| Distillers’ grains are a byproduct of distilling process.Guizhou has a rich brewing industry and huge production of distillers’ grains,which are rich in nutrients and are a good alternative to feeding livestock and poultry with concentrate.However,the high cellulose content of distillers’ grains makes it difficult for animals to digest and absorb,and the high moisture content easily leads to mold and decay,which is not suitable for long-term storage,limiting the reuse of distillers’ grains resources.Probiotics can ferment and utilize cellulose,produce various enzymes and play competitive antagonism,etc.to improve cellulose feed quality,inhibit mold growth,extend shelf life and improve feed utilization,thus promoting animal growth and enhancing animal immunity.At present,domestic and foreign research on the use of probiotic fermentation of distillers’ grains is mostly focused on the fermentation process and the promotion of livestock growth,but less attention is paid to the fermentation of distiller’s grains itself and livestock gastrointestinal flora structure and immunity,thus limiting the resource exploitation of distillers’ grains.Therefore,the present study was conducted to optimize the conditions of fermented sauce-flavor distillers’ grains of Maotai(hereinafter referred to as distillers’ grains),to investigate the dynamics of the change in the bacterial flora structure of fermented distillers’ grains with compound probiotics,and to analyze the effects of feeding fermented distillers’ grains with compound probiotics on the average daily weight gain,immunity-related indexes and rumen flora structure of finishing cattle,in order to provide some theoretical basis for the promotion and application of distillers’ grains feedification in ruminant farming.1.Optimization of conditions for solid fermentation of distillers’ grains by compound probiotic bacteriaIn order to determine the optimal conditions for solid-state fermentation of distiller’s grains by probiotics bacteria,four probiotic bacteria,including Aspergillus niger,Enterococcus faecalis,Lactobacillus plantarum and Saccharomyces cerevisiae were selected in this experiment,and the antagonistic relationship analysis was performed by the cross-hatch method,and the base material with different proportions of corn flour,bran and rapeseed meal added to distiller’s grains as the substrate was prepared,The inoculum amount,initial p H value,fermentation time and fermentation temperature of the distiller’s grains fermented by the compound probiotics bacteria were screened by single-factor test and orthogonal test,using crude protein,viable count,neutral detergent fiber and acid detergent fiber content of the distiller’s grains fermented by the compound probiotics bacteria as the judgment criteria,and the results showed that:(1)There were no antagonistic effects of the four probiotic strains.(2)The optimal fermentation base combination of compound probiotics for fermentation of distiller’s grains is 77% distiller’s grains+ 6% corn meal + 5% bran + 12%rapeseed meal.(3)The optimal fermentation conditions were 8%(V/m)inoculum of compound probiotics bacteria,initial p H 6 and fermentation time of 5 d.(4)Compared with unfermented distiller’s grains,the crude protein content of distiller’s grains increased by34.46%,neutral detergent fiber content decreased by 35.28%,acid detergent fiber content decreased by 54.02%,and viable count was 9.45 lg(CFU/g)after fermentation with compound probiotics;meanwhile,the antinutritional factor tannase activity of basal rapeseed meal increased by 23.07%,thioglucoside and phytic acid content decreased by 22.92% and22.19%,respectively.These results showed that optimized fermentation with compound probiotics could effectively improve the quality of distiller’s grains and reduce the content of anti-nutritional factors in the base material rapeseed meal.2.Analysis of the structural changes of bacterial flora in distiller’s grains after fermentation with compound probioticsIn order to understand the changes in the structure of distiller’s grains fermented with compound probiotics,this experiment established pure distiller’s grains control group(i.e.KB group),distiller’s grains base material group(i.e.DZ group)and probiotic fermentation group(0d,1d,3d,5d,15 d,30d and 60 d fermentation respectively,i.e.ZJ0,ZJ1,ZJ3,ZJ5,ZJ15,ZJ30 and ZJ60 groups).The probiotic fermentation groups were fermented with compound probiotic tonne bags under optimal conditions,and samples were collected for different fermentation time periods,and the amount of live bacteria of Lactobacillus,Saccharomyces and Aspergillus niger was determined by plate counting method,and the structural changes of bacterial flora were analyzed by 16 S r DNA high-throughput sequencing method,and the results showed that:(1)Compared with KB,CK and JZ0 groups,the number of viable Lactobacillus and Saccharomyces in JZ1～JZ15 groups showed an increasing trend,JZ15 and JZ30 groups remained basically stable,and JZ60 group showed a decreasing trend;compared with KB and CK groups,the number of viable Aspergillus niger in JZ0 and JZ1 groups showed an increasing trend,while JZ1 and JZ3 groups decreased,and no viable bacteria were seen by ZJ5,ZJ15,ZJ30 and ZJ60 groups.(2)The distiller’s grains samples collected for sequencing all met the quality control requirements,and the effective sequences were all maintained at about 70 000～80 000 tages,meeting the requirements for subsequent analysis.(3)Compared with KB and CK groups,Chao1,ACE and Shannon index were significantly lower(P < 0.05)in JZ5,JZ15,JZ30 and JZ60 groups,and remained basically stable from d 5to 60 of fermentation,suggesting that the community species richness and the degree of species diversity were reduced in the fermented distiller’s grains by compound probiotics.(4)Compared with the KB,CK and JZ0 groups,the relative abundance of Firmicutes in the JZ5,JZ15,JZ30 and JZ60 groups was significantly higher(P < 0.05);the relative abundance of Proteobacteria,Bacteroidota and Actinobacteriota were significantly lower(P < 0.05)and remained basically stable from d 5 to 60 of fermentation.(5)Compared with the KB and CK groups,the relative abundance of Lactobacillus was significantly higher(P < 0.05)in the JZ5,JZ15,JZ30 and JZ60 groups,and The relative abundance of Muribaculaceae,Acetobacter,Lachnospiraceae＿NK4A136＿group and Helicobacter all decreased significantly(P < 0.05),and basically remained stable from d 5 to 60 of fermentation.The results showed that with the extension of the fermentation time,the live counts of Lactobacillus and Saccharomyces in the compound probiotics fermented distiller’s grains remained basically stable in the 15th-30 th d of fermentation,and the late stage of fermentation was not adapted to Aspergillus niger growth.may be related to the p H drop and anoxic environment in the late fermentation period;The community species richness and species diversity of compound probiotics fermented distiller’s grains decreased,the relative abundance of beneficial bacteria(Firmicutes and Lactobacillus)increased,and the relative abundance of harmful bacteria(Proteobacteria,Acetobacter and Helicobacter)decreased,and the community structure remained basically stable at d 5～60.3.Effect of feeding compound probiotic fermented distiller’s grains on daily weight gain and immune-related indexes of finishing cattleTo clarify the effect of feeding compound probiotic fermented distiller’s grains on daily weight gain and related immune indexes of fattening cattle.In this experiment,30 healthy Simmental crossbred fattening bulls(local yellow cattle × Simmental cattle;265.0 ± 22.48 kg)were selected and randomly divided into three groups,the basal diet group(Control),15 %fermented distiller ’s grains group(PFDG-15 %)and 30 % fermented distiller ’s grains group(PFDG-30 %).The distiller ’s grains added therein were selected from those fermented for 5d with compound probiotics.On the 0 th and 30 th day of feeding,the average daily gain was calculated by weighing before morning feeding.Blood samples of 4 finishing cattle were randomly collected from each group before morning feeding on the 45 th day.Serum antioxidant indexes and serum immune indexes were determined by micro method and ELISA.and the results showed that:(1)Compared to the Control group,the average daily weight gain of cattle in the PFDG-15% group increased but was not statistically significant(P > 0.05),while it was significantly lower in the PFDG-30% group(P < 0.05).(2)Compared with the Control group,the bovine serum Malondialdehyde levels were significantly lower in both the PFDG-15% and PFDG-30% groups(P < 0.05),and the bovine serum Superoxide dismutase levels were significantly increased in the PFDG-30% group(P < 0.05).(3)Compared with the Control group,the levels of bovine serum Immunoglobulin G,were significantly higher in both the PFDG-15% and PFDG-30% groups(P < 0.05),while the levels of bovine serum anti-inflammatory factor Interleukin-10 were highly significant lower in the PFDG-30% group(P < 0.01).The results showed that feeding 15% compound probiotic fermented distiller’s grains improved the antioxidant capacity and immunity status of finishing cattle to some extent,while reducing the average daily weight gain and anti-inflammatory capacity of finishing cattle when added at 30%.4.Effect of feeding compound probiotic fermented distiller’s grains on rumen flora structure of finishing cattleIn order to explore the effects of fermented distiller’s grains fermented by compound probiotics on enzyme activity and microflora structure in rumen fluid of finishing cattle,the rumen fluid of finishing cattle was randomly selected before morning feeding on the 45 th day of feeding,with 4 cattle in each group.The enzyme activity and microflora structure of rumen fluid of finishing cattle were determined by ELISA and 16 S r DNA high-throughput sequencing,respectively.and the results showed that:(1)The cellulase,lipase and protease activities in the rumen of cattle in the PFDG-15% and PFDG-30% groups were highly significantly increased compared to the Control group(P < 0.01).(2)The rumen fluid samples collected for sequencing all met the quality control requirements,and the valid sequences were basically kept above 50,000 tages,meeting the requirements for subsequent analysis.(3)Compared with the Control group,there were no statistical differences in Chao1,ACE,Shannon and Simpson indices of rumen bacterial community in PFDG-15% and PFDG-30%groups(P > 0.05),suggesting that feeding compound probiotic fermented distiller’s grains had little effect on rumen bacterial diversity and abundance in finishing cattle.(4)Compared with Control group,there was no significant difference between PFDG-15% and PFDG-30% group bacterial species at the gate level(P > 0.05),suggesting that feeding compound probiotic fermented distiller’s grains had little effect on the rumen bacterial gate of finishing cattle.(5)Compared with the Control group,the relative abundance of the dominant genus F082 was significantly higher(P < 0.05)and Selenomonas was significantly lower(P < 0.05)at the genus level in the PFDG-15% and PFDG-30% groups;the relative abundance of Lachnospiraceae＿UGG-009 showed an increasing trend,with a significant difference(P <0.05)in the PFDG-30% group was significantly different(P < 0.05).The relative abundance of genus p＿251＿o5 was significantly higher(P < 0.05)and the relative abundance of NED5E9 was significantly lower(P < 0.05)in the PFDG-15% group;the relative abundance of Prevotellaceae＿UGG-001,probable＿genus＿10 and Lachnoclostridium was significantly higher(P < 0.05)in the PFDG-30% group.(6)Correlation analysis showed significant correlations(P<0.05)between mean daily weight gain,serum antioxidant capacity,immunity and rumen enzyme activity and changes in rumen bacterial genus in finishing cattle.The results showed that feeding compound probiotic fermented distiller’s grains did not affect the diversity and abundance of rumen bacteria in finishing cattle,but increased the enzyme activity,increased the relative abundance of fatty acid synthesis flora(F082)and cellulolytic flora(Lachnospiraceae＿UGG-009),and decreased the relative abundance of disease-related flora(Selenomonas)in the rumen of finishing cattle.In summary,this study optimized the fermentation conditions of the compound probiotics distiller’s grains,which effectively improved the nutritional value of distiller’s grains;Compound probiotic fermentation can increase the relative abundance of beneficial bacteria and decrease the relative abundance of harmful bacteria on distiller’s grains;Feeding compound probiotic fermented distiller’s grains can,to a certain extent,improve rumen enzyme activity,antioxidant capacity and immunity status of finishing cattle,increase the relative abundance of fatty acid synthesis flora and cellulolytic flora of finishing cattle,and reduce the relative abundance of disease-related flora. |
PDF Full Text Request