Rumen microbial growth rates and yields: effect of amino acids and protein.

Effects of amino acids upon microbial growth, optimum ratio of nonprotein to amino acid nitrogen for microbial growth, and incorporation of amino acids into microbial cells were determined with washed cell suspension in vitro as were rumen microbial cells. Rumen microbial dry matter, nitrogen, ribonucleic acid, deoxyribonucleic acid, and substrate disappearance was greatest when a mixture of 18 amino acids was substituted for urea. Substitutions of mixtures of 10 essential amino acids, 8 nonessential amino acids, and sulfur containing amino acids and glutamate also stimulated microbial growth. Acid hydrolyzed casein markedly improved microbial growth. Branched amino acid addition did not affect growth. The optimum ratio of nonprotein to amino acid nitrogen for microbial growth was 75% urea nitrogen and 25% amino acid nitrogen. With this amount of amino acids, an average of 53% of added amino acid was incorporated into microbial cells, 14% was fermented to carbon dioxide and volatile fatty acids, and 33% remained in the supernatant. Both 100% urea and 100% amino acid in growth media were unfavorable for maximal microbial growth. With all carbohydrate substrates, 100% urea nitrogen supported the growth of 9 mg bacterial dry matter per 100 mg of substrate. Substitutions of amino acids for urea increased yields to over 20 mg/100 mg. Microbial growth yields in incubations under carbon dioxide were less than when flasks were flushed with nitrogen. However, yield of bacterial dry matter per unit of substrate was less under nitrogen than under carbon dioxide.     

Stimulatory and inhibitory effects of protein amino acids on growth rate and efficiency of mixed ruminal bacteria

Mixed ruminal bacteria were incubated in vitro with glucose, xylose, cellobiose, and various protein amino acids replaced isonitrogenously with 25% (i.e., 25 mg of N/L) of ammonia-N, to determine the growth rate and the amount of sugar consumed in the exponential growth phase. The growth rate and efficiency (grams of bacteria per gram of sugars) increased by 46 and 15%, respectively, when a mixture of 20 amino acids was added. On the other hand, neither growth rate nor efficiency increased when any one of these amino acids was added singly, except for Glu and Gln, each of which produced significant but small improvements. The stimulatory effect of the combined amino acids on bacterial growth declined when each of Leu, Trp, Tyr, Glu, Met, Phe, and Val was removed from the original group of 20. When a mixture of only these seven amino acids was used as a supplement, their stimulatory effects on growth rate and efficiency were only 21 and 25%, respectively, of the effects that the mixture of 20 amino acids showed. The effects increased to 76 and 72% on growth rate and efficiency, respectively, when Gly, Cys, and His were supplied in addition to the seven amino acids. The growth rate and efficiency of the ruminal bacteria were inhibited by an addition of each of Ile, Thr, Cys, Phe, Leu, Lys, or Val to ammonia-N, and the effects of the first five of these amino acids were highly significant. Isoleucine, threonine, and phenylalanine were each inhibitory even at a low concentration (1 mg of NL), while cysteine and leucine showed inhibitory effects at higher concentrations (more than 10 mg of N/L). A higher growth rate of the ruminal bacteria when supplemented with amino acid mixtures was accompanied with a higher growth efficiency, which was attributable to a relatively smaller proportion of energy expended on maintenance according to the Pirt derivation.     

Sources of variation in rates of in vitro ruminal protein degradation

Rates and extents of ruminal protein degradation for casein, solvent soybean meal (SSBM), expeller soybean meal (ESBM), and alfalfa hay were estimated from net appearance of NH3 and total amino acids in in vitro media containing 1 mM hydrazine and 30 mg/L of chloramphenicol. Protein was added at 0.13 mg of N/mL of medium, and incubations were conducted for 4 to 6 h, usually with hourly sampling. Inocula were obtained from ruminally cannulated donor cows fed diets of grass silage or alfalfa and corn silages plus concentrates. Preincubation or dialysis of inocula was used to suppress background NH3 and total amino acids; however, preincubation yielded more rapid degradation rates for casein and SSBM and was used in subsequent incubations. Preincubation with added vitamins, VFA, hemin, or N did not alter protein degradation. Protein degradation rates estimated for SSBM, ESBM, and alfalfa were not different when computed from total N release or N release in NH3 plus total amino acids, regardless of whether amino acids were quantified using ninhydrin colorimetry or o-phthalaldehyde fluorescence. Accounting for the release of peptide-N also did not affect estimated degradation. However, casein degradation rates were more rapid when using total N release or accounting for peptide-N, indicating significant accumulation of small peptides during its breakdown. Rates also were more rapid with inocula from lactating cows versus nonlactating cows with lower feed intake. Protein degradation rates were different due to time after feeding: casein rate was more rapid, but SSBM and ESBM rates were slower with inocula obtained after feeding. Several characteristics of ruminal inoculum that influenced breakdown of the rapidly degraded protein casein did not appear to have direct effects on degradation of protein in soybean meal.     

Effect of inhibitor concentration and end-product accumulation on estimates of ruminal in vitro protein degradation

Effects of varying the concentrations of hydrazine sulfate (HS) and chloramphenicol (CAP), inhibitors of microbial-N uptake and protein synthesis, on rates of protein degradation estimated from net appearance of NH3 and total amino acids (TAA) were studied in a ruminal in vitro fermentation system. Without inhibitors, recoveries of N added as NH3 and TAA were 4 and 6% after 4-h incubations, and apparent degradation rates estimated from release of NH3 and TAA for casein, solvent soybean meal (SSBM), and expeller soybean meal (ESBM) approached 0. Increasing inhibitor concentrations from the standard amounts of 1 mM HS plus 30 mg of CAP/L to 2 mM HS plus 90 mg of CAP/ L gave rise to numerically greater N recoveries and degradation rates, but these differences were not statistically significant. Compared with the standard inhibitor concentrations, use of 2 mM HS, without CAP, yielded similar recoveries and rates, but 30 or 90 mg of CAP/L, without HS, was not satisfactory. Versus that with 1 mM HS plus 30 mg of CAP/L, media containing 2 mM HS plus 90 mg of CAP/L gave increased TAA recoveries and higher rates for casein, but not SSBM, in the presence of added starch. Faster degradation rates were obtained for casein, but slower rates for SSBM and ESBM, in Sweden versus Wisconsin using inocula from cows fed different diets but with similar CP and energy contents. Differences in microbial catabolism of peptides may account for differences in degradation rates observed between Sweden and Wisconsin. Adding NH3 plus free and peptide-bound amino acids to the inoculum reduced apparent degradation rates, possibly via end-product inhibition. Analysis of data from multiple time-point incubations indicated that casein degradation followed simple, first-order kinetics, while a biexponential model fitted degradation patterns for both SSBM and ESBM.     

Acquisition of amino acids by Lactobacillus delbrueckii subsp. bulgaricus 2038 when grown in the presence of casein

The acquisition of amino acids by Lactobacillus delbrueckii subsp. bulgaricus 2038 (Lb. bulgaricus 2038) when grown in the presence of bovine casein, the major protein in bovine milk, was investigated by examining the expression of genes related to proteolysis and amino acid biosynthesis. To support the growth on bovine casein, Lb. bulgaricus 2038 has to synthesise five kinds of amino acids de novo, as proteolysis from casein does not provide these. The incomplete hydrolysis in combination with amino acids biosynthesis may explain the slow growth of Lb. bulgaricus 2038 in a casein environment. Meanwhile, it was determined that Lb. bulgaricus 2038 uses different intracellular peptidases when grown in casein or whey medium, and initially yields the important amino acid glutamate from the C-terminal or N-terminal end of peptides imported into the cell.     

Factors influencing rumen microbial growth rates and yields: effect of amino acid additions to a purified diet with nitrogen from urea.

Effects of isonitrogenous urea and amino acid additions upon microbial growth in rumen contents from a cow fed a purified diet in which urea was the sole nitrogen source were studied. Incorporation of amino acids into microbial cells, volatile fatty acids, and carbon dioxide was estimated. Rates of microbial growth, volatile fatty acid production, and effects of amino acids upon microbial nitrogen yields were highest right after feeding and decreased with time after feeding. Microbial growth and amounts of amino acids incorporated into microbial cells, volatile fatty acids and carbon dioxide were related closely to quantity of starch remaining in the rumen. High amounts of starch increased microbial protein synthesis from carbon-14 labeled amino acids and reduced amounts of amino acid fermentation. Estimated microbial protein yields per day were 326.0, 444.4, 497.3, and 527.3 g when 0, 15, 30, and 45 mg amino acid nitrogen replaced urea nitrogen during incubation. Respective values for microbial cells per mole estimated adenosine triphosphate were 15.2, 19.2, 21.0, and 24.5. Microbial cell yields per kg carbohydrate digested were 139.0, 189.5, 212.0, and 224.8 g for 0, 15, 30, and 45 mg amino acid nitrogen. Addition of small amounts of amino acids to a diet containing urea as the sole nitrogen source improved considerably rumen microbial protein yields.     

Metabolism of amino acids, dipeptides and tetrapeptides by Lactobacillus sakei

The microbial degradation of proteins, peptides and amino acids generates volatiles involved in the typical flavor of dry fermented sausage. The ability of three Lactobacillus sakei strains to form aroma compounds was investigated. Whole resting cells were fermented in phosphate buffer with equimolar amounts of substrates consisting of dipeptides, tetrapeptides and free amino acids, respectively.Dipeptides disappeared quickly from the solutions whereas tetrapeptides were only partially degraded. In both approaches the concentration of free amino acids increased in the reaction mixture but did not reach the equimolar amount of the initial substrates. When free amino acids were fed to the bacteria their levels decreased only slightly. Although peptides were more rapidly degraded and/or transported into the cells, free amino acids produced higher amounts of volatiles.It is suggested, that after transport into the cell peptides are only partially hydrolyzed to their amino acids, while the rest is metabolized via alternative metabolic pathways. The three L. sakei strains differed to some extend in their ability to metabolize the substrates to volatile compounds. In a few cases this was due to the position of the amino acids within the peptides. Compared to other starter cultures used for the production of dry fermented sausages, the metabolic impact of the L. sakei strains on the formation of volatiles was very low.     

纳他霉素与山梨酸复配改良MRS培养基的研究

对不同浓度纳他霉素（Natamycin）与山梨酸（Sonic Acid）及其复配后在MRS培养基中防止杂菌生长的情况进行了研究,结果表明,单独使用10mg／L纳他霉素可防止MRS培养基中真菌的生长;单独使用2000mg／L山梨酸可抑制真菌及常见细菌的生长;纳他霉素与山梨酸复配后可适当降低二者的使用浓度并具有协同增效的作用;但均没有抑制乳酸菌的生长,由此初步得到了一种新的改良MRS培养基。     

Effect of soy peptide on brewing beer

Here, we examined the effect of soy peptides (SPs) on the fermentation and growth of Yeast Bank Weihenstephan 34/70 (W34/70), a bottom-fermenting yeast. We compared fermentation for SP with that for a free amino acid (FAA) mixture having the same amino acid composition as SP, as a nitrogen source. Maltose syrup was used as a carbon source, and the medium contained excess amounts of essential minerals and vitamins. We observed that SP was better than FAA mixture at promoting fermentation and growth and that much more beta-phenylethyl alcohol was produced during fermentation with SP than with FAA mixture. Subsequently, we compared fermentations with the FAA mixture and selected mixtures containing various dipeptides of Phe as a nitrogen source. We found that the rates of Phe metabolism and beta-phenylethyl alcohol generation were much higher when Phe was presented as a dipeptide (Phe-Asp, Phe-Leu, or Phe-Phe) than when presented as FAA. These results show that amino acids such as Phe are absorbed more rapidly when presented as a peptide than as FAA, resulting in a more rapid production of beta-phenylethyl alcohol.     

Use of bacterial extracts to enhance amino acid breakdown in dry fermented sausages

The effect of the intracellular cell-free extracts (ICFEs) of two bacterial strains (Lactobacillus sakei GO and Bacillus pumilus) on the amino acid catabolism and the sensory properties of dry fermented sausages, was investigated. Extracts were added to sausages alone or in combination with a protease, papain. Amino acid breakdown was monitored by the changes in free amino acids, ammonia and amine content during the ripening process. A 15% decrease in the content of free amino acids was observed in sausages added with the ICFE from L. sakei GO. Furthermore, the extract of L. sakei GO significantly reduced (54-68%) the content of the amino acids considered as precursors of the typical ripened flavour, i.e., valine, leucine and isoleucine. Chemical changes were not reflected in a significant improvement of the sensory quality of sausages added with the ICFEs. The potential use of the bacterial ICFEs studied in the present work for the manufacture of dry fermented sausages, and its comparison with the use of fungal extracts, are discussed.     

Effect of carbon-4 and carbon-5 volatile fatty acids on growth of mixed rumen bacteria in vitro

Mixed ruminal bacteria (400 mg cells/liter) were incubated in artificial media containing ammonia, sodium carbonate, macrominerals, vitamins, sulfide, microminerals, acetate, propionate, and butyrate. When mixed carbohydrates (equal parts glucose, maltose, sucrose, cellobiose, and soluble starch) were added at 155 mg/liter per h for 10 h, average bacterial growth rate was slow, and dry weight yield was greater than 23%. Additions of isobutyrate, valerate, isovalerate, and 2 methylbutyrate had little influence on synthesis of bacterial dry weight, deoxyribonucleic acid, ribonucleic acid, or carbohydrate. When a timothy hay inoculum was used, isovalerate and 2 methyl-butyrate increased protein synthesis by 11.2 and 16.4%, but isobutyrate and valerate alone were without effect. All four acids combined increased bacterial protein by 18.7%. Responses with an inoculum of 60% concentrate and mixed hay were smaller and not statistically different from control incubations. Low concentrations of Trypticase (less than 250 mg/liter) improved efficiency of microbial protein synthesis from organic matter, but more was associated with decreased efficiency and utilization of extracellular ammonia.     

Formation of bitter peptides in cheese and from casein]

Whereas a slightly bitter taste is desirable in certain foods, it is an off-flavour in cheese which may even lead to unfitness for consumption. Bitter principles from cheese have been found to be peptides with molecular weights ranging from 2000 to 3000. For the purpose of further characterization, bitter peptides were isolated from enzymatic casein hydrolysates as well as from bitter cheese and purified. 30 proteases from different origins proved to be able to form peptides with bitter taste of varying intensity from casein. Present experience shows that the formation of bitter peptides during casein hydrolysis can be inhibited only to a very small measure. Bitter peptides are extrmely resistant to proteases, which is probably attributable to their high contents of hydrophobic amino acids and hydrophobic bonds. The detection of only N- or C-terminal amino acid in each of 11 different bitter peptides shows that peptide chains are present and not cyclic peptides as repeatedly assumed. It must be aimed at avoiding the cheese defect "bitter" by using appropriate starter cultures and rennet substitutes as little disposed as possible to produce bitter peptides.     

Dilution of Cow's Milk and Egg Proteins with Glutamic Acid and the Effect on the Protein Efficiency Ratio

SUMMARY– The effect of dilution of egg proteins and cow's milk proteins with varying levels of L-glutamic acid (GA) on the growth of young rats and protein efficiency ratio of the blends was studied. Addition of glutamic acid to diets containing 8.5% to 5.0% egg proteins to maintain the nitrogen content of the diet constant at 1.6% (equal to 10% protein) did not cause any increase in the growth rate of rats as compared to that on corresponding diets without added glutamic acid. The protein efficiency ratios progressively decreased from 4.74 for 10% egg protein diet to 2.88 for 5% egg protein + 8.4% glutamic acid diet. Addition of glutamic acid to diets containing 8.5 to 5.0% milk proteins to maintain the nitrogen content of the diet constant at 1.6% level caused a significant decrease in the growth rate as compared to that on corresponding diets without added glutamic acid. The protein efficiency ratios also progressively decreased from 3148 for 10% milk proteins to 1.46 for a mixture of 5% milk proteins + 8.4% glutamic acid. The results show that both egg proteins and milk proteins contain adequate amounts of non-essential amino acids for maximum utilization of essential amino acids present in them for the growth of young rats.     

亚硝酸钠对自然发酵哈尔滨风干肠微生物生长、脂肪氧化及挥发性化合物的影响

研究不同添加水平的亚硝酸钠（NaNO2）对自然发酵哈尔滨风干肠微生物生长、脂肪氧化和挥发性化合物的影响。结果表明,添加NaNO2对风干肠发酵过程中的pH值、水分活度、总菌落数和乳酸菌菌落数无显著影响（P＞0.05）,但添加0.10?g/kg以上的NaNO2可以显著降低风干肠中大肠杆菌菌落数（P＜0.05）。色差分析表明当NaNO2添加量达到0.10?g/kg时可以起到良好的发色作用,过氧化物值和硫代巴比妥酸反应产物显示NaNO2具有良好的抗氧化作用,NaNO2残留量随发酵时间的延长而逐渐降低,发酵结束后,各实验组间差异不显著（P＞0.05）。与此同时,挥发性化合物的分析结果显示,NaNO2添加量不影响风干肠中由碳水化合物发酵和氨基酸降解所产生的挥发性化合物的相对含量（P＞0.05）,但显著影响由脂肪氧化所产生的醛类和酮类化合物的相对含量（P＜0.05）,NaNO2添加量越大,对脂肪氧化产物的抑制作用越显著。     

Culture of Aspergillus parasiticus in apple juice. I. The influence of sodium benzoate and potassium sorbate on fungal growth and aflatoxin biosynthesis

Sodium benzoate or potassium sorbate (100, 200, 300 and 400 mg/l) were added to cultures of Aspergillus parasiticus NRRL 2999 on apple juice (from syrup) and incubated quiescently at 25 degrees C for 3, 6, 9, 12 or 15 days. The cultures were analyzed for pH, mycelial dry weight and accumulation of aflatoxin B1 and G1. The initial pH of 2.5 remained constant in all instances throughout the incubation period. Sodium benzoate, at all concentrations, suppressed fungal growth and stimulated the biosynthesis of G1, whereas little influence was exerted upon the accumulation of B1. Potassium sorbate stimulated fungal growth at 100 mg/l, while at all concentrations it considerably inhibited toxin production (no detectable amounts of B1 and 3 to 5 times less G1 than in controls). The concentration of G1 surpassed that of B1 without exception.     

前体氨基酸对赤霞珠干红葡萄酒中生物胺含量的影响

以新疆地产赤霞珠果实为原料,在葡萄汁中分别加入不同浓度的7种前体氨基酸,进行酒精发酵与苹果酸-乳酸发酵。通过高效液相色谱法的分析检测,了解前体氨基酸对葡萄酒中8种生物胺含量的影响。结果表明,前体氨基酸对葡萄酒中生物胺的含量产生一定的影响,当加入质量浓度为40 mg/L和100 mg/L的组氨酸时,组胺含量由1.04 mg/L减少为0.20 mg/L和0.35 mg/L;而当加入色氨酸、精氨酸和鸟氨酸时,会使色胺和腐胺的含量有所降低,当加入300 mg/L质量浓度的精氨酸时,腐胺含量由1.45 mg/L减少至0.63 mg/L。当葡萄酒中加入同一种氨基酸时,对葡萄酒中不同的生物胺含量的影响也不尽相同。     

Chemical changes in casein heated with and without d-glucose in the powdered state or in an aqueous solution

Chemical changes in casein, heated either in the presence or the absence of d-glucose at 50 or 75°C in a powdered state at 75% relative humidity (RH), or in an aqueous solution, were investigated. During heating, colour development, the formation of polymerised products and severe decomposition of amino acid residues occurred. In the powder reaction system, mainly basic amino acids decomposed whereas, in the solution system, all amino acids were uniformly decomposed. In addition, small amounts of free amino acids and low molecular weight peptides (below MW 500) were detected, these amounts being greater in the solution system, suggesting that non-enzymic cleavage of peptide linkage occurred. These changes were observed both in the absence and in the presence of glucose but glucose promoted such changes.     

Iron-binding properties, amino acid composition, and structure of muscle tissue peptides from in vitro digestion of different meat sources

ABSTRACT:  Background: The enhancing effect of meat on nonheme iron bioavailability in humans is thought to be due to the release of low-molecular-weight (LMW) iron-binding peptides during digestion. Objective: To better characterize the LMW iron-binding peptides from meat digests. Methods: Cooked beef, chicken, cod, lamb, and pork myofibrillar or sarcoplasmic protein extracts, casein, and egg albumin were digested in vitro with pepsin or pepsin/pancreatin. Ultrafiltrates were analyzed for N and iron and further characterized by gel filtration with added ⁵⁹Fe, amino acid analysis, and LC-MS. Results: 84% to 98% of total iron in enzymic digests was associated with soluble LMW peptides (< 10 kDa) of the myofibrillar proteins compared to only 2% to 20% in the corresponding sarcoplasmic protein digests. Pepsin digestion alone of the myobrillar proteins generated > 80% soluble LMW iron, compared to < 5% with casein and egg albumin. Iron-binding peptides from myofibrillar protein with an estimated 2 kDa molecular mass were separated by gel filtration. Peptides in this fraction were enriched in aspartic and glutamic acid residues and included potential peptide fragments of myosin. Conclusion: LMW (< 10 kDa) peptides in enzyme digests of myofibrillar proteins were the major facilitators of iron solubility. Unlike with casein, egg albumin, and most sarcoplasmic proteins, these LMW peptides were generated on pepsin digestion. One group of iron-binding peptides had a mass of approximately 2 kDa and was enriched in glutamic and aspartic acids. Such early generation of a multitude of LMW iron-binding peptides could explain the enhancing effect of muscle tissue on iron absorption.     

添加微量元素对黑豆发芽过程中成分的影响

目的:研究微量元素对黑豆发芽过程中成分变化的影响。方法:采用HPLC法对添加一定量微量元素Zn、Mn、Cu、Fe发芽的黑豆中大豆苷、染料木苷、大豆苷元和染料木素进行含量测定;采用紫外分光光度法对黑豆中总黄酮的含量进行测定;采用高效凝胶过滤色谱法对肽分子量分布进行测定;采用比色法对多肽、游离氨基酸和植酸进行含量测定;采用火焰原子吸收光谱法、电感耦合等离子体原子发射光谱法对微量元素进行含量测定。结果表明:添加微量元素对黑豆发芽过程中成分影响较明显,最终选定的微量元素浓度为:硫酸锌30 mg/L、硫酸锰20 mg/L、硫酸铜10 mg/L、硫酸亚铁20 mg/L。通过定量分析,黑豆添加微量元素发芽与清水发芽的黑豆16 d相比,异黄酮苷元增长了88.24%;游离氨基增加了87.29%;植酸含量则减少了30.86%。结论:黑豆添加微量元素发芽有利于异黄酮苷元、游离氨基酸等营养成分的生成,营养价值更高。     

Influence of polyethylene glycol on the excretion and digestibility of amino acids in casein and tannic acid‐fed broilers

Using the precision feeding technique, an experiment was conducted to evaluate the influence of tannic acid (TA) and polyethylene glycol (PEG) on the excretion of amino acids and the apparent and true digestibilities of casein protein in broilers. Seventy‐two 9‐week‐old broiler cockerels grouped in nine treatments of eight replicates were fed warm water (50 mL, control birds), casein alone (12 or 18 g) or casein (12 or 18 g) with TA solution (4.5 g per 10 mL) or/and PEG solution (2 g per 10 mL). Total excreta were collected for 48 h and freeze‐dried. The amino acid content of casein and excreta was determined by reverse phase high‐performance liquid chromatography. In the absence of TA the digestibility of casein was almost complete. TA increased the excretion of amino acids to a varying extent (P < 0.01). Although the digestibility of all essential and non‐essential amino acids was adversely affected by the presence of TA, raising the amount of casein from 12 to 18 g improved significantly (P < 0.05) the apparent and true digestibilities of all amino acids. PEG reduced significantly (P < 0.01) the excretion of amino acids and improved significantly (P < 0.01) the amino acid digestibility of casein in TA‐dosed birds. However, the improvement was greater when the lower level of casein (12 g) was fed. Thus PEG might play an important role in inactivating dietary tannins in the gastrointestinal tract of birds and improving protein digestibility and utilisation, particularly when the diet is low or intermediate in protein. Copyright © 2007 Society of Chemical Industry