The Preparation Of Tryptone From Hermetia Illucens L.larvae

The black soldier fly,Hermetia illucens L.,is a kind of diptera stratiomyiidae Hermetia,larvae is saprophytic.Black soldier fly is strong resistance,wide adaptabilit and easy breeding.Its larvae contains high oil and protein,so many researchers use the larva for antimicrobial peptide extraction,preparation of chitosan,skin caring products,the development of biodiesel conversion and so on.Therefore,the black soldier fly is extensive farming in south China and other areas,and has good prospects for market development.Peptone is a kind of water soluble mixture which is made up of proteose,peptone,peptide,amino acid which is hydrolysised by plant,animal and vegetable protein by enzyme.After Naegeli applied to bacterial culture and obtain good training effect for the first time in 1880,peptone has gradually become the main source of nitrogen nutrition microbial culture,isolation and identification,as well as an integral part of medicine,biochemical reagent,immune detection and fermentation research.In this paper Hermetia illucens L.larvae was treated as raw material,processed by drying,crushing,deoiling by way of enzymatic hydrolysis.Hermetia illucens L.was degreased by Soxhlet extraction method,and determine the optimal extraction of organic reagent and extraction time.Taking the content of amino nitrogen as the index,the suitable protease,buffer and its concentration were optimized.The single factor（pH of the buffer solution,hydrolysis temperature,trypsin dosage,hydrolysis time,substrate dosage）was test to determine the appropriate level of L₁₆（4⁵）orthogonal test for optimization,and obtain the best solution about the enzymatic hydrolysis of defatted Hermetia illucens L.The hydrolysate was obtained by centrifugation,and the final product was obtained after drying and pulverizing.The physical and chemical indexes and microbial indexes of the final product were analyzed so that compare with another product we bought in the supermaket.Research results were as follows:（1）Acetone was more suitable for Hermetia illucens L.degreasing compared with aether and petroleum benzin,and the best extraction time was 6h.Compared to papain,bromelain,neutral protease,alkaline protease,Trypsin was significant effects,the content of amino nitrogen in the product was 15.68,8.43,20.12,and 6.50 times,respectively.Further experimental results show that the optimal environment about bottom fluid and its concentration were NaH₂PO₄-Na₂HPO₄ buffer and 0.025 mol/L.（2）Appropriate level were identified by test of trypsin Hermetia illucens L.defatted powder of single factor:NaH₂PO₄-Na₂HPO₄ buffer pH（7,7.5,8,8.5,9）,hydrolysis temperature（45,50,55,60℃）,adding amount of trypsin（4000,5000,6000,7000 U/g）,hydrolysis time（3,3.5,4,4.5 h）,add the amount of substrate（5,10,15,20 w/v）;（3）We designed the L₁₆（4⁵）orthogonal optimization experimentation.The results showed that when the conditions came to pH 7.5,temperature was 50℃,amount of trypsin was 4000 U/g,hydrolysis time was 4.5 h,substrate concentration was 15 w/v,the result of the hydrolysis reaction was best,and the content of amino nitrogen in supernatant hydrolysate was 2.03 g/100mL.According to the mean value of orthogonal test,when the conditions came to pH 7.5,temperature was 55℃,amount of trypsin was 7000 U/g,hydrolysis time was 4.5 h,substrate concentration was 15 w/v,the content of amino nitrogen was 2.74 g/100mL;（4）After freeze drying and pulverizing of the supernatant hydrolysate,we mensurated its physicochemical indexes.The moisture content of dry powder was 5.25%,ash content was 6.73%,amino nitrogen content was 8.11%,total nitrogen content was 13.56%,the pH of 2%aqueous solution was 6.78,E.coli was negative.Amino acid content was up to90.05%,the essential amino acids accounted for 50.14%,no detecting in Escherichia coli.The content of amino acids in the supernatant was up to 90.05%,and the essential amino acids accounted for about 50.14%,containing about 17 amino acids.The contents of glutamic acid,leucine,lysine,alanine,arginine and valine were more than 5.00%;（5）The supernatant powder was used as peptone to replace the peptone culture medium on the market.Microbial growth test showed that the supernatant powder was more suitable for the growth of E.coli and Bacillus subtilis.Therefore,the effect of the supernatant powder was close to that of the tryptone,and the related indexes met the standard of HK/QW-SJS-35.Conclusion:This study shows that Hermetia illucens L.can be used as the raw material preparation of peptone,its physical and chemical indicators satisfy the industry standard.The microbial growth test showed that the peptone prepared by this method was more suitable for the characteristics of peptone.The optimized process parameters are simple and easy to operate.