Development Of Compound Probiotics For Pig And Its Application

This study was aimed to screen out a better strain of lactic acid bacteria that could form a good synergistic effect with Bacillus licheniformis YB-DY-1 and Saccharomyces cerevisiae P-1 preserved in our laboratory,and to develop compound probiotics.The research contained four parts,screening of probiotic lactic acid bacteria,developing compound probiotics and evaluating its feeding effect on growing pigs,pregnant sows and their offspring.They are as follows in detail.1.Screening,isolation and identification of lactic acid bacteria from pig.In order to obtain highly efficient lactic acid bacteria.Firstly,we collected health pig's intestine,then isolated lactic acid bacteria from intestine mucosa and purify them.Next,with acid and bile salt tolerance experiments,pathogenic bacteria inhibition experiment in vitro and mice safety experiment in vivo,several useful bacteria were chosen.At the end,identify those strains by phylogenetic tree of 16SrDNA gene sequence.The result showed a highly effective strain RS-018 from the ileum was successfully screened among 44 strains.The survival rate of RS-018 tolerating 0.3%pig bile salt and pH2.5was 86.21%and 85.06%,respectively.The diameter of the inhibitory circle of RS-018against Escherichia coli K88,Salmonella and Staphylococcus aureus was 21.64 mm,13.01 mm and 16.29 mm,respectively.16S rDNA sequence length of RS-018 was 1490bp and it was identified as Lactobacillus reuteri.It is safe for application through mice safety experiment in vivo.The results suggested that the screened Lactobacillus reuteri RS-018 can be used as a candidate strain for the development of porcine probiotics.2.Preparation of compound probiotics.Compound probiotics were prepared with Lactobacillus reuteri RS-018,Bacillus licheniformis YB-DY-1 and Saccharomyces cerevisiae P-1.According to viable count,conditions of co-culture and solid-state fermentation?SSF?for compound probiotics,and the method of drying process of SSF products?freeze-drying and oven-drying?were optimized.The result of optimizing co-culture conditions were as follows:firstly,inoculate 1%RS-018 and 1%P-1simultaneously,statically culture at 35?for 12 h;then inoculate 5%YB-DY-1,and culture at 37?,150 r/min for 24 h.As a result,the total viable count could reach 1.24×10⁹ cfu/ml,furthermore,each stain was over 10⁸ cfu/ml.The optimum conditions of SSF were as follows:5%incubation amount of seed liquid,60%initial moisture,cultured at36?for 36 h.As a result,the total viable count in the probiotic could reach 1.12×10¹⁰cfu/g,moreover,more than 10⁹cfu/g in each stain.The optimum drying method is freeze-drying,after drying,the total viable count in the probiotic preparation could reach6.23×10⁹cfu/g,moreover,more than 3×10⁸cfu/g in each stain.The results suggested that the compound probiotic could meet the basic requirements for feeding animals and could be further used for pigs to determine its feeding effect.3.Effects of feeding probiotics on growth performance,serum biochemical and immune indexes of growing pig.In order to evaluate the effect of compound probioticson growing pig,72 healthy binary?Yorshire×Landrace?female pigs?16.07±1.02 kg?were randomly divided into 4 groups,3 replicates of each group,each replicate with 6 pigs.The control group was fed with basal diet.0.1%,0.2%and 0.3%probiotics was supplemented in the other three experimental groups based on basal diet,respectively.The experiment period lasted for 35 days.At the end of trial,the data of body weight and food intake were collect to determine growth performance.2 pigs were randomly selected from each replicate to sample blood for serum biochemical and immunological parameters.The results showed that probiotics increased the average daily gain?ADG?and average daily feed intake?ADFI?in a dose-dependent manner.Comparing with the control group,the ADG and ADFI in 0.3%group and the serum albumin?ALB?in 0.2%and 0.3%group were significantly higher?P<0.05?.ALB content was the highest in 0.3%group,but there were no significant differences of alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,total protein?TP?,globulin?GLB?,urea nitrogen?BUN?and immunoglobulin M?IgM?in serum among groups?P>0.05?,while immunoglobulin A?IgA?andimmunoglobulin G?IgG?in serum increased in a dose-dependent manner.In addition,IgA and IgG in 0.3%group were significantly higher than those of control group?P<0.05?.The results indicated that the compound probiotics could improve the growth performance and immune function of growing pigs,and the optimum concentration is 0.3%.4.Effects of probiotics on production performance of pregnant sow and growth performance of their offspring.In order to evaluate the feeding effect of compound probiotics on pregnant sows and their offspring,24 healthy pregnant sows?pregnancy stage ranging from 81 to 85 days?were randomly divided into 4 groups,each group with6 pigs.The control group was fed with basal diet.0.1%,0.2%and 0.3%compound probiotics was supplemented in the other three experimental groups based on basal diet,respectively.On the day of delivery,the litter weight,litter size and live litter size were recorded.The piglets were weaned at 28 days of age,then collect the data of litter weight,litter size,survival rate during weaning period and their diarrhea rate in laction period.One pig was randomly selected from each litter to sample blood for determining immune parameters.Next 24 litters of weaned piglets?total 227 piglets?were fed separated from sows with the same concentration of probiotics as their mothers'.The experiment period was 28 days.At the end of the trial,the data of diarrhea rate,litter weight and feed intake were collected.The results showed that there were no significant differences of total litter size,live litter size,survive rate,individual weight and litter weight?P>0.05?of new birth piglets;during suckling period,the live litter size,survive rate,weight of weaned litter,IgM in serum and diarrhea rate?P>0.05?of sucked piglets have no significant differences,but IgA and IgG in serum increased in a dose-dependent manner.Comparing to the control group,IgA in 0.2%group and IgA and IgG in 0.3%group were significantly higher?P<0.05?.As for weaned piglets,with the supplement of probiotics,ADG and ADFI increased,while feed/gain?F/D?decreased in a dose-dependent manner.Comparing with the control group,when the supplement reached 0.2%,ADG and ADFI were significantly higher?P<0.05?,and diarrhea rate was significantly lower?P<0.05?.When the supplement concentration reached 0.3%,F/D was significantly lower than that of control group?P<0.05?.The results suggested that feeding compound probiotics didn't affect the production performance of pregnant sows,but can also improve immune function of its suckling piglets.Moreover,feeding compound probiotics could improve the growth performance of weaned piglets and decrease their diarrhea rate,and the optimum concentration was 0.3%.