Combined Mutagenesis Of Bacillus Licheniformis With Two Light Sources And Solid-state Fermentation Of Chicken Feather Powder And Okara

The unsafe food from animals caused by excessive antibiotic and other drug use has attracted worldwide attention.A large number of scientific research have shown the effectiveness of fermented feed high in polypeptides in improving animal immunity and nutrition,and thus it has become one of the attractive choices for resolving the problem of antibiotic overuse in feed.At present,several industries are using poultry feathers to make polypeptide feed for non-poultry animals.Keratinase is one of the important enzymes for converting animal feather protein into an active polypeptide.As a safe strain,Bacillus licheniformis can produce a certain amount of keratinase during fermentation.Therefore,use of B.licheniformis PWD-1 to carry out solid-state fermentation on animal feathers can produce biological feed enrich with polypeptides.However,the original strain of B.licheniformis produces low-activity enzymes,impairing the key performance parameters such as efficiency,yield,and productivity during poultry feather fermentation.Therefore,in this study,a strain of B.licheniformis with a high yield of keratinase was obtained by mutagenesis with two light sources.Then,the chicken feather powder mixed with okara was subjected to solid-state fermentation by using mutagenic strains to prepare the high-activity biological feed.The main research contents and results are as follows:(1)Mutagenesis of B.licheniformis PWD-1with two light sources for enhanced keratinase production:To improve keratinase production,two light sources,ultraviolet(UV)and laser irradiations,were applied on PWD-1strain both individually and in combination.The final combination selected was 60 min of laser irradiation,followed by 10 min of UV treatment at a distance of 4 cm that provided 94%lethality.Out of 176 isolated mutants,58 were studied,and the most keratinase-producing mutant(LU11)was finally chosen due to its greatest K:C ratio(1.43),which boosted activity 2.7-fold.The purified keratinase(65 k Da)from LU11 showed 40%yield and 1.7-fold purity,compared to the wild strain's 29%yield and 1.38-fold purity.The highest keratinase activity was observed at 55°C and p H 8,with a Z-value of 15.78°C for LU11 and 19.72°C for wild strain.In the catalytic analysis,the mutant strain's V_max and specific constant(K_cat/K_m)were 357.17 U/ml and 33.11 min^-1m M^-1,respectively,which were significantly higher than the respective wild strain's values(102.04 U/ml and 28.36 min^-1m M^-1,respectively).In summary,the mutant keratinase from LU11 outperformed the wild keratinase and can be used to degrade valuable keratin in the food,feed,medical,or cosmetic industries.(2)Optimization of the solid-state fermentation(SSF)conditions for higher keratinase and antioxidant scavenging by using the LU11 strain:The LU11 strain was subsequently cultured in solid-state fermentation(SSF)using two agro-food industrial waste products,namely chicken feather powder(CFP)and okara,to valorize them into value-added peptides.Three critical parameters were optimized using both single-factor and response surface methodology(RSM):the okara to CFP ratio was 0.7(7:10),the inoculum size was 15%,and the time was 90 h for peptide,keratinase,and DPPH scavenging activity.Under the optimum conditions,the activity of keratinase and antioxidant scavenging were 910.12 U/g and 85.03%,respectively,while peptide content was 185.99 mg/g,which was 5.05%higher in comparison with the peptide yield by the wild strain.SEM and EDX analyses were used to determine the SSF effect caused by wild-type and mutant strains on substrates.The percent crystallinity index(%Cr I)of the composite substrates increased to mutant and wild strain 31.24 and 18.26%compared to the control.Principal component analysis(PCA)of the mutant strain data set revealed a 99.17%PC score,demonstrating the efficacy of the SSF condition utilizing mutant strain.These results and characterization investigations demonstrated the mutant strain's superiority to the wild strain for peptide production during SSF using CFP and okara composite substrates.(3)Low-intensity magnetic field(MF)assisted SSF(MSSF)for higher keratinase activity and peptide production:To further improve peptide production and keratinase activity,a low-intensity magnetic field(MF)was employed during SSF with LU11.Initially,the magnetic field-assisted SSF(MSSF)conditions were optimized by one factor at a time approach,which provided the optimized conditions as the number of treatments 3 at every 24 h(24,48,and 72 h)with 4 h holding time at 120 Gs of magnetic intensity(m I).Under these optimum conditions,keratinase activity and peptide production increased by 10.31%and 13.77%,respectively.Further,in order to determine the effect of MSSF on the keratin as the main substrate,MSSF experiments were also done under m I different conditions(40,80,120,and 160 Gs),and keratin was extracted after MSSF to analyze its structural changes.In micrograph analysis by SEM,the keratin surface appeared loose and porous as the m I increased,and Na was detected as an additional element at80,120,and 160 Gs m I,whereas it was absent at 40 Gs and control.Similarly,aggregated keratin collapses with increasing m I to generate needle-like protrusions,as indicated by AFM nanomaterial analysis.At 120 Gs,the root means square of keratin surface roughness(Rq)increased to 1.41%,whereas?-and?-keratin surface roughness followed a dropping and increasing trend,respectively,which later enhanced antioxidant activity.In comparison to the control,the rate of-OH,DPPH scavenging,and Fe²⁺chelation increased by 26.98,14.60,and32.72%,respectively.(4)Feed quality compositions analysis of the different fermentation strategies:In the final study,the feed quality of MSSF-treated okara-CFP composite samples was determined and compared with individual substrates(CFP and okara);mixed substrates without inoculum were considered as control.Optimized mixed substrates with or without inoculum of LU11(hereafter,mixed11 and mixed C,respectively)were used,while individual CFP and okara were treated with LU11(hereafter,CFP11 and okara11,respectively).Total amino acids contents were found to be 84.29%in mixed11,59.29%in mixed C,18.67%in okara11,and 49.72%in CFP11.The methionine contents(an important amino acid for feed)in the raw CFP and okara were 0.49 and 0.21%,respectively,while mixed11 showed 1.09%methionine.Another sulfur-containing amino acid(SCAA),cysteine,in raw CFP and okara were 8.43%and 0.05%,respectively,which increased to9.25%and 0.13%after MSSF with LU11,whereas this amino acid content was 6.14%in mixed C and 7.04%in mixed11.However,the presence of toxic nitrogen in amino acids might contribute to the catabolism of some malodorous volatile molecules,which were analyzed by GC-MS.It was found that the number of volatile compounds in Mixed11 was higher,but the quantity was lower than those of other samples.In mixed11,a total of 9 groups of volatile compounds were found together with some other miscellaneous compounds.The aldehyde group was absent in mixed C and CFP11,while,in okara11,the alcohol group was also absent in addition to the aldehyde group.In summary,the mixed11,with its balanced amino acids and low volatile compounds,could be considered an excellent feed for non-poultry livestock feed.