Mutagenesis And Fermentation Kinetics Of A High Yielding Strain C23221 Of Pediocin

Among food pathogenic bacteria,Listeria monocytogenes is a powerful foodborne pathogen that can cause life-threatening listeriosis in infants,pregnant women,the elderly,and people with weakened immune systems.Although antibiotics and others have been effective on the treatment of Listeria monocytogenes,there is still a lack of effective means to safely prevent Listeria monocytogenes contamination in the food industry.The search for safe and efficient natural biological preservatives is currently an international research hotspot.Bacteriocins have gradually become the focus of research because of their ability to inhibit harmful bacteria in food.Bacteriocin is a biologically active precursor peptide or protein produced by some bacteria in vivo after metabolism through ribosomes,and when they accumulate to a certain extent,they can inhibit or kill other microorganisms in the same or similar environment,and usually the bacteriocins they produce do not harm the bacteria itself.Pediocin is a kind of lactic acid bacteriocin with good heat resistance and wide p H adaptability,which has a strong inhibitory effect on pathogenic bacteria and spoilage bacteria in food,and can be applied to food processing in various complex environments,so it is considered to be a new type of biological preservative with the most promising new class of biological preservatives other than nisin for the control of Listeria monocytogenes.However,the existing natural fermentation technology of bacteriocins has the problem of low yield,which seriously restricts the application of bacteriocins in the food industry.Therefore,in this study,the original strain of Pediococcus pentosaceus C-2-1 obtained from pre-laboratory screening was used to improve the bacteriocin yield by various methods.Firstly,the expression vector was constructed by using the penocin synthesis gene,followed by ultraviolet mutagenesis of Pediococcus pentosaceus C-2-1,the differences between the genomes of the highyielding mutant strain and the original strain were analyzed by comparative genomics,and finally the growth of the high-yielding strain in whey was analyzed by combining fermentation kinetics,and the amplification culture of the fermenter was carried out to improve the bacteriocin yield.The specific results of the study are as follows:1.Cloning and expression verification of the pediocin-like pen A geneAnti SMASH and BAGEL4 were used to mine the whole genome data of Pediococcus pentosaceus C-2-1,and a bacteriocin synthesis gene cluster was obtained.The amino acid sequence was compared by NCBI and the bacteriocin synthesized by the core gene of this gene cluster was the pediocin-like penocin.Firstly,primers(Pedio F,Pedio R)were designed,the pen A target gene was obtained by PCR,the cloning vector p UCm T–pen A was constructed by TA cloning,then the target gene was connected to the expression vector plasmid p ET28a(+)after sequencing.The recombinant plasmid was transferred into the expression vector BL21(DE3),then induced with IPTG(final concentration of 1 mmol/L)at 30 °C for 4 h,and finally the successfully expressed recombinant protein was obtained.The recombinant proteins were purified using Ni2+ affinity chromatography,eventually most of the heteroproteins were removed and collected to a single fraction.The antibacterial activity was determined by in situ inhibition and agar diffusion,and the results showed that the antibacterial activity of the recombinant protein(264.1648 IU/m L)was higher than that of the cell-free fermentation supernatant of C-2-1(170.8834 IU/m L),which was 1.54-fold higher in comparison.2.UV mutagenesis and differential gene analysis of Pediococcus pentosaceus C-2-1The original strain C-2-1 was treated with 8 rounds of UV mutagenesis using 60 s as the optimal UV irradiation time.The potency of the selected mutant strain C23221(1448.605 IU/m L)was 8.47 times higher than that of the original strain C-2-1(170.81IU/m L).The second-generation whole genome sequencing of mutant strain C23221 showed that the genome of mutant strain C23221 was composed of chromosomes of1742268 bp,which was 79769 bp less than the original strain C-2-1,with 2052 proteincoding genes,4 r RNA operons and 47 t RNA genes.A total of 19 putative proteins involving 47 genes were unique to C23221 by GO database analysis;specific ped genes associated with pediocin biosynthesis in mutant C23221 were identified using anti SMASH,suggesting that mutant C23221 produced new bacteriocins under mutagenic conditions.3.Research on fermentation kinetics of mutant strain C23221 and high-density fermentationSoybean whey,which was mainly composed of gypsum halogen,was identified as the most suitable medium for the growth of mutant strain C23221.Using Logistic,Luedeking-Piret,Luedeking and other mathematical models,the bacterial growth,product production and substrate consumption of mutant C23221 in the whey fermentation process were predicted and the model was verified.The correlation coefficients of the fitted models were all R2 > 0.99,indicating that the bacteriocin production of mutant C23221 could be predicted and described by establishing a fermentation kinetic model;the bacteriophage volume and bacteriocin production of mutant C23221 were partially correlated,and it was determined that the replenishment from 16 h met the growth requirement of bacteria.The effect of different primary sugar concentrations on bacteriocin yield was investigated,and the initial sugar concentration was 70 g/L during fermentation in the fermenter.In a 10 L fermenter,fermentation is carried out by exponential flow plus complementary sugars.At 27 h fermentation time,the bacteriostatic activity was up to 788.0885 IU/m L,which was 4.61 times higher than that of the original strain in MRS culture,and 1.15 times higher compared with the whey shake flask fermentation of the mutant strain.The above results showed that heterologous expression of the pediocin synthesis gene by constructing an expression vector could increase the production of pediocin.Mutagenesis breeding by ultraviolet mutagenesis can screen out the high-yielding strain C23221 with high yield and stability,and analyze the reasons for the increase of bacteriostatic activity in combination with comparative genomics.Finally,high-density fermentation based on industrial waste whey not only saves costs but also improves antibacterial activity.In summary,the yield of pediocin can be increased by the above three methods,providing a certain theoretical basis for industrial production.