Study On The Isolation And Identification Of Pichia Anomala And Its Effect On Growth And Health Of Weaned Piglets

This study was to isolate and identify a Pichia anonmala?P.anonmala?AR₂₀₁₆from solid distiller's yeast of Anren county,Hunan province.Furthermore,we studied its biochemical characteristics,optimum culture conditions and safety evaluation.At the same time,this experiment was investigated the effects of supplementing diet with P.anonmala AR₂₀₁₆016 on growth and health of weaned piglets.The research content is divided into the following three experiments:In experiment one,we isolated and identified a P.anonmala from solid distiller's yeast by traditional taxonomic and molecular biological methods,and its crude protein,amino acid and the killer toxin were determined.And the culture conditions were optimized.The results shown that:?1?Using 26S rDNA analysis,a high protein strain was identified as P.anonmala and named as AR₂₀₁₆.?2?The strain AR₂₀₁₆016 contained59.56%crude protein and 17 amino acids.The optimum culture condition is:initial pH5.0,28oC,shaking speed of 180 r/min.In experiment two,the effectiveness of P.anonmala AR₂₀₁₆016 was evaluated by measuring its ability to heat tolerance,bile salt tolerance,artificial gastric and intestinal juice tolerance.To evaluate the safety of P.anonmala AR₂₀₁₆,firstly,a total of 20Kunming mouse were randomly assigned to 2 groups?10 in each group?,half male and half female.The 7-day oral toxicity test was performed at a dose of 20 mL/kg body weight,control group supplemented with normal saline,and experiment group supplemented with 2×10⁹ cfu/mL P.anonmala AR₂₀₁₆.Secondly,a total of 40 Kunming mouse,randomly assigned to one of four groups?10 in each group?,half male and half female.The 28-day oral toxicity test was performed at a dose of 20 mL/kg body weight,control group supplemented with normal saline,low-dose group,middle-dose group,high-dose group supplemented with P.anonmala AR₂₀₁₆016 strain at concentration of2×10⁷,2×10⁸,2×10⁹ cfu/mL,respectively.The results shown that:?1?In the resistance testing,the survival rate of P.anonmala AR₂₀₁₆016 reached 88.90%after 0.5 h treatment at45oC and under the high salt concentration of 0.5%stilled alive.In the artificial intestinal juice experiment,the living bacterium number of 6.0,8.0 and 10 h increased significantly,comparing with 0 h?P<0.05?,viable count increased with increasing time.?2?Compared with the control group,there was no obvious poisoning and no death in the test mice,which were classified as“non-toxic”?MTD>20 g/kg?,and the serum level of interferon-?was significantly increased in the high-dose group?P<0.05?.In experiment three,a total of 30 28-d Rongchang ternary(?Large White×Landrace×Rongchang?weaned piglets?7.48±0.22 kg?were randomly assigned into three groups,each group had 10 replicates and with 1 piglets in it.Piglets in control group supplemented with basal diets?CK?,piglets in Candida utilis group supplemented diet with the 1×10⁹ cfu/mL C.utilis?C.utilis?,piglets in P.anonmala group supplemented diet with the 1×10⁹ cfu/mL P.anomala?P.anomala?,at a dose of1 mL each piglet for every time,and the experiment lasted 28 d.The results shown that:?1?Supplementing diet with C.utilis or P.anomala increased final weight,average daily gain and average daily intake significantly,meanwhile significantly decreased diarrhea rate of weaned piglets?P<0.05?,the effect of P.anomala wasbetter than C.utilis?P<0.05?.?2?Compared with CK group,the glucose and total protein concentration,total superoxide dismutase,glutathione peroxidase and total antioxidative capacity activity in serum of piglets fed C.utilis or P.anomala were increasedsignificantly,whereasaspartateaminotransferaseactivity,alanine aminotransferaseactivity,malondialdehyde,D-lactic acid and endotoxin content were decreased significantly?P<0.05?,the effect of P.anomala was slightly better than C.utilis.?3?The trypsin,amylase and lipase activity in jejunal contents in C.utilis group and P.anomala group were significantly higher than in CK group,and the activity of trypsin and lipase in ileal contents were significantly higher than CK group.?4?The villus height and villus height/crypt depth of ileum of P.anomala group were significantly higher than CK group and C.utilis group?P<0.05?.?5?C.utilis group and P.anomala group significantly increased the protein expression level of occludin and zonula occluens-1 of ileum mucosa?P<0.05?,P.anomala group was slightly better than P.anomala group.?6?Compared with CK group,C.utilis group and P.anomala group increased mRNA expression level of ALP and TNF-?of jejunum and ileum mucosa,and reduced mRNA expression level of APN of ileum mucosa,significantly?P<0.05?.The relative expression of TLR-2 mRNA in jejunal mucosa was increased significantly in the P.anomala group?P<0.05?.?7?At the phylum level,the relative abundance of Bacteroidetes and Actinobacteria in P.anomala group were increased significantly,and Proteobacteria were decreased significantly.The relative abundance of Actinobacteria in C.utilis group was increased significantly compared with CK?P<0.05?.And there was no significant difference in Firmicutes among 3 groups?P>0.05?.At the family level,The relative abundance of Succinivibrionaceae,Lactobacillaceae and Lachnospiraceae in P.anomala group were increased significantly,whereas Clostridiaceae,Campylobacteraceae and Vibrionaceae decreased significantly?P<0.05?.The relative abundance of Lachnospiraceae and Lactobacillaceae in C.utilis group was increased significantly,Vibrionaceae and Clostridiaceae was decreased significantly?P<0.05?.At the genus level,the relative abundance of Prevotellaceae in C.utilis group and P.anomala group were increased significantly,Bacillus and Pseudon were decreased significantly?P<0.05?.To sum up,P.anomala AR₂₀₁₆016 could tolerate high temperature,high acidity and high bile salts environment,meanwhile high survivability in the artificial gastric intestinal juice environment,and high safety.Supplementing diet with C.utilis or P.anomala can significantly improve the growth performance and digestive enzyme activity,reduce diarrhea rate,enhance intestinal barrier function and improve microflora abundance of weaned piglets,the effect of P.anomala was better than C.utilis.