| Chinese Sauerkraut is a kind of time-honored vegetable product by cold processing.It has a unique flavor.Currently,enterprises for sauerkraut production still employ traditional natural fermentation technology.In this way,strains are random and unidentified,fermentation conditons are difficult to regulate,product quality are unstable,hidden danger in food safety exists,all these problems have severely restricted the development of our nation’s sauerkraut industry.Considering these realistic problems,breeding and preparation of probiotic lactic acid bacteria speicific for vegetable fermentation and the probiotic properties were studied.The strains with excellent performance in fermentation were selected and used for vegetable fermentation.For one hand,it can solve various problems in sauerkraut industry of our nation,for another,sauerkraut product with excellent health function was developed which hit the target of transforming large bulk of vegetables with high value and high efficiency.This study was able to establish an industrial generic technology innovation platform for sauerkraut industry in our country,and reverse the passive situation of relying on imported fermented product,making contributions to economic construction and solving three dimensional rural issues.The main research contents and results were as follows:1、Changes and population dynamics of lactic acid bacteria flora throughout spontaneous fermentation of Chinese sauerkraut were analyzed in this study.It has been shown that Enterococcus faecalis,Lactococcus lactis subsp.lactis,Leuconostoc mesenteroides subsp.mesenteroides,Lactobacillus plantarum,L.casei and L.zeae dominated the fermentation process.E.faecalis and L.mesenteroides subsp.mesenteroides were present in the brine as soon as the vegetable was pickled(0d)while other four species were isolated successively during fermentation.E.faecalis and L.lactis subsp.lactis were mainly present in the early stage of fermentation and died in the middle stage;L.mesenteroides subsp.mesenteroides did not die until the 5th day;L.zeae existed in the middle stage and disappeared at the 5.5th day.L.plantarum and L.casei were present in the middle and later stages and predominated the final stage of fermentation.In summary,the fermentation process was initiated by L.mesenteroides subsp.mesenteroides,which belonged to heterofermentative lactic bacteria and served as a CO2 supplier for Lactococcus lactis,followed by E.faecalis,L.lactis,L.zeae during the middle stage of fermentation,which was typical homofermentative process and finally was succeeded by L.plantarum and L.casei.2、Functional lactic acid bacteria(LAB)specific for vegetable fermentation were screened from Chinese sauerkraut brine by designing purpose media.The selected LAB strains showed excellent tolerance to high concentrations of bile salt and acid.They can also survive passage through the simulated gastric fluid and reach the intestine.Through the performance measurements of vegetable fermentation,NCU116,one of these strains,was found to have the best fermentation ability,and was further subjected to a series of analysis including identification of physiological and biochemical characteristics,16S rRNA and pheS sequencing.NCU116 was identified as Lactobacillus plantarum subsp.plantarum.3、Response surface methodology was applied to optimize the medium composition for L.plantarum,and study on optimized high density cultivation of L.plantarum was carried out by neutralization and exponential fed method.SAS’s central composite design and response surface methodology were introduced to optimize the fermentation medium based on the single-factor test.The optimized formula was as follows:5.43%Glucose,0.98%Peptone,0.59%K2HPO4.High-density Cultivation technology of lactobacillus plantarum was researched,ammonia neutralization and fed carbon and nitrogen sources were operated in a 15L fermentator under the optimal conditions(3%inoculation,pH 6.5 and 35℃),the final cell concentration reached 9.3×109CFU/mL.4、The protective agent formula during freeze-drying process for Lactobacillus plantarum NCU116 was optimized by single factor experiment and orthogonal experiment.And effect of freeze-dried conditions,such as centrifugal condition,fermented liquid pH before centrifugation,material thickness and pre-freezing time,on cell activity was studied.Its eutectic points and disintegrating temperature were determined,and the effect for rapid freeze-drying and slow freeze-drying were compared with each other.Besides,two-step vacuum freeze drying technology was studied and compared with traditional freeze drying technology.The optimum parameters of two-step vacuum freeze drying technology were obtained as follows:trehalose 5%,skim milk 10%,glycerol 2%,fermented liquid pH before centrifugation 6.0,centrifuged for 15min at 4000r/min,eutectic points-31℃ and-30.8℃,disintegrating temperature between-32.9℃ and-27℃,the effect for rapid freeze-drying was better,material thickness 4mm,vacuum dried for 2h at 34℃,pre-freezed for 2h at-50℃,and then vacuum freeze dried for 18h.With two-step vacuum freeze drying,cell concentration of lyophilized material reached 9.8 X 1011CFU/g,the moisture content of lyophilized material fell down to only 0.61%,compared to 5.8 × 1011CFU/g and 1.94%for one-step vacuum freeze drying.Apparently the effect of two-step vacuum freeze drying was better,it had a great potential in application and development.5、In the direct vat set fermentation,Lactobacillus plantarum NCU116 was used as starter culture.In this study,pH value,acidity and changes of pathogenic bacteria(Coliform,Salmonella and Staphylococcus aureus)of natural fermentation were compared with that of direct vat set fermentation.It was indicated that with direct vat set fermentation,acid production rate and capacity increased a lot,fermentation rate was faster,plus fermentation time was shortened.In addition,the counts of three pathogens were significantly fewer in direct vat set fermentation(P<0.05)and remained at a low level,with the pathogens all disappeared since the 2nd day.In conclusion,Lactobacillus plantarum NCU116 had noteworthy inhibition to the food-borne pathogens during pickle fermentation.6、Lactobacillus plantarum NCU116 was investigated for tolerance in artificial gastric fluid,artificial intestinal fluid,bile salt and high concentration salt environment.It was shown that the survival rate of Lactobacillus plantarum NCU116 were 32.62%and 45.76%respectively after being treated in artificial gastric fluid(pH 1.5/2.5)for 3h.Higher survival rate was achieved after 3h in pH above 3.5.The survival rate achieved 49.63%after 4h in artificial intestinal fluid.After 24h in ox bile salt(0.03-1.00g/100mL),viable bacteria counts reduced with the increase of salt concentration,but still remained at 107CFU/mL.In salt environment with sodium chloride of 1-8 g/100mL for 24h,viable bacteria counts lowered slightly with the increase of mass fraction of sodium chloride.When the mass fraction was 8 g/100mL,viable bacteria number was still above 108 CFU/mL.It was obvious that Lactobacillus plantarum NCU116 had an excellent tolerance to artificial gastric fluid,artificial intestinal fluid,bile salt and high concentration of salt and maintained high survival rate,making a bright future for its application in food industry.7、The adherence of probiotics to human epithelial cells and mucosal surfaces is an important criterion for the selection of probiotics.Human intestinal tract environment was simulated,colon cancer cell line HT-29 was selected to test the adhesive ability of Lactobacillus plantarum NCU116 in vitro.Furthermore,the impact of different factors on the adherence was studied.It can be found that the adhesion(4.78±0.21 CFU/cell)of Lactobacillus plantarum NCU116 was very excellent.The adhesion of NCU116 was better with its concentration increase,but the growth tended to saturation when the concentration was up to 1.0×108CFU/ml.The adhesion of the bacteria in stationary phase was the best.The adhesion grew when prolonging the incubated time with colon cancer cell line HT-29.But the growth leveled off when the incubation time was up to 2.0h.The environmental pH had a significant impact on the adhesion of NCU116,it had a better adherence in acid environment.It didn’t have significant effects of Ca2+and Mg2+ on the adherance of Lactobacillus plantarum NCU116 on HT-29 cells.In summary,Lactobacillus plantarum NCU116 has an excellent adherence in host and thus is promising in food and healthcare industries.8、L.plantarum NCU116 fermentation liquor,strains and metabolic products were studied to detect the antibacterial activity in vitro based on oxford plate assay system,using 9 species of normal pathogen and nonpathogens as indicator organisms.It was found that L.plantarum NCU116 has the strongest inhibition to Escherichia coli ATCC 25922,but has no inhibition to 3 species of probiotics.Further study included the effect of samples of different concentrations,different organic acids,eliminating interference of organic acid and hydrogen peroxide,pH adjustment,heat and enzyme treatment on anti-E.coli ATCC 25922 activity.It was shown that no significant differences were seen with samples of different concentrations.The antibacterial substance contains other active materials besides organic acid,such as low molecular weight peptide bacteriocin and hydrogen peroxide.Low pH is a necessity for antibacterial activity.In conclusion,L.plantarum NCU116 bore strong antibacterial activity,main antibacterial substances are strains themselves,all sorts of organic acids and low molecular weight peptide bacteriocin. |
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