Effects of inoculating feruloyl esterase-producing Lactiplantibacillus plantarum A1 on ensiling characteristics,in vitro ruminal fermentation and microbiota of alfalfa silage

Background Ferulic acid esterase(FAE)-secreting Lactiplantibacillus plantarum A1(Lp A1)is a promising silage inoculant due to the FAE’s ability to alter the plant cell wall structure during ensiling,an action that is expected to improve forage digestibility.However,little is known regarding the impacts of Lp A1 on rumen microbiota.Our research assessed the influences of Lp A1 in comparison to a widely adopted commercial inoculant Lp MTD/1 on alfalfa’s ensilage,in vitro rumen incubation and microbiota.Results Samples of fresh and ensiled alfalfa treated with(either Lp A1 or Lp MTD/1)or without additives(as control;CON)and ensiled for 30,60 and 90 d were used for fermentation quality,in vitro digestibility and batch culture study.Inoculants treated silage had lower(P<0.001)pH,acetic acid concentration and dry matter(DM)loss,but higher(P=0.001)lactic acid concentration than the CON during ensiling.Compared to the CON and Lp MTD/1,silage treated with Lp A1 had lower(P<0.001)aNDF,ADF,ADL,hemicellulose,and cellulose contents and higher(P<0.001)free ferulic acid concentration.Compared silage treated with Lp MTD/1,silage treated with Lp A1 had significantly(P<0.01)improved ruminal gas production and digestibility,which were equivalent to those of fresh alfalfa.Realtime PCR analysis indicated that Lp A1 inoculation improved the relative abundances of rumen’s total bacteria,fungi,Ruminococcus albus and Ruminococcus flavefaciens,while the relative abundance of methanogens was reduced by Lp MTD/1 compared with CON.Principal component analysis of rumen bacterial 16S rRNA gene amplicons showed a clear distinction between CON and inoculated treatments without noticeable distinction between Lp A1 and Lp MTD/1 treatments.Comparison analysis revealed differences in the relative abundance of some bacteria in different taxa between Lp A1 and Lp MTD/1 treatments.Silage treated with Lp A1 exhibited improved rumen fermentation characteristics due to the inoculant effects on the rumen microbial populations and bacterial community.Conclusions Our findings suggest that silage inoculation of the FAE-producing Lp A1 could be effective in improving silage quality and digestibility,and modulating the rumen fermentation to improve feed utilization.

Adsorption,in vitro digestion and human gut microbiota regulation characteristics of three Poria cocos polysaccharides

Poria cocos(PC)is a famous traditional Chinese medicine(TCM)and a widely used healthcare ingredient,which has antiobesity,enhancing immunity and improving sleep effects.Traditionally,only water-soluble poria polysaccharide(WSP)is extracted and applied for clinical application,while insoluble polysaccharide(alkali-soluble poria polysaccharide,ASP)is discarded as herb residue.However,the whole PC has also been historically utilized as functional herbal food.Considering the beneficial role of dietary fiber and the traditional use of PC,ASP may also contribute substantially to the therapy function of PC.Compared to WSP,little attention has been paid to ASP and ASP modified product carboxymethyl poria polysaccharide(CMP)which has been used as an antitumor adjuvant drug.In this study,the oil,cholesterol,metal ions and polyphenols adsorption ability,in vitro simulated digestive and the gut microbiota fermentation characteristics of WSP,ASP and CMP were studied to evaluate the functional values of three P.cocos polysaccharides(PCPs).The results showed that all three PCPs had good adsorption capacity on cholesterol,polyphenols and metal ions(Cd^(2+)/Zn^(2+)/Mg^(2+)),among which ASP showed the highest capacity than WSP and CMP.The adsorption capacity of all three PCPs on heavy metal ions(Cd^(2+)/Zn^(2+))was stronger than that of non-heavy metal ions(Mg^(2+));The in vitro digestibility of all three PCPs was very low,but WSP was slightly higher than ASP and CMP;Moreover,the indigestible residue of all three PCPs could improve the richness and diversity of gut microbiota,among which ASP had the greatest influence.In general,ASP and CMP could significantly promote the proliferation of some probiotics and inhibit the growth of some harmful bacteria.The gut microbiota diversity of CMP was reduced,but the richness of probiotics,especially Parabacteroides distasonis was significantly enhanced compared with the ASP group,and the growth of harmful bacteria Klebsiella pneumoniae was inhibited after CMP treatment.The short-chain fatty acids(SCFAs)analysis results showed that all three PCPs could significantly promote the production of acetic acid,propionic acid and the total acid content compared with blank control group,and SCFAs producing activity was positively correlated with the proliferative capacity of probiotics.Taken together,the good adsorption characteristics and gut microbiota regulatory activity of ASP may lay foundation for its lipid-lowering and immune-improving function.Additionally,the probiotic effect of CMP and ASP indicated that except for only use the water extract of PC in clinic,CMP and ASP also can be used in healthcare to take full advantage of this valuable medicine.

Impacts of the unsaturation degree of long-chain fatty acids on the volatile fatty acid profiles of rumen microbial fermentation in goats in vitro

This study investigated the impacts of the degree of unsaturation （unsaturity） of long-chain fatty acids on volatile fatty acid （VFA） profiles of rumen fermentation in vitro. Six types of long-chain fatty acids, including stearic acid （C18：0, control group）, oleic acid （C18：1, n-9）, linoleic acid （C18：2, n-6）, a-linolenic acid （C18：3, n-3）, arachidonic acid （C20：4, n-6） and eicosapentaenoic acid （C20：5, n-3）, were tested. Rumen fluid from three goats fitted with ruminal fistulae was used as inoculum and the inclusion rate of long-chain fatty acid was at 3% （w/w） of substrate. Samples were taken for VFA analysis at 0, 3, 6, 9, 12, 18 and 24 h of incubation, respectively. The analysis showed that there were significant differences in the total VFA among treatments, sampling time points, and treatment×time point interactions （P〈0.01）. a-Linolenic acid had the highest total VFA （P〈0.01） among different long-chain fatty acids tested. The molar proportion of acetate in total VFA significantly differed among treatments （P〈0.01） and sampling time points （P〈0.01）, but not treatment×time point interactions （P〉0.05）. In contrast, the molar proportion of propionate did not differ among treatments during the whole incubation （P〉0.05）. However, for butyrate molar proportions, significant differences were found not only among sampling time points but also among treatments and treatment×time point interactions （P〈0.01）, with eicosapentaenoic acid having the highest value （P〈0.01）. Additionally, no statistically significant differences were found in the acetate to propionate ratios among treatments groups （P〉0.05）, even the treatments stearic acid and a-linolenic acid were numerically higher than the others. The inclusion of 3% long-chain unsaturated fatty acids differing in the degree of unsaturation brought out a significant quadratic regression relation between the total VFA concentration and the double bond number of fatty acid. In conclusion, the a-linolenic acid with 3 double bonds appeared better for improving rumen microbial fermentation and the total VFA concentration.

Rumen methane output and fermentation characteristics of gramineous forage and leguminous forage at differing harvest dates determined using an in vitro gas production technique

An in vitro rumen gas production technique was employed to determine the methane production and fermentation characteristics of Leymus chinensis and Medicago ruthenica at differing harvest dates（May 15,May 30,June 30,July 30,August 30 and September 30）,which are sequential phases within a single continuous growth of two 10-year-old pastures.To quantify the rate of degradation and compare in vitro rumen fermentation characteristic,a logistic-exponential model,where initial gas volume was zero（LE_0）,was used to fit gas production and methane output results.Dried,milled forage samples were incubated in vitro for 72 h at 39℃ and gas production was recorded intermittently throughout the incubation and gas samples were collected to measure methane production.Results showed that there were significant interactions between species and harvest for all chemical composition variables（P〈0.001） and condensed tannin content（P〈0.001）.L.chinensis produced more total gas and methane than M.ruthenica（P〈0.001）.Both total gas and methane production decreased lineally（P〈0.001） with advancing harvest date.The degradation rates of L.chinensis and M.ruthenica harvested on September 30 were lower than those on the other harvest dates（P〈0.01）.M.ruthenica fermented fluid had higher concentration of ammonia N（P〈0.05） and molar proportions of isobutyrate（P〈0.01）,valerate（P〈0.001） and isovalerate（P〈0.01） in total volatile fatty acids than L chinensis.Furthermore,concentration of isovalerate decreased cubically with advancing harvest date（P〈0.05）.In conclusion,M.ruthenica produced less methane than L.chinensis and the total gas and methane production decreased with advancing harvest date for both species,which may be due to the changes in contents of chemical compositions and condensed tannin in forages.

Evaluation of nutritive value and in vitro rumen fermentation gas accumulation of de-oiled algal residues

Background：Algae are widely recognized for their high oil content and for exponentially accumulating biomass with particular potential to provide single cell protein for human consumption or animal feed.It is believed that along with biodiesel from algae,the high protein de-oiled algal residue may become an alternative feed supplement option in the future.This study was conducted to investigate de-oiled algal residue obtained from the common Chlorella species,Thalassiosira weissflogii,Selenarstrum capricornutum,Scenedesmus sp.,and Scenedesmus dimorphus for assessment as potential feed supplements for ruminants by comparing with soybean（Glycine max） meal and alfalfa（Medicago sativa） hay.Results：With the exception of T.weissflogii,algal residue had higher concentrations of Cu,Zn,and Mn and lower concentration of Ca,Mg,and K than soybean meal and alfalfa hay.The algal residue CP（crude protein）concentrations ranged from 140 to 445 g/kg DM and varied among the de-oiled residues.In vitro rumen fermentation gas accumulation curves indicated that algal biomass degradation potential was less than that of soybean meal or alfalfa hay by up to 41.7%.The gas production curve,interpreted with a dual pool logistic model,confirmed that the fraction sizes for fast fermenting and slow fermenting of de-oiled algal residues were smaller than those in soybean meal and alfalfa hay,and the fermenting rate of the fractions was also low.Conclusions：Inferior in vitro rumen gas accumulation from the five de-oiled algal residues suggests that these algal byproducts are less degradable in the rumen.

Effects of inclusion of purple prairie clover (Dalea purpurea Vent.)with native coolseason grasses on in vitro fermentation and in situ digestibility of mixed forages

Background:Incorporation of legume species into native North American pastures is considered an effective method to increase native pasture productivity and improve the nutritive value of forage.This study evaluated the effects of inclusion of purple prairie clover(PPC,Dalea purpurea Vent.),a native legume forage,with native coolseason grasses on the in vitro fermentation and in situ digestibility of mixed forages.Methods:Whole plant PPC and mixtures of cool-season grasses were harvested when the PPC reached the vegetative(VEG),full flower(FL)and seedpod(SP)stages,and were combined in ratios(DM basis)of 0:100,25:75,50:50,75:25 and 100:0 at each maturity.In vitro ruminal incubations using these mixtures were conducted for 48 h to determine gas production(GP),in vitro DM disappearance(IVDMD),total volatile fatty acids(VFA)and ammonia-N production.Mixtures of forages harvested when the PPC reached the FL stage and 50:50 mixture of forages harvested at VEG,FL and SP stages were incubated in the rumen of three heifers for 0,2,6,12,24,48,72 and 96 h to determine in situ degradabilities of DM,neutral detergent fibre(aNDF)and crude protein(CP).Results:Contents of aNDF and ADF increased(P<0.01),while CP decreased(P<0.001)as PPC matured.Concentrations of extractable condensed tannins in PPC ranked as FL>VEG>SP(P<0.05).Regardless of PPC proportions in the mixture,GP decreased(P<0.05)with increasing PPC maturity.Increasing PPC proportions linearly increased(P<0.001)GP,IVDMD and total VFA at VEG,but linearly decreased(P<0.001)them at SP.Irrespective of PPC maturity,ammonia-N production linearly increased(P<0.01)with increasing proportions of PPC and the concentration was higher(P<0.05)at VEG than at FL and SP stages.Increasing proportion of PPC at either maturity linearly increased(P<0.001)molar percentage of acetate(A)and branched-chain VFA,but linearly decreased(P<0.001)molar percentage of propionate(P),resulting in a linearly increase(P<0.001)in the A:P ratio.Increasing FL PPC in the mixture linearly and quadratically(P<0.01)increased a(soluble fraction),but linearly and quadratically decreased(P<0.01)b(potentially degradable fraction)for DM and aNDF,resulting in linear(P<0.05)and quadratic(P<0.01)increases in DM and aNDF maximum potential degradabilities(a+b).Effective degradabilities of DM and aNDF were also linearly and quadratically increased(P<0.05),and CP was quadratically increased(P<0.05)with increasing FL PPC,with the greatest effective degradability being observed with ratios between 50:50 and 75:25.Ruminal maximum potential degradabilities of DM and aNDF decreased(P<0.001)as the forage matured.Effective degradability of DM ranked as VEG>FL>SP(P<0.001),whereas the effective degradability of aNDF was similar between VEG and FL and both were greater(P<0.01)than SP.Conclusions:Inclusion of vegetative PPC in a mixed forage diet resulted in the greatest digestibility and incorporation of PPC before seedpod stage with native grasses had a positive effect on ruminal fermentation.Effects of PPC on ruminal digestion depend on both the stage of maturity and its proportion in mixed legume-grass pastures.Pastures containing 50%of PPC in full flower stage would likely provide the greatest quality diet to grazing ruminants subject to potential animal selectivity.

Structural Characteristics of Cellulose and Xylan during in vitro Fermentation by Pig Fecal Bacteria

In this study,cellulose and xylan were in vitro fermented by pig fecal bacteria.Rapid fermentation(40 h) and extended fermentation(eight weeks)were performed.The properties and ultra-structure changes of post-fermented solid residues were studied.In the end effluent,acetic acid,propionic acid,and butyric acid were observed to be the principal short-chain fatty acids(SCFAs) produced by anaerobic fermentation.Xylan was more accessible to bacteria than cellulose,leading to higher SCFA and lactic acid production.In addition,the crystalline structure of cellulose changed,leading to 16.3% and42.1% increases in crystallinity index for rapid and extended fermentation,respectively.Through this research,a systematic and advanced method to study the degradation chemistry of cellulose and xylan during fermentation was developed.

Methodological factors affecting gas and methane production during in vitro rumen fermentation evaluated by meta-analysis approach

Effects of some methodological factors on in vitro measures of gas production（GP, mL/g DM）, CH4production（mL/g DM） and proportion（% CH4 on total GP） were investigated by meta-analysis. These factors were considered：pressure in the GP equipment（0 = constant; 1 = increasing）, incubation time（0 = 24; 1 = ≥ 48 h）, time of rumen fluid collection（0 = before feeding; 1 = after feeding of donor animals）, donor species of rumen fluid（0 = sheep; 1 =bovine）, presence of N in the buffer solution（0 = presence; 1 = absence）, and ratio between amount of buffered rumen fluid and feed sample（BRF/FS; 0 = ≤ 130 mL/g DM; 1 = 130–140 mL/g DM; 2 = ≥ 140 mL/g DM）. The NDF content of feed sample incubated（NDF） was considered as a continuous variable. From an initial database of 105 papers, 58 were discarded because one of the above-mentioned factors was not stated. After discarding 17 papers,the final dataset comprised 30 papers（339 observations）. A preliminary mixed model analysis was carried out on experimental data considering the study as random factor. Variables adjusted for study effect were analyzed using a backward stepwise analysis including the above-mentioned variables. The analysis showed that the extension of incubation time and reduction of NDF increased GP and CH4 values. Values of GP and CH4 also increased when rumen fluid was collected after feeding compared to before feeding（＋26.4 and ＋9.0 mL/g DM, for GP and CH4）,from bovine compared to sheep（＋32.8 and ＋5.2 mL/g DM, for GP and CH4）, and when the buffer solution did not contain N（＋24.7 and ＋6.7 mL/g DM for GP and CH4）. The increase of BRF/FS ratio enhanced GP and CH4production（＋7.7 and ＋3.3 mL/g DM per each class of increase, respectively）. In vitro techniques for measuring GP and CH4 production are mostly used as screening methods, thus a full standardization of such techniques is not feasible. However, a greater harmonization of analytical procedures（i.e., a reduction in the number of available protocols） would be useful to facilitate comparison between results of different experiments.

Study on prebiotic effectiveness of neutral garlic fructan in vitro

Garlic is traditionally believed to have many health benefits including prevention of gastrointestinal diseases.One of the major components,garlic fructan(GF),was evaluated for its prebiotic effectiveness on human intestinal microflora.Garlic fructans A(DP 16)and B(DP 21)were prepared by ethanol fractionation precipitation.Then,they were added to an in vitro fermentation system as the sole carbon source inoculated with human fecal suspension.The total anaerobic bacteria,Bacteroides and Bifidobacteria,were enumerated by plate counting on selective media.Terminal restriction fragment length polymerization(tRFLP)was used to analyze DNA extracted from the in vitro cultures.The results indicated that the log CFUs of both Bacteroides(GF A 6.96,GF B 7.15)and Bifidobacteria(GF A 7.74,GF B 7.74)grown in the GF cultures at 24 h were significantly higher than those at 0 h(Bacteroides 4.93,Bifidobacteria 4.78)(P<0.05).Terminal restriction fragments(TRFs)256–258 bp(ascribed to Bifidobacterium)in the profiles were also observed higher in the TRFLP profiles from the garlic fructans’cultures.In this study,GFs were found to selectively stimulate the growth of beneficial Bifidobacteria from human fecal microflora.The prebiotic effectiveness of GFs supports the use of garlic as a way to prevent some gastrointestinal diseases.©2013 Beijing Academy of Food Sciences.Production and hosting by Elsevier B.V.All rights reserved.

Structural characterization of oligosaccharide from Spirulina platensis and its effect on the faecal microbiota in vitro

In the present study,an oligosaccharide SPO-1 from Spirulina platensis was prepared by glycosidase from a marine bacterium.The prebiotic activity of SPO-1 on the growth of Lactobacillus paracasei and Bifidobacterium animalis,and its effect on human gut microbiota were examined in vitro.The molecular weight of the tetrasaccharide SPO-1 was 650.2 Da,and it was mainly composed of glucose with α-type glycosidic linkages.The prebiotic activity score of SPO-1 was the highest for the growth of probiotic strains L.paracasei and B.animalis.Furthermore,as fermentation proceeded,SPO-1 was gradually degraded and utilized by intestinal bacteria.The results showed that after treatment with SPO-1,carbohydrate consumption and short-chain fatty acids levels were increased,especially those of i-butyric and i-valeric acids.Moreover,SPO-1 significantly promoted the abundance,diversity and composition of gut microbiota,especially stimulating the growth of Bacteroides,Escherichia-Shigella and Megamonas.In addition,the change in intestinal microbiota function predicted by phylogenetic investigation of communities by reconstruction of unobserved states(PICRUSt)after treatment with SPO-1 is mainly related to the terms“carbohydrate metabolism”and“amino acid metabolism”.These results suggest that SPO-1 is a potential oligosaccharide in regulation of intestinal microbiota.

Characterization of a novel bifunctional enzyme from buffalo rumen metagenome and its effect on in vitro ruminal fermentation and microbial community composition

To efficiently use lignocellulosic materials in ruminants,it is crucial to explore effective enzymes,especially bifunctional enzymes.In this study,a novel stable bifunctional cellulase-xylanase protein from buffalo rumen metagenome was expressed and characterized,CelXyn2.The enzyme displayed optimal activity at pH 6.0 and 45℃.The residual endoglucanase and xylanase activities were 90.6%and 86.4%after a 60-min pre-incubation at 55℃.Hydrolysis of rice straw,wheat straw,sheepgrass and sugar beet pulp by CelXyn2 showed its ability to degrade both cellulose and hemicellulose polymers.Treatment with CelXyn2 improved the hydrolysis of agricultural residues with an evident increase in production of total gas,lactate and volatile fatty acids.The results of 16S rRNA and real-time PCR showed that the effect on in vitro ruminal microbial community depended on fermenta-tion substrates.This study demonstrated that CelXyn2 could strengthen lignocellulose hydrolysis and in vitro ruminal fermentation.These characteristics of CelXyn2 distinguish it as a promising candidate for agricultural application.

Ruminal fermentation and microbial community differently influenced by four typical subtropical forages in vitro

The present study evaluated the effects of 4 typical subtropical forages on ruminal microbial community composition to formulate a better diet for buffalo.Corn straw silage,elephant grass,cassava residues and sugarcane tail silage were used as substrates for in vitro fermentation.Eight replicates were set up for every substrate,and fermentation was carried out in a 100-m L glass syringe,using buffalo rumen inoculum.Every replicate was anaerobically dispensed with 10 m L of rumen inoculum,20 m L of Mc Dougall's buffer and 200 mg of dried substrate,and placed in a water bath at 39C.Gas production was recorded at 0,2,6,12,24,36,48 and 72 h of incubation.After 24 h,fermentation was ceased for 4 replicates and samples were collected.Volatile fatty acids(VFA) concentrations were measured using gas chromatography.Microbial populations were quantified using quantitative real-time PCR(q RT-PCR),and microbial community was analyzed using high throughput sequencing technology.The results showed,cassava residues as substrate had the highest gas production,acetate,propionate and total VFA concentrations(P < 0.05),and corn straw silage had the lowest acetate:propionate ratio(P < 0.05).The lowest numbers of fungi,Ruminococcus albus and Fibrobacter succinogenes,and the highest number of protozoa were observed with cassava residues(P < 0.05).The least abundances of bacterial phyla Firmicutes,Bacteroidetes and genus Prevotella,and substantially higher abundance of phylum proteobacteria(56%) and genus Succinivibrio(52%) were observed with cassava residues.The most abundances of Methanobrevibacter gottschalkii and Entodinium were observed with cassava residues.Spearman's correlations analysis showed,Succinivibrio had strong positive correlations with propionate,butyrate,Metadinium and M.gottschalkii,indicating fermentation products were related to microbial community.In conclusion,incubation with cassava residues resulted in lower number of fiber degrading microbes but higher protozoal population because of its low fiber contents.The microbial community was highly altered by in vitro incubation with cassava residues,whereas remained similar for the other 3 high fiber containing substrates.

Indigestible fraction of guava fruit:Phenolic profile,colonic fermentation and effect on HT-29 cells

Guava fruit is rich in phenolic compounds(PCs),whose metabolic fate has not been explored.Non-digestible carbohydrates and PCs in whole guava(WG)and seedless guava(SG)were submitted to an in vitro colonic fermentation,followed by evaluation of the anti-proliferative activity of the fermentation extracts in HT-29 cancer cell lines.The main PCs in both samples were(+)-gallocatechin and gallic acid,while procyanidin B was the most abundant one associated with soluble indigestible fraction and quercetin predominated in the insoluble indigestible fraction.The fermentability index at 24 h was 78.84%in WG and 84.74%for SG,near to the value for raffinose used as reference,with butyric acid as the main short chain fatty acids(SCFA)produced.The greatest antiproliferative effects were observed in the SG at 12 h of fermentation and WG at 24 h of fermentation.These results allow to suggest consumption of guava fruit,either with or without seeds,as a feasible way to maintain colonic health.