Research Of Novel Lactic Acid Bacteria Fermentation Feed On Pork Quality And Its Mechanism

Pork is the most important meat source in China.With the improvement of people's living standard,the public's demand for meat quality is increasing day by day.Additionally,as a limiting factor in the quality and acceptability of meat products,oxidation plays a pivotal role in modulating the color,taste,and nutritional value of meat.Therefore,it is very necessary to adopt effective nutrition regulation in the process of breeding,so as to maintain appropriate content of intramuscular fat and fatty acid in pork and prevent muscle from lipid oxidation.On the other hand,there are anti-nutrient factors in the diet,which affect the utilization rate of feed raw materials.Studies have shown that the content of anti-nutrient factors decreases and small molecules such as small peptides increase in the feed after fermentation by probiotics.Moreover,the feed after fermentation are rich in probiotics and their metabolites,which is more conducive to improving the health level of animal bodies.The purpose of this study was to screen lactic acid bacteria with antioxidant capacity,and then combine with Bacillus to ferment corn and soybean meal compound feed to explore its effects on growth performance,meat quality and flavor substances of finishing pigs.On the basis of clarifying the effect of FMF on improving the meat,the changes of nutrient composition of the feed before and after fermentation by microorganisms were revealed firstly.Furthermore,the antioxidant capacity of LAB in vivo was evaluated through animal experiments,and the possible mechanism of improving meat quality in FMF was analyzed,so as to provide theoretical basis for the regulation of pork quality by means of nutrition.The main results are as follows:1.Screening of lactic acid bacteria with antioxidant capacity in vitroThe purpose of this study was to screen lactic acid bacteria with excellent antioxidant capacity through preliminary screening of hydrogen peroxide tolerance and re-screening of free radical scavenging capacity.(1)Four lactic acid bacteria had strong hydrogen peroxide tolerance,showing that the growth trend was almost not affected by the concentration of hydrogen peroxide in the medium.Based on the scavenging ability of superoxide anion free radical,DPPH free radical,hydroxyl free radical and,ZJUAF-4 was selected as the lactic acid bacteria with high antioxidant capacity.(2)Further analysis of antioxidant capacity showed that ZJUAF-4 had higher antioxidant enzyme activity and could produce vitamin E,vitamin C,glutathione.(3)Physiological and biochemical analysis and 16S rRNA identification showed that ZJUAF-4 was Pediococcus pentosaceus.2.Study on the preparation of ZJUAF-4 combined with Bacillus subtilis BS12 fermentation feed and its application in finishing pigsThe feed was fermented by Pediococcus pentosaceus ZJUAF-4 and Bacillus BS12 to explore the application of the fermented mixed feed(FMF)in finishing pigs.In experiment 1,600 finishing pigs(Duroc × Large white × Yorkshire)with an average initial weight of 90.53±0.75 kg were randomly divided into 3 treatment groups.Each treatment consisted of 4 replicates of 50 pigs(half male and half female).Treatment 1 was the basal diet(control group);5%replacement of protein from rice bran in the basal diet by protein from FMF was treatment 2;10%replacement of protein from rice bran and rice bran meal in the basal diet by protein from FMF was treatment 3.The test period was 39 days.At the end of the test,the growth performance of each group was evaluated,and 5 finishing pigs with an average weight of 124.50±2.15kg were randomly selected from each group for slaughter sampling.In order to verify the application effect of FMF,the second experiment was carried out.In experiment 2,400 finishing pigs(Duroc × Large white × Yorkshire)with an average initial weight of 89.54 ±0.61 kg were randomly divided into 4 treatment groups.Each treatment consisted of 4 replicates with 25 pigs(the ratio of male to female is basically the same)each replicate.Treatment 1 was the basal diet(control group);2%replacement of protein from rice bran in the basal diet by protein from FMF was treatment 2;4%replacement of protein from rice bran in the basal diet by protein from FMF was treatment 3;8%replacement of protein from rice bran and rice bran meal in the basal diet by protein from FMF was treatment 4.The test period was 35 days.At the end of the test,the growth performance of each group was evaluated.At the same time,12 finishing pigs with an average weight of 120.52 ± 0.33 kg were randomly selected from each group for slaughter sampling.The results showed that(1)dietary supplementation with FMF significantly increased the ADG of the finishing pigs(P<0.05),reduced the ratio of feed to weight(P<0.05),significantly increased the apparent digestibility of crude protein,dry matter,crude fat,phosphorus and amino acid in the feces(P<0.05),and then improved the growth performance of the pigs.(2)Compared with the control group,dietary supplementation with FMF significantly improved the meat color,tenderness,water-holding capacity,and intramuscular fat content of the finishing pigs(P<0.05),thus improving the meat quality.(3)According to the analysis of flavor substances,adding FMF in the diet significantly increased the content of unsaturated fatty acids(C20:4 ?-6,C18:2 ?-6c,C18:1 ?-9c,C18:3?-3 and C20:2)in pork(P<0.05).The contents of flavour amino acids(glutamic acid,aspartic acid,threonine,serine,alanine,glycine)and essential amino acids(phenylalanine,leucine,methionine,valine,lysine)in pork were significantly increased(P<0.05).The content of alcohol substances(isooctyl alcohol,nonyl alcohol),aldehyde substances(nonaldehyde,octanal,heptanal,hexanal,benzaldehyde)and ketone substances(acetophenone,3-hydroxy-2-butanone,methyl heptenone)in pork were significantly increased(P<0.05)to improve pork flavor.(4)Antioxidant index detection indicated that the activities of GSH-Px,CAT and SOD in the serum of the finishing pigs were significantly increased,and the contents of MDA were decreased in the FMF group(P<0.05).Additionally,the activities of SOD and GSH-Px in the muscle were increased in the FMF group(P<0.05),indicating that FMF could improve the antioxidant levels of the serum and muscle of the finishing pigs.3.Analysis of nutrient composition changes of fermented mixed feed with ZJUAF-4 and Bacillus BS12The changes of nutrient composition of the feed before and after fermentation were investigated by using the co-fermentation feed of ZJUAF-4 and BS12.The results showed that(1)fermentation significantly reduced the content of ?-Conglycinin,Glycinin,acid detergent fiber and neutral detergent fiber in the fermented mixed feed(FMF)(P<0.05).The contents of acid soluble protein,essential amino acids(lysine,leucine,histidine,phenylalanine,isoleucine,threonine,valine)and unsaturated fatty acids(C20:1,C18:3?-3,C18:2?-6 and C18:1?-9)in the FMF were significantly increased(P<0.05).(2)After fermentation,the FMF was rich in probiotics(ZJUAF-4 and BS12),and fermentation significantly increased the content of organic acids(propionic acid,formic acid,butyric acid and acetic acid)in the FMF(P<0.05),reduced the pH value of the FMF,and improved the acidity of the FMF.(3)Antioxidant capacity analysis showed that fermentation significantly increased the content of antioxidant substances(glutathione,carnosine,folic acid)and the activity of antioxidant enzymes(SOD,GSH-Px)in the FMF(P<0.05).(4)In vitro digestibility test results showed that fermentation significantly improved the in vitro digestibility of amino acids in the FMF(P<0.05),which may be related to the degradation of antigen proteins.4.Study on the effect and mechanism of ZJUAF-4 on Diquat-induced intestinal oxidative stress in miceDiquat was used to construct a mouse intestinal oxidative stress model,and vitamin C was used as a positive control to explore the influence of ZJUAF-4 on intestinal barrier oxidative stress and the changes of intestinal flora in mice.(1)Diquat-induced oxidative stress significantly increased intestinal permeability to FD4,serum DLA content and DAO activity in mice(P<0.05),indicating that intestinal oxidative stress caused serious damage to intestinal barrier.ZJUAF-4 pretreatment significantly inhibited the increase of serum FD4,DLA and DAO levels(P<0.05),and relieved the oxidative damage of intestinal barrier.(2)Intestinal morphology analysis of mice showed that the jejunal villi and microvilli of Diquat-treated mice were damaged,while ZJUAF-4 pretreatment could improve the morphology of the villi and microvilli.(3)Intestinal oxidative stress significantly reduced the SOD activity and increased the MDA concentration in serum and jejunum of mice(P<0.05),while ZJUAF-4 pretreatment significantly inhibited the increase of SOD level and decreased MDA level in serum and jejunum of mice(P<0.05).The changes of ROS content in the jejunum of mice also showed a similar trend,which indicated that ZJUAF-4 pretreatment significantly inhibited the increase of ROS content in the jejunum of mice caused by Diquat(P<0.05).(4)Western blot analysis showed that the expression of Nrf2 and its downstream antioxidant proteins NQO1 and HO-1 in jejunum was significantly reduced(P<0.05)in the Diquat group,while ZJUAF-4 pretreatment significantly inhibited the reduction of Nrf2 and its downstream antioxidant proteins NQO1 and HO-1(P<0.05).(5)Diquat improved the the abundance of Bacteroidetes and decreased the abundance of Firmicutes at the phylum level,and improved the the abundance of Bacteroides and Helicobacter and decreased the abundance of Pediococcus,Enterococcus and Dubosiella at the phylum level.Whereas ZJUAF-4 pretreatment reversed these changes in microbiota,which indicated that ZJUAF-4 pretreatment improved the abundance of Firmicutes and decreased the abundance of Bacteroidetes at the phylum level,and improved the the abundance of Pediococcus,Enterococcus and Dubosiella and decreased the abundance of Bacteroides and Helicobacter at the phylum level under Diquat stimulation.In summary,this study screened and obtained a lactic acid bacteria ZJUAF-4 with excellent antioxidant capacity.Furthermore,study of the application effect of the feed fermented with ZJUAF-4 and BS12 on finishing pigs indicated that FMF could improve the growth performance of pigs.Dietary supplementation with FMF significantly increased the meat color,water power,tenderness,intramuscular fat content,and then improve meat quality.The accumulation of unsaturated fatty acids,flavour amino acid,essential amino acids,volatile flavor substances,such as alcohols,aldehydes and ketones was increased in finishing pigs fed FMF.Adding FMF in the diet significantly increased the antioxidant levels of serum and muscle in finishing pigs.On the basis of clarifying the effect of FMF on improving the meat quality,the changes of nutrient composition of the feed before and after microbial fermentation were further revealed.The result indicated that the FMF with ZJUAF-4 and BS12 contributed to improving the content of small molecular nutrients in the feed,such as acid soluble protein,amino acid and fatty acid.And the probiotics,organic acid and antioxidant capacity were also increased in the FMF.Subsequently,the oxidative stress model was used to evaluate the effect and mechanism of ZJUAF-4 on Diquat-induced intestinal oxidative stress in mice,and the results showed that ZJUAF-4 alleviated Diquat-induced oxidative stress by activating Nrf2 pathway and restoring the intestinal flora in mice.The results indicated that the FMF,rich in ZJUAF-4,had strong antioxidant capacity and could improve the meat quality and flavor of finishing pigs.