Cloning Expression,Enzymatic Properties And Application Of Lactobacillus Fermentum 4,6-?-Glucanotransferase

As a derivative of starch,maltodextrin is often used in the production of food,health care products and medicine.The modified maltodextrin prepared by the enzymatic method with maltodextrin as the substrate can improve its original physical and chemical properties,and at the same time it can reduce its calories.It is a kind of dietary fiber with more application prospects.4,6-?-glucanotransferases(4,6-?-GTases),as a class of enzymes newly discovered in recent years,has the function of cutting?(1-4)bond to form?(1-6)bond.Therefore,these enzymes have great application potential in the preparation of modified maltodextrin.The main findings of this paper are as follows:(1)Screening and truncated expression of 4,6-?-GTases gene.According to the conserved motifs of reported 4,6-?-GTases,degenerate primers were designed and a putative4,6-?-GTase gene(gtf16)was successfully screened by PCR using the genome of the screened lactic acid bacteria as the template.The full-length gene could not be expressed in E.coli BL21(DE3),so the target gene(gtf16)was truncated by different truncation methods.The highest enzyme activity of the target protein after truncation was 83.7 U·m L^-1,and the optimum pH was 5.0,the optimum temperature was 40?,and the half-life at 40?was 30min;the enzyme could use maltotriose and malto-oligosaccharides with a higher degree of polymerization as donor substrates;The?-glucan product prepared with amylose was composed of the reductive end of continuous?(1-4)bond and the non-reductive end of continuous?(1-6)bond(type?),in which the proportion of?(1-6)bond was up to 75.0%.(2)Application of recombinant 4,6-?-GTase in the preparation of modified maltodextrin.According to the content of the anti-digestive component(RS)in the digestive enzymes hydrolyzed products,the preparation process was optimized.The optimal conditions for the reaction were determined as follows:temperature was 45?,pH was 4.5,the enzyme concentration was 2000 U·g^-1 maltodextrin,reaction time was 6 h.In the modified maltodextrin prepared under the best condition,the RDS content was 22.1%,the SDS content was 4.9%,and the RS content was 73.0%.(3)In vitro probiotic benefits of modified maltodextrin.In the medium with modified maltodextrin,the concentration of probiotics reached 0.5-0.9(OD₆₀₀)in 48 h,and the pH dropped from 7.0 to 4.5-5.3;the medium with glucose,the concentration of probiotics reached 0.3-1.1(OD₆₀₀)in 48 h,and the pH dropped from 7.0 to 4.0-5.5;the basal medium,the concentration of probiotics reached 0.04-0.16(OD₆₀₀)in 48 h,and the pH dropped from7.0 to 6.4-6.7;In contrast,probiotics could use modified maltodextrin as a carbon source material and could further synthesize short-chain fatty acids.(4)The effect of modified maltodextrin on flour processing quality.Analyzed from two aspects of powder quality and starch gelatinization,the addition of modified maltodextrin could enhance the gluten strength of flour and delay the retrogradation of starch.For the high-gluten flour with 7%modified maltodextrin,the stability time was extended from 22.6min to 29.5 min,the softening was reduced from 31.0 Fu to 8.0 Fu,and the starch retrogradation value decreased by about 25.6%;the medium-gluten flour with 5%modified maltodextrin,the stability time was extended from 8.9 min to 11.7 min,the softening was reduced from 103.0 Fu to 41.0 Fu,and the starch retrogradation value decreased by about28.1%.(5)The effect of modified maltodextrin on the quality of bread and steamed bread.The hardness of bread with 3%modified maltodextrin was 81.7%lower than the control on the first day,after 7 d of storage at room temperature,the hardness was 22.1%lower than the control;the hardness of steamed bread with 7%modified maltodextrin was 53.2%lower than the control on the first day,after 7 d of storage at 4?,the hardness was 54.1%lower than the control.