High-temperature Solid-state Fermentation Of Soybean Meal And Its Strengthening Study By Low Intensity Alternating Magnetic Field

Excessive addition of antibiotics into animal feed can cause serious hidden dangers of food safety among animal-derived food products.Soybean meal（SBM）,as a by-product of edible oil industry,is much suitable for production of peptide-containing biological feed due to its superior attributes of wide sources,low cost and abundant proteins.The fermentation technology of soybean meal（SBM）has a broad development prospect.However,there are many unsolved problems which may limit the development of SBM in solid-state fermentation（SSF）industry,such as high fermentation cost,low fermentation efficiency and low intelligent level of fermentation.Thus,in order to skip the sterilization process of soybean meal fermentation,to increase the fermentation efficiency and to realize the precise control of fermentation process,the technologies of the high-temperature solid-state fermentation,the low intensity alternating magnetic field and the near-infrared real-time monitoring were studied in this work.Specific researches and corresponding conclusions are as follows:（1）Seed liquid production of Geobacillus stearothermophilus by fermentation was carried out using the protease content and biomass as evaluation indexes.The tryptone soybean medium with the highest protease yield（123.61 U/mL）was selected as the fermentation seed liquid.The optimum culture conditions of the strain optimized by orthogonal design were 55°C of incubation temperature,3%（v/v）of inoculum size,7.0 of initial pH value and 180 r/min of shaking speed.Under these conditions,compared to control group,the biomass of bacterium increased by 24.2%and reached up to 7.49×10⁸ CFU/mL.The modified Gompertz model was more suitable for fitting the growth process of Geobacillus stearothermophilus,and the correlation coefficient R² of this model was 0.9894.（2）Optimization experiment for the conditions of non-sterile high-temperature fermentation was carried out with the peptide content of fermented soybean meal as the index.Optimum fermentation conditions were as follows:50.04 h of fermentation time,55.33°C of fermentation temperature,1.34 of water to material ratio and 12.46%（v/v）of inoculum size.Under such conditions,compared to raw SBM,the contents of peptide,crude protein,soluble protein,total phenol of HFSBM increased by 148.58%,5.26%,37.47 and 42.96%,respectively,while the TI,crude fiber fat contents and pH value decreased by 77.43%,14.63%,19.42%and 22.90%,respectively.The nutritional values of SBM increased significantly.The results exhibited thatα’,αandβsubunits ofβ-conglycinin（90.5,71.5&55.2 kDa）,acid and basic subunits of glycinin（37.6&19.8 kDa）,Gly m bd 30k（30 kDa）and Kunitz TI（24 kDa）,which are food allergens with molecular weight（MW）>20 kDa,were degraded,while the contents of peptides（200-500 Da）and amino acids（AA）（<200 Da）increased significantly.Results of the pilot scale verification test using an 8 m³ rotary fermenter showed that high-temperature fermentation could achieve satisfactory results without autoclaving and pulverization steps and reduce the production cost.（3）Low intensity alternating magnetic field（MF）-assisted seed liquid fermentation and solid-state fermentation of soybean meal were determined using the biomass of Geobacillus stearothermophilus and peptide content of HFSBM as the indexes.The orthogonal test results of MF-assisted treatment conditions for seed liquid were as follows:48 h of total fermentation time,80 Gs of intensity,intervention at the 6^th h after inoculation and 2 h of treatment time.Under these conditions,the total number of colonies in seed liquid assisted by MF was 36.94%higher than that in untreated group.The orthogonal test results of MF-assisted treatment conditions for SSF were as follows:48 h of total fermentation time,80 Gs of intensity,intervention at the 14^th h after inoculation and 5 h of treatment time.Under these conditions,the peptide content of HFSBM further increased by 12.32%（reaching up to 70.86 g/kg）compared to untreated control.The observation results of transmission electron microscope（TEM）exhibited that,compared to control,MF-induced cells had normal appearances,the content of ribosome increased,and their membrane tended to be thicker after MF treatment.It increased the synthesis abilities of secretory proteins and its extracellular transport,which might be one of the main reasons of the MF-induced enhancement of peptide production of HFSBM.（4）Results of transcriptome study of Geobacillus stearothermophilus showed that 408 differential genes were found after MF treatment,containing 156up-regulated and 252 down-regulated genes.Furthermore,it was found that the expression levels of key genes involving in cell signaling,movement and membrane transport,protein and amino acid metabolisms,nucleotide metabolism and carbohydrate metabolism were changed by MF through GO and KEGG enrichment analyses.The levels of genes involved in ribosome（rplE/F/N/O/P/Q/R/V/X,rpsC/E/h/K/M and rpmD）,RNA polymerase（rpoA/B/C）and AAs（hisA/B/D/F/H/I/Z,argB/D/F,carA/B,gltD,rocA,murD and ilvC）metabolisms and syntheses were up-regulated by MF treatment,which made the nitrogen cycle of Geobacillus thermophilus cells in a relatively active status.The increase of the secretion levels of extracellular proteins and enzymes was conducive to the degradation of substrate proteins and enhancement of peptide amounts.In addition,the expressing level of holB gene（DNA polymerase IIIδsubunit）that directly involving in the DNA synthesis decreased after MF treatment,reducing the possibility of a significant increase rate in cell replication rate.Compared to the control group,only a 36%growth rate of the bacterium biomass after MF treatment might be related to this.（5）Results of proteome study of Geobacillus stearothermophilus showed that there were 25 differentially expressed proteins after MF treatment,including 12up-regulated and 13 down-regulated proteins.All differentially expressed proteins involved in biological activities of membrane proteins,protein metabolism,glycometabolism and oxidation protection.As shown in the proteome results,the up-regulated S-layer protein increased the cell adhesion and colony forming ability,making it easier for bacterium to anchor with the fermentation substrates.MF decreased the ribosome dormancy which enhanced the synthesis rates of proteins and aminopeptidase.Results of the comprehensive analysis of proteome and transcriptome showed that increased efficiency of glucose metabolism could provide more energy for cells after MF treatment,accelerating the RNA synthesis and the transport of amino acyl tRNA to the corresponding site of ribosome,improving the peptide chain extension efficiency.Accumulated proteins and aminopeptidase induced by MF in cells could be released by cell secretion and autolysis during SSF,further enhancing the proteolysis of fermentation substrate and subsequently increasing the peptide content of HFSBM.（6）The dynamic changes of peptide,crude protein and trypsin inhibitor contents in SBM were monitored in real time by a probe NIR spectrometer.The optimal synergy intervals of peptide,crude protein and TI of fermented SBM were5,604⁶,001,6,807⁷,205,8,010⁸,408 and 9,615¹0,000 cm^-1;4,000⁴,663,4,667⁵,330,6,001⁶,665 and 6,669⁷,332 cm^-1,respectively.The corresponding calibration and prediction models of peptide contained 0.9494 of R_c（correlation coefficient）and 4.45 of RMSECV（root mean square error of cross validation）,as well as 0.9570 of R_p（correlation coefficient）and 4.06 of RMSEP（root mean square error of prediction）,respectively.The corresponding calibration and prediction models of crude protein contained 0.9385 of R_c and 3.45 of RMSECV,as well as 0.9346 of R_p and 3.56 of RMSEP,respectively.The corresponding calibration and prediction models of TI contained 0.9446 of R_c and 1.13 of RMSECV,as well as 0.9492 of R_p and 1.09 of RMSEP,respectively.The real-time monitoring system of Fourier near-infrared spectroscopy combined with Si-PLS algorithm could quickly monitor the variations of the key indicators in the high-temperature SSF of SBM,which could provide information for the design of the intellectualized system for high-temperature solid-state fermentation of SBM.（7）In vivo experiments using broilers that fed with 50,100 and 150 g/kg HFSBM（HFSBM diet groups）were carried out and the basic and antibiotic diets were selected as control groups.Compared to these two groups,chickens fed with HFSBM diets had no negative effects in growth performance,but had an upward trend in the relative weights of thymus（p=0.111 and 0.156）and bursa of Fabricius（p=0.274 and 0.051）,increasing from 0.08 g/100 g to 0.11⁰.12 g/100 g body weight（BW）and from 0.09⁰.10 g/100 g to 0.10⁰.12 g/100 g BW,respectively.Birds fed with HFSBM diets also had a decline trend（p=0.112 and p=0.024）in GOT levels,respectively,decreasing from 478 and 598 U/L to 334⁴29 U/L.Furthermore,duodenal absorptive capacity improved（p=0.02&0.001 and p=0.052&0.027）,VH and V/C also increased from 1,358 and 1,430μm to 1,508¹,512μm and from 7.69and 7.99 to 9.03¹0.54,respectively.Meanwhile,a significant increase in the number（from 4.35 and 4.24 to 5.61⁶.63 log CFU/mL）of Bacillus subtilis in jejunum was observed.Addition of HFSBM instead of SBM in broiler diets could not only reduce the amount of antibiotics,but also enhance the immune system,protect liver cells,promote intestinal absorption and improve intestinal microorganisms.