Technological Parameters Of Rapeseed Meal Fermentation With Enzymolysis And Its Evaluation Of Nutritional Value For Pigs

Rapeseed meal（RSM）is an ideal protein source for animal feed for its relatively high content of crude protein（35-45%）and balanced amino acid composition.However,the high level of anti-nutritional substances such as cellulose（8.1-9%）and glucosinolate（4-6%）in RSM has led to the limited application of RSM in monogastric animals.Microbial fermentation and enzymolysis are commonly used in production to improve the nutritional value of RSM.However,the nutritional value of the substance is related to the fermentation and enzymolysis parameters.In this study,the fermented parameters of fermentation and enzymolysis were optimized.The chemical composition and physicochemical changes of RSM before and after treatment were compared.Then,the nutritional value of fermented rapeseed meal（FRSM）for growing pigs was evaluated.This study provides the reference and basic data for improving the utilization efficiency of RSM.Experiment 1:Optimization of parameters of fermentation and enzymolysis of rapeseed meal and physicochemical changes of fermented rapeseed mealA five-factor four-level orthogonal experiment L₁₆（4⁵）was used to investigate the effects of co-fermentation with Bacillus subtilis and enzyme complex（containing cellulase,pectinase,amylase,etc.）on the content of glucosniolates,acid soluble protein（TCA-SP）under different conditions of fermentation time,temperature,enzyme amount,inoculum amount and material-to-water ratio to determine the optimal fermentation conditions.Furthermore,the physicochemical properties and microbial composition before and after fermentation were compared.The results were as follows:（1）The amount of bacterial inoculum and fermentation temperature significantly affected the content of glucosinolates;the amount of enzyme addition and inoculum,fermentation time,temperature and fermentation material-to-water ratio significantly affected the contents of TCA-SP.The optimal parameters of joint treatment were:fermentation time,48 h;fermentation temperature,45℃;enzyme amount,1.25 mg/g;inoculum amount,10%;material to water ratio,1:0.9.（2）Under optimal conditions,compared to the RSM,the contents of CP,TCA-SP and EE contents of FRSM were increased（P<0.05）by 13.71%,247.47%and 8.94%,respectively;the contents of glucosinolates,crude fiber（CF）,netural detergent fiber（NDF）and acid detergent fiber（ADF）decreased（P<0.05）by 50.43%,40.06%,25.98%and 21.22%;the contents of Val,Tyr,Phe,Cys and Met increased（P<0.05）by 7.3%,17.35%,6.8%,83.3%and 19.15%.（3）Microstructure of RSM and FRSM were different.Unfermented RSM was mainly observed a smooth surface and a tightly structure,while FRSM showed a rough and porous surface.（4）By FTIR,the untreated RSM had absorption peaks in the protein marker band(1600-1700 cm^-1)and the polysaccharide marker band at 3440 cm^-1and 1080 cm^-1.Compared to RSM,joint treatment with Bacillus subtilis and enzyme complexes increased the FTIR spectrum absorption peaks at 1660 cm^-1,3440 cm^-1 and 1080 cm^-1.The quantitative analysis of FTIR revealed that the secondary structure of the FRSM protein was changed.Compared to RSM,the ratio ofα-helix andα-helix toβ-sheet ratio in FRSM increased（P<0.05）by 32.15%and 34.10%,and theβ-turn content decreased by 6.84%（P<0.05）.（5）Microbial composition changes between RSM and FRSM were observed by16s RNA sequence analysis.The Chao＿1,Dominance and OTU index in FRSM showed a decreasing trend.In contrast,the Simpson index was higher than that of raw RSM,indicating that the microbial community in the FRSM tends to be uniform.Fimictures（80.37%）were the main microogranisms in the RSM,followed by Bacteroidota and Proteobacteria.Compared with RSM,the abundance of Fimicutes in FRSM increased to 89.64%,while the abundance of Bacteroidota and Proteobacteria decreased to 0.7%and 3.7%,respectively.The same results were also observed at the genus level,with the abundance of Bacillus in FRSM increasing to87.58%while the abundance of Muribaculaceae decreasing to 0.4%.The study showed that the treatment with Bacillus subtilis fermentation and enzymolysis was effective in reducing the content of anti-nutritional factors such as glucosinolate,CF,NDF and ADF in RSM,increasing the content of CP and TCA-SP in RSM,and changing the surface morphology and the secondary structure of the protein in RSM.Experiment 2:Nutritional value of rapeseed meal by fermentation and enzymolysis for growing pigsThe aim of this study was to evaluate the nutritional value of pre-treated RSM by Bacillus subtilis and enzyme complexes for growing pigs.40 Duroc×Landrace×Yorkshire（DLY）barrows（initial body weight were 17.46±0.44 kg）were randomly assigned to 4 diets with 10 replicates per diet.Pigs were fed a nitrogen-free diet（NFD）,a corn-soybean meal diet（CSD）,a rapeseed meal diet（RSD,RSM replaces35%of the nitrogen in CSD）,and a fermented rapeseed meal diet（FRSD,FRSM replaces 35%of the nitrogen in CSD）.Each diet was supplemented with 0.4%Cr₂O₃.The experiment lasted 30 days,including a 5 d adaption period,a 21 d growing period and a 4 d metabolism period.All feces and urine were collected during the metabolic period to determine nutrient digestibility and energy values.On day 31,the blood samples were collected from the jugular vein,centrifuged and dividend to determine immunoglobulins（Ig）and hormones.After blood sampling,pigs were slaughtered,and the digesta samples from the end of the ileum were collected in a sample bag for the determination of amino acid（AA）digestibility.The results showed as follows:（1）Compared with the RSM group,the final body weight（FBW）,average daily feed intake（ADFI）and average daily gain（ADG）were significantly higher（P<0.05）,and the feed to gain ratio（F/G）was significantly lower（P<0.05）in the FRSM group.（2）The serum IgA,IgG and leptin levels in the FRSM group were significantly higher（P<0.05）than in RSM group.In contrast,the ghrelin levels were significantly lower（P<0.05）than RSM group.FRSM had no significant effect on serum glucagon-like peptide-1（GLP-1）and IgM level.（3）Compared with RSM,the total intestinal apparent digestibility（ATTD）in FRSM of CP,CF,NDF and ADF in FRSM was significantly increased（P<0.05）by11.44%,122.61%,8.56%,20.62%and 27.15%,respectively.The digestive energy（DE）and metabolic energy（ME）values were higher（P<0.05）in the FRSM（13.76MJ/kg vs 13.40 MJ/kg）than in RSM（11.20 MJ/kg vs 10.79 MJ/kg）.The apparent N digestibility,true N digestibility,N utilization and true N utilization of FRSM were significantly higher（P<0.05）than RSM,which were 83.47%,84.84%,78.45%and81.29%,respectively.（4）Compared with RSM,the apparent ileal digestibility（AID）and standard ileal digestibility（SID）of CP were significantly higher in FRSM（P<0.05）;the AID of Lys,Arg,Leu and Phe were significantly higher in FRSM than in RSM（P<0.05）;the SID of Lys,Asp,Leu and Phe in FRSM were higher（P<0.05）than RSM,which were 72.36%,72.54%,71.98%and 71.98%,respectively.In conclusion,joint treatment with Bacillus subtilis and enzyme complexes could improve the nutritional value of RSM.Under the optimal treatment conditions,fermentation and enzymolysis treatment increased the TCA-SP content and changed the secondary structure of proteins,and reduced the anti-nutritional factors such as glucosniolates,CF and NDF,there by improving nutrient digestibility,AA digestibility,energy value and nitrogen utilization.