Two protein sources, casein and textured vegetable protein, were fed at both high (10%) and low (6%) levels of protein. Analysis of variance of PER values shows a significant (P less than 0.05) laboratory by protein level by assay length interaction.

The influence of dietary fiber source on human intestinal transit and stool output.

Wheat bran ground to a coarse and fine particle size, purified cellulose and ethanol-extracted cabbage fiber, fed to 24 adult males during an 80-day metabolic trial, were examined for effects on intestinal transit time, laxation and stool composition. Brilliant blue, plastic pellets, polyethylene glycol (PEG)-4000 and Cr(III) mordanted onto isolated bran fiber were simultaneously administered for transit measurements. Intersubject variability in response to fiber source was highly significant for all transit and stool measurements. Only coarse bran or cellulose addition increased transit speed (decreased transit time) over basal rates. Grinding of bran significantly reduced fecal output because of reduced fecal water. Only subjects consuming cellulose or fine bran reported difficult or uncomfortable defecations. Though cabbage produced the smallest fecal output, stools had a high moisture content comparable to those obtained from coarse bran, which suggests a large microbial output in response to a fermentable substrate. Significant negative correlations were produced when changes in dry matter or cell wall intakes were regressed with Cr (III) transit. These findings suggest that the level of either food or fiber in the diet are variables that influence intestinal transit time and should be controlled in studies measuring it. Increases in fiber intake linearly increased fecal output of water and dry matter. Regression slopes were characteristic of each fiber source.

The influence of starvation and refeeding on the lipoprotein lipase activity of skeletal muscle and adipose tissue of lean and obese Zucker rats.

Lipoprotein lipase (LPL) activities of skeletal muscles, heart and adipose tissue were investigated during feeding, prolonged food restriction, and refeeding. The influence of the duration of starvation on adipose tissue LPL activity was to cause it to decrease throughout starvation, whereas heart LPL activity increased during the first 24 hours of fasting and then declined for the remainder of the fast. Starvation of 10-week-old female, lean and obese rats to 80% of initial body weight required 5 and 9 days, respectively. In fed controls, no differences between phenotypes were found for any tissue in the LPL activities expressed per gram tissue. However, obese rats exhibited significantly smaller muscle mass and a resulting 29% lower total skeletal muscle LPL activity. No phenotype differences were detected for tissue LPL activities during starvation or refeeding. During caloric restriction, the LPL activities were reduced in heart (-18%) and adipose (-52%) tissues, but skeletal muscle was unchanged except for the slow-twitch, oxidative soleus muscle, which was increased approximately two-fold. After refeeding to initial body weight, the LPL activity of heart returned to normal, but adipose tissue was dramatically increased (+300%) for both lean and obese Zucker rats. These data suggest that reduced skeletal muscle mass with normal LPL activity per gram tissue may contribute to an increased availability of plasma triglyceride fatty acids to adipose tissue of the genetically obese rat.

Assessment of rat growth methods for estimating protein quality: interlaboratory study.

An interlaboratory study involving protein efficiency ratio (PER), net protein ratio (NPR), relative NPR (RNPR) and relative nitrogen utilization (RNU) was carried out. Six collaborators assayed 6 samples, including casein plus methionine which was used as a reference protein. Collaborators prepared their own diets and analyzed the diets for nitrogen. Test proteins were added at the 8% level (N X 6.25). PER values varied more than NPR values which varied more than either RNPR or RNU. RNU and RNPR produced almost identical values, but RNPR remains the official method of choice, because it is a well established method.

Protein digestibility of the same protein preparations by human and rat assays and by in vitro enzymic digestion methods.

The apparent and true digestibilities of the same preparations of six proteins (spray dried whole egg, cottage cheese, canned tuna, peanut flour, soy isolate, and wheat gluten) were estimated in four to five men and in rats and compared to estimates of digestibility from three different in vitro enzymic digestion procedures. For all six proteins, the correlation coefficient was 0.46 between true digestibility in humans and in rats; with values for tuna excluded, r = 0.96. With all six proteins, none of the in vitro values was significantly correlated with values from humans or rats. However, with either the three animal proteins alone or the three plant proteins alone, correlations were high (r greater than 0.90) between one or more of the in vitro estimates and the observed true or apparent human and rat digestibilities. The differences in the relationship between enzymic digestion estimates and the human digestibility estimates for plant or animal proteins suggest that for accurate prediction of protein digestibility in humans by these enzymic methods, different equations would have to be used for plant and animal proteins. For protein sources containing both plant and animal protein, use of the in vitro enzymic procedures would give only an approximate estimate of digestibility in humans.

Production of higher alcohols during indonesian tapé ketan fermentation.

A study was made of the higher alcohols (fusel oils) produced during the Indonesian tapé ketan fermentation using Amylomyces rouxii as the principal mold, alone or in combination with yeasts belonging to genera commonly found in the tapé ketan fermentation (Endomycopsis, Candida, and Hansenula). Total fusel oils increased with length of fermentation. Fusel oils detected in the product distillate included isobutanol and isoamyl and active amyl alcohols. No n-propanol was detected. Isobutanol and isoamyl alcohols were formed in the largest amounts. A. rouxii alone produced nearly the same quantity of fusel oils (total production, 275 mg/liter at 192 h) as it did in combination with Endomycopsis burtonii (total production, 292 mg/liter at 192 h).A. rouxii and Endomycopsis fibuliger produced fusel oils totaling 72 mg/liter at 32 h and 558 mg/liter at 192 h. A. rouxii in combination with Candida yeasts produced somewhat more fusel oils, ranging from 590 to 618 mg/liter at 192 h. A. rouxii in combination with Hansenula yeasts produced the least fusel oils, totaling 143 to 248 mg/liter at 192 h. During the first 36 h, production of fusel oils was higher at 30 and 35 degrees C than at 25 degrees C. At 48 h fusel oil production was slightly higher at 30 degrees C than at 35 degrees C. Beyond 48 h, production of fusel oils was higher at 25 degrees C. A. rouxii in combination with Hansenula anomala and Hansenula subpelliculosa produced considerable ethyl acetate, ranging from 145 to 199 mg/liter at 36 h and 354 to 369 mg/liter at 192 h.

Changes in the crude fiber content of the American diet.

The intake of crude fiber in the American diet was assessed for 7 time periods between 1909 and 1975 using food consumption and composition tables. Crude fiber intake dropped 28% from 6.8 g/day in 1909 to 4.9 g/day in 1957 to 1959 and has remained at that level until the present. The intake of fiber from vegetables has remained relatively constant from 1909 to 1975 while potatoes, fruit, cereals, dry peas, and dry bean consumption have declined. The trends shown for crude fiber consumption in the United States support the hypothesis that fiber intake has decreased coincidentally with increases in degenerative diseases.

Indonesian tapé ketan fermentation.

Indonesian tapé ketan is a fermentation in which a mold, Amylomyces rouxii Calmette (Chlamydomucor oryzae Went and Prinsen Geerligs), in combination with one or more yeasts such as Endomycopsis burtonii converts steamed rice to a sweet-sour, slightly alcoholic paste. A study was made to determine the biochemical changes that occur in the substrate during fermentation. It was found that the product was ready for consumption after fermentation at 30 degrees C for 36 to 48 h. A. rouxii used about 30% of the total rice solids, resulting in a crude protein of 12% in 96 h, whereas the combination of the mold with E. burtonii reduced total solids by 50% in 192 h, causing crude protein to increase to 16.5%. Soluble solids increased from 5 to about 67% in 36 h and decreased to 12% at 192 h with A. rouxii alone, whereas soluble solids fell to about 8% at 192 h in the fermentation with both the mold and the yeast. The mold, by itself, reduced the starch content of the rice from 78 to 10% in 48 h and to less than 2% in 144 h. The mold plus yeast reduced the starch content to about 18% in 48 h; however the "starch" content did not fall below 6% even at 192 h, presumably because the yeast was producing glycogen, which was determined along with the residual starch. With both the mold and the mold plus yeast fermentations, reducing sugars increased from less than 1% to approximately 5% in 24 h and reached maximum concentration, 16 to 17%, between 36 and 48 h. A. rouxii by itself produced a maximum of about 5.6% (vol/vol) ethanol at 96 h. The highest concentration of ethanol (8%, vol/vol) was produced by the mold plus E. burtonii at 144 h. The mold by itself reduced the starting pH from 6.3 to about 4.0 in 48 h. The combination of the mold and yeast reduced the pH to 4.1 in 144 h. The mold increased total acidity to approximately 6.2 meq of H per 100 ml, and the combination of the mold and yeast increased the total acidity to 7.8 meq of H per 100 ml in 192 h. At 48 h there was practically no difference in the volatile acidity (0.20) for the combined fermentation compared with 0.26 meq of H per 100 ml for the mold fermentation. The mold and at least one species of yeast were required to develop the rich aroma and flavor of typical Indonesian tapé.

Factors affecting production of mold mycelium and protein in synthetic media.

The effects of certain cultural conditions on the yield of dry mycelium, protein, and total amino acid content of Rhizopus oligosporus Saito (NRRL 2710), Rhizopus rhizopodiformis (Cohn apud Lichtheim) Zopf (NRRL 6246), and Absidia corymbifera (Cohn) Sacc. et Trotter (NRRL 6247) were studied. The yield of mycelium was found to significantly increase as the spore inoculum was increased from 187,500 to 2,250,000 spores. But the total amino acids (grams/liter) did not change significantly, whereas the percentage of crude protein decreased. An inoculum containing approximately 750,000 spores/ml was used in all of the other experiments. Mycelial production was highest at 37 degrees C for all three molds. However, the best temperature for percentage of crude protein and total amino acids varied with the organism. The mycelial yield and total crude protein of R. oligosporus showed some significant changes as the C/N ratio was increased in 3% glucose medium. In a synthetic medium having a 15:1 C/N ratio, the strains of R. oligosporus, R. rhizopodiformis, and A. corymbifera had better yields from falactose than glucose, not only in dry mycelium but also in total crude protein (grams/liter) and total amino acids (grams/liter). R. oligosporus grew very well on several ammonium salts. but the maximum yield of dry mycelium, total crude protein (grams/liter), and total amino acids (grams/liter) occurred with ammonium sulfate. The optimum pH for both Rhizopus species was 4.0, although R. oligosporus grew equally well at pH 3.0 and slightly less at pH 5.0. The highest yield of mycelium for A. corymbifera was obtained in a medium with an initial pH of 8.0. It was calculated that a fermenter chanrged with an adequate medium and 1,000 lb (about 450 kg) of R. oligosporus or A. corymbifera cells could produce 88 or 90 lb of protein (on a dry-weight basis) per h if the product was removed continuously.