Construction Of Lactic Acid Bacteria Fusion And Evaluation Of Fermented Pickled Pepper Performanc

Pickled pepper production is the main way to improve the economic benefits of deep processing of fresh peppers,but at present its processing level is low,most of them are made by natural fermentation soaking.At the same time,due to the lack of special lactic acid bacteria for fermentation of pickled pepper,some companies use strain compounding to improve the quality of pickled pepper.However,the compounding method has a ratio that is difficult to control,and the dominant strains will compete with each other,which in turn leads to unstable quality of each batch of pickled pepper.In order to obtain lactic acid bacteria with excellent flavor and fermentation performance,two excellent strains of lactic acid bacteria were isolated and screened as parental strains from the old Sichuan pickled pepper brine.Using protoplast fusion technology,we finally obtained fusion strains with better fermentation performance and flavor than the parental strains,and evaluated and analyzed their kimchi fermentation performance,thus providing some scientific and theoretical basis for the mass production of pure fermented kimchi.The specific results are as follows:1.In this study,388 strains of bacteria were isolated,38 strains of lactic acid bacteria were screened according to the calcium solubility circle,physiological and biochemical experiments,and 10 strains of lactic acid bacteria with acid yield greater than 11 g·kg^-1 of commercial strain CICC6239 were screened according to acid yield,among which the acid yield of strain 6Y-15 was the largest at 17.93 g·kg^-1.The organic acid content of the 10 strains was determined and it was concluded that strain 6Y-15had the highest malic acid and citric acid content of 5667.559μmo L/L and 242.69μmo L/L,respectively,while strain 1Y-35 had the highest lactic acid content of 12111.2μg/m L,and the OD_600nm value of this strain when incubated for 24 h at 80 g/L salt concentration was 0.486,which was significantly higher than that of strain 6Y-15（P<0.05）.Meanwhile,the growth rate of strain 6Y-15 was significantly higher than that of strain 1Y-35（P<0.05）,and both strains belonged to Lactobacillus plantarum as identified by physiological,biochemical,morphological and molecular biology.2.In order to obtain fusion strains superior to the parental strains,the protoplast preparation,inactivation time and fusion conditions of strain 1Y-35 and strain 6Y-15were investigated in this paper using protoplast fusion technique,respectively.The results showed that strain 1Y-35 showed the best protoplast formation and regeneration rates of 87.5%and 16.86%,respectively,at a lysozyme concentration of 1 mg/m L.At the optimum enzyme concentration,strain 1Y-35 showed the best protoplast formation and regeneration rates of 88.75%and 14.08%,respectively,after 75 min of enzymatic digestion.At the optimum enzyme concentration,strain 6Y-15 showed the best protoplast formation and regeneration rate of 80.67%and 15.7%,respectively,after120 min of enzymatic digestion.Meanwhile,the optimal inactivation conditions for strains 1Y-35 and 6Y-15 were UV inactivation for 25 min and heat inactivation for 35min.A total of 52 fusions were obtained by multiple rounds of fusion after holding the completely inactivated parental strains for 10 min at 37°C and coating them with regeneration medium.From the above obtained fusions,seven salt-tolerant strains were screened,among which strain R-5 and strain R-40 were more salt-tolerant,with OD_600nm of 1.48 and 1.3at 80 g/L salt concentration,respectively.The acid production of these seven strains was measured,and the maximum acid production of strain R-5 was 14.5 g·kg^-1.The organic acid contents of strains R-5 and R-40 were measured,and the contents of R-5were higher than those of R-40.The contents of malic acid,citric acid and lactic acid of strain R-5 were 649.7μmo L/L,9794μmo L/L and 27358μg/m L,respectively,which were higher than those of the parental strain,and the growth rate of strain R-5 was higher than that of the parental strain 1Y-35.Therefore,R-5 was finally screened as the optimal fusion strain,and then R-5 was subjected to morphological and molecular biological characterization,and the strain remained Lactobacillus plantarum.3.The total acid content,color value,antioxidant activity and organic acid content of the fermented pepper solution were measured after 25 d.The acid production of strain R-5 was 7.13 g·kg^-1,which was higher than that of parental strain 6Y-15.In terms of taste and morphology,strain R-5 scored higher than the parental strain 1Y-35,and the flavonoid content of strain R-5 was 234.24μg/m L,which was significantly higher than that of the parental strain 6Y-15.The highest scavenging rate of DPPH radicals was 78.95%in the pickled pepper solution of fusion strain R-5,and the values were higher than those of the parental strain.In the determination of organic acid content,the lactic acid content of strain R-5 was 10402.93μg/g,which was higher than that of parental strain 6Y-15,and the malic acid content was 12.3μg/g,which was significantly higher than that of parental strain 1Y-35.In summary,the fermentation performance and organic acid content of the fusion strain R-5 are improved on the basis of the parental strain,and can be used as a pure fermentation strain in kimchi fermentation.