Technical note: Methodological and feed factors affecting measurement of protein A,B, and C fractions,degradation rate,and intestinal digestibility of rumen-undegraded protein

When formulating dairy cow rations, characterization of protein in feeds requires estimation of protein degradation in the rumen and digestion in the intestine. The objective of this work was to evaluate experimental and feed-related factors that affect characterization using in situ, in vitro, or mobile bag techniques, of 0-h washout (A), potentially degradable (B), and undegradable (C) protein fractions, protein degradation rate (K_d), and digestibility of rumen undegradable protein (dRUP). Data sets of 136 studies on A, B, C, and K_d and 113 studies on dRUP were amassed from the literature. Mixed-effect linear models were used to relate these variables to methodological and feed factors while accounting for random differences among studies. Predictions of A, B, and C protein fractions were significantly influenced by crude protein and neutral detergent fiber interactions with bag pore size, incubation time, bag area, and sample-to-bag area ratio. For example, a 20.0% decrease in crude protein of a theoretical legume silage sample would increase A fraction prediction by 20.1%, but 34.7% with bag incubation time ?1 standard deviation below the mean. Similarly, reported K_d values were significantly influenced by crude protein interactions with bag area and sample-to-bag area ratio and by neutral detergent fiber interaction with pore size. Feed variables and measurement variables influencing protein digestibility measures suggest that these analytical factors are likely associated with variance among differing methodologies and within unique samples of the same feed. When predicting dRUP, the use of mobile bag method produced significantly different estimates compared with the in vitro 3-step method. The use of mobile bag resulted in an 8.9% (±3.8%) higher estimate of dRUP compared with the in situ technique. In 618 and 977 samples, sample variation to sample mean ratio for acid detergent fiber and pepsin-acid incubation time was 63.0 and 58.0%, respectively. Variation in feedstuff content and lack of standardization of methods used to measure protein disappearance led to a lack of robustness in the measurements commonly employed.     

中性大蒜果聚糖体外发酵产短链脂肪酸

大蒜果聚糖是大蒜深加工后废弃蒜渣中的主要成分,不能被人体消化利用,可选择性增殖肠道益生菌,具有开发成新型益生元的潜力。体外发酵产短链脂肪酸的分析对进一步评价大蒜果聚糖对肠道微生态的影响有重要意义。方法:本研究通过体外模拟人体肠道系统的pH、温度和营养条件,接种人体新鲜粪便悬浮液进行间歇式静态厌氧培养,采用高效液相色谱法(HPLC)分析不同聚合度(平均DP为11、16、21)的中性大蒜果聚糖为唯一碳源,培养0、4、8、12、24 h产短链脂肪酸的情况。结果:发酵12 h时短链脂肪酸浓度达到最高,之后便开始下降。其中样a(平均DP为11)的发酵液中总SCFA浓度最高,达86.38μmol/mL。总短链脂肪酸产量为样a(DP 11)>样b(DP16)>样c(DP 21)。24 h时,各发酵液的短链脂肪酸中丁酸和乳酸的比例较乙酸和丙酸高。结论:大蒜果聚糖能被人体肠道微生物发酵利用在体外产短链脂肪酸。其中丁酸和乳酸的摩尔浓度较乙酸和丙酸高。聚合度低的大蒜果聚糖产酸高于聚合度高的果聚糖。     

Effect of quantifying peptide release on ruminal protein degradation determined using the inhibitor in vitro system

The aim of this work was to compare use of an o-phthaldialdehyde (OPA) colorimetric assay (OPA-C), which responds to both free AA and peptides, with an OPA fluorimetric assay (OPA-F), which is insensitive to peptides, to quantify rates of ruminal protein degradation in the inhibitor in vitro system using Michaelis-Menten saturation kinetics. Four protein concentrates (expeller-extracted soybean meal, ESBM; 2 solvent-extracted soybean meals, SSBM1 and SSBM2; and casein) were incubated in a ruminal in vitro system treated with hydrazine and chloramphenicol to inhibit microbial uptake of protein degradation products. Proteins were weighed to give a range of N concentrations (from 0.15 to 3 mg of N/mL of inoculum) and incubated with 10 mL of ruminal inoculum and 5 mL of buffer; fermentations were stopped after 2 h by adding trichloroacetic acid (TCA). Proteins were analyzed for buffer-soluble N and buffer extracts were treated with TCA to determine N degraded at t = 0 (FD0). The TCA supernatants were analyzed for ammonia (phenol-hypochlorite assay), total AA (TAA; OPA-F), and TAA plus oligopeptides (OPA-C) by flow injection analysis. Velocity of protein degradation was computed from extent of release of 1) ammonia plus free TAA or 2) ammonia plus free TAA and peptides. Rate of degradation (k_d) was quantified using nonlinear regression of the integrated Michaelis-Menten equation. The parameters K_m (Michaelis constant) and k_d (Vmax/K_m), where Vmax = maximum velocity, were estimated directly; k_d values were adjusted (Ak_d) for the fraction FD0 using the equation Ak_d = k_d − FD0/2. The OPA-C assay yielded faster degradation rates due to the contribution of peptides to the fraction degraded (overall mean = 0.280/h by OPA-C and 0.219/h by OPA-F). Degradation rates for SSBM samples (0.231/h and 0.181/h) and ESBM (0.086/h) obtained by the OPA-C assay were more rapid than rates reported by the National Research Council (NRC). Both assays indicated that the 2 SSBM differed in rumen-undegradable protein (RUP) content; the more slowly degraded SSBM had RUP content (35% by OPA-C) similar to that reported by the NRC. The RUP content of ESBM (42% by OPA-C) was lower than the NRC value. Preliminary studies with 4 additional protein concentrates confirmed that accounting for peptide formation increased degradation rate; however, a trend for an interaction between assay and protein source suggested that peptide release made a smaller contribution to rate for more slowly degraded proteins. The OPA-C assay is a simple and reliable method to quantify formation of small peptides.     

利用体外发酵模型研究叶绿素对儿童肠道菌群的影响

通过不同年龄儿童粪便的体外发酵,研究叶绿素的生物代谢及对肠道菌群的影响,探明其生物活性,明确摄入绿叶蔬菜的健康意义。体外发酵试验菌群测序分析结果表明:叶绿素降低了儿童肠道菌群的多样性,添加叶绿素体外发酵24 h后,2岁儿童菌群多样性指数由2.6~3.2降至2.4~2.7,5岁儿童菌群多样性指数由2.5~2.8降至1.8~2.4。属水平上,柯林斯菌属、双岐菌属相对丰度降低,乳酸菌属的相对丰度升高。菌群代谢物检测结果表明,叶绿素促进同年龄儿童粪便代谢物乳酸、乙酸水平的增加,说明叶绿素具有一定的生物活性,影响肠道菌群组成及其代谢产物含量的变化。     

体外发酵番茄红素的益生元作用

采用体外模拟肠道微生态发酵体系,研究番茄红素的益生元作用。结果表明,番茄红素经体外发酵,显著提高了乳酸杆菌属、双歧杆菌属、拟杆菌属、普氏菌属和阿克曼氏菌属的数量,约可提高1~2个数量级,减缓了瘤胃球菌属和罗斯氏菌属数量的减少,显著增加了乙酸、丙酸、丁酸、戊酸、异戊酸等短链脂肪酸的产量。相关性分析表明,随着发酵时间的延长,上述菌群与乙酸、丙酸、丁酸、戊酸等短链脂肪酸产量的相关性呈逐渐增加的趋势,且在番茄红素发酵24 h时,益生菌群的数量与短链脂肪酸的产量呈极显著正相关。由此可见,番茄红素具有显著的益生元作用,发酵产生的益生菌数量与短链脂肪酸产量间的相互作用随发酵时间的延长逐渐增加,这种互作可能在番茄红素抗炎、抗氧化、抗肿瘤方面发挥重要作用,研究结果为番茄红素的开发应用提供数据支撑。     

Effect of an intramammary lipopolysaccharide challenge on the hindgut microbial composition and fermentation of dairy cattle experiencing intermittent subacute ruminal acidosis

Feeding grain-rich diets often results in subacute ruminal acidosis (SARA), a condition associated with ruminal dysbiosis and systemic inflammation. Yet, the effect of SARA on hindgut microbiota, and whether this condition is aggravated by exogenous immune stimuli, is less understood. Therefore, the aims of this study were to determine the effects of an intermittent high-grain SARA model on the hindgut microbial community, and to evaluate whether the effects of SARA on the fecal microbiome and fermentation were further affected by an intramammary lipopolysaccharide (LPS) challenge. A total of 18 early-lactating Simmental cows were divided into 3 groups (n = 6); 2 were fed a SARA-inducing feeding regimen (60% concentrate), 1 was fed a control (CON) diet (40% concentrate). On d 30, 1 SARA group (SARA-LPS) and the CON group (CON-LPS) were intramammarily challenged with a single dose of 50 µg of LPS from Escherichia coli O26:B6, whereas the remaining 6 SARA cows (SARA-PLA) received a placebo. Using a longitudinal randomized controlled design, with grouping according to parity and days in milk), statistical analysis was performed with baseline measurements used as a covariate in a mixed model procedure. The SARA-inducing feeding challenge resulted in decreased fecal pH and increased butyrate as a proportion of total short-chain fatty acids in the feces. On d 30, SARA-challenged cows had decreased fecal diversity as shown by the Shannon and Chao1 indices and a decrease in the relative abundance of Euryarchaeota and cellulolytic genera, and numerical increases in the relative abundance of several Firmicutes associated with starch and secondary fermentation. The LPS challenge did not affect the fecal pH and short-chain fatty acids, but increased the Chao1 richness index in an interaction with the SARA challenge, and affected the relative abundance of Verrucomicrobia (1.13%), Actinobacteria (0.19%), and Spirochaetes (0.002%), suggesting an effect on the microbial ecology of the hindgut during SARA conditions. In conclusion, the SARA-inducing feeding regimen promoted important microbial changes at d 30, including reduced diversity and evenness compared with CON, whereas the external LPS challenge led to changes in the microbial community without affecting fecal fermentation properties.     

改性葡萄皮渣膳食纤维体外发酵特性研究

采用人粪便提取液为接种物,通过体外发酵技术研究不同改性处理的葡萄皮渣膳食纤维在模拟人体大肠环境条件下的发酵过程,测定不同发酵时间的pH值和短链脂肪酸(SCFA)的含量。结果表明：葡萄皮渣膳食纤维是一种理想的可被大肠微生物所利用的碳源,能够在体外模拟环境下发酵产生乙酸、丙酸、丁酸和异丁酸等短链脂肪酸;葡萄皮渣膳食纤维经过不同改性处理后对其发酵液pH值的变化无明显影响,但对发酵液中短链脂肪酸的总量和各组分含量均有一定影响。     

高链玉米淀粉-不饱和脂肪酸复合物的体外大肠发酵特性及其对肠道菌群的影响规律

该研究以高直链玉米淀粉（High-Amylose Maize Starch,HAMS）与四种饱和度不同的脂肪酸为原料,制备淀粉-不饱和脂肪酸复合物,研究不同复合物体外大肠发酵特性及菌群调控的影响。结果表明,随着脂肪酸不饱和度的增加,除淀粉-油酸复合物（34.87%）外,形成的复合物V型结晶越少（30.42%~32.93%）。淀粉-不饱和脂肪酸复合物在整个发酵过程中发酵速率无显著性差异（p>0.05）,最终产气量基本一致（13.8~14.2 mL）。淀粉-不饱和脂肪酸复合物产丁酸浓度（14.83~17.91 mmol/L）低于HAMS （22.42 mmol/L）,但产较多乙酸（60.25~63.73 mmol/L）和丙酸（21.22~24.81 mmol/L）。淀粉-不饱和脂肪酸复合物均能显著促进Prevotella的相对丰度,这与其较高的丙酸产量有关。综上,淀粉-不饱和脂肪酸复合物大肠发酵特性受脂肪酸饱和度的影响较小,主要与淀粉-脂质复合物自身结构有关,复合物中脂肪酸的饱和度对高链玉米淀粉-不饱和脂肪酸复合物菌群结构具有一定影响。     

不消化性葡聚糖体外酵解液对RAW 264.7巨噬细胞的免疫调节作用

为探究不消化性葡聚糖（indigestible glucans,IGs）体外肠道菌酵解液的免疫调节活性,采用厌氧发酵模型利用健康人体粪便菌群分别对6 种IGs（大麦β-葡聚糖、海带多糖、酵母β-葡聚糖、茯苓多糖、抗性淀粉和聚葡萄糖）进行24 h 体外酵解,除菌过滤获得IGs 酵解液,对pH 值和短链脂肪酸含量进行测定。并以RAW 264.7巨噬细胞为研究对象,分为正常组、阳性组和IGs 酵解液组,采用CCK-8 试剂盒测定细胞活力,一氧化氮（nitric oxide,NO）检测试剂盒测定细胞NO 释放量,流式细胞仪检测细胞活性氧（reactive oxygen species,ROS）含量,酶联免疫吸附测定法测定细胞因子分泌量。结果表明,相比于正常组,6 种IGs 的24 h 酵解液均显著提高了RAW 264.7巨噬细胞活力、NO释放量、ROS 产生量和小鼠肿瘤坏死因子α 分泌量。各IGs 24 h 肠道菌酵解液对RAW 264.7巨噬细胞均具有免疫调节的作用。     

低分子量硫酸软骨素体外酵解特征及其对肠道菌群的调节作用

硫酸软骨素（Chondroitinsulfate,CS）具有调节肠道微生物的作用,但不同分子量对其益生活性的影响鲜有报道。该研究比较了硫酸软骨素（80 ku）及其光催化降解产物（Low Molecular Weight Chondroitin Sulfate,LWCS,4 ku）体外酵解特征及其对肠道代谢物的影响。通过化学法、气相色谱质谱联用、高通量测序等方法测定了不同时间发酵液中p H值、CS和LWCS含量、还原糖含量、短链脂肪酸浓度以及肠道微生物的变化。结果显示,在发酵48 h内,CS、LWCS以及还原糖含量均显著降低（P<0.05）,说明肠道微生物能够降解利用硫酸软骨素及其降解产物。且降解后的硫酸软骨素更显著地增加了拟杆菌门和副杆菌属等有益菌的丰度（P<0.05）,更明显的抑制了潜在致病菌型埃希氏-志贺氏菌属（P<0.05）。此外,LWCS发酵能维持24 h的酸性环境,并产生更多的乙酸、丙酸和总短链脂肪酸（分别增加1.15、1.03和1.09倍）。以上结果表明,光催化降解后的硫酸软骨素具有更强的肠道益生作用,该研究为硫酸软骨素的开发应用提供了科学依据。     

基于体外模拟肠道微生态体系比较不同果蔬全粉的益生元功效

体外模拟肠道系统逐渐成为研究肠道菌群及其代谢产物的一种操作简便、重现性好的方法。本研究通过建立体外模拟肠道微生态体系,对丝瓜、桑葚、枸杞和香菇冻干粉的益生元功效展开研究。结果表明,经24 h体外发酵后,丝瓜冻干粉对乳酸杆菌属、普氏菌属、拟杆菌属数量和丙酸产量增加的促进作用显著高于其它果蔬组,桑葚对双歧杆菌数量和乙酸产量增加的促进作用显著高于其它果蔬组。以上结果说明基于体外模拟肠道微生态体系,丝瓜和桑葚冻干全粉均具有较为显著的益生元功效。