| Lactic acid bacteria (LAB) are widely used for the production of fermented foods, such as dairy, meat, cereal and vegetable foods. Compared to the spontaneous fermentation, the industrialized fermentation with LAB as starter cultures can initiate rapid acidification of the raw material, and facilitate the control of the whole fermentation processes, resulting in standardisation of the end product, due to higher concentration and activity of LAB contained in the starter cultures. Jerusalem artichoke (JA) is a plant rich in inulin, which can grow well in non-fertile land and is resistant to plant diseases, not competing with grain crops for arable land. JA was attractive as a material of functional foods, and the tuber of JA always had been used primarily in pickling in some Asia regions. Because JA can’t be fermented well spontaneously when the room temperature was lower than10℃, the present work focused on the isolation and characterization of the low-temperature LAB from traditional natural fermented vegetable, Na-cai (mainly fermented in winter). Concurrently, in order to obtain high quality of LAB starter culture for the pickling of Jerusalem artichoke, the effects of nutrient components on the growth and fermentative activity of Lactobacillus plantarum starter culture were systematically investigated. Next, investigation on manganese supplement in the activities of some key enzymes of LAB metabolism pathway was carried out in order to clarify the impacts of manganese supplement on the metabolism pathway of LAB. Gas chromatography coupled with mass spectrometry (GC/MS) metabonomic technologies and proteomics technologies were also used to investigate metabolie variations with or without manganese addition.(1) In this study, a total of17lactic acid bacteria (LAB) strains were isolated, characterized and identified from Na-cai for their suitability for JA pickling under low temperature. The obtained results showed that the selected strain DY-2, which was considered to be belonging to Lactobacillus plantarum group by16S rDNA identification, was the most effective at improving the fermentation quality of JA pickle under lower temperature, as indicated by lower pH values, less nitrite residuals, higher viable cells in fermented broth and better sensory evaluation for the final products.(2) Profiles of organic acids and volatile compounds produced by individual LAB during JA pickling under different temperature were compared by GC/MS, which further indicated that the selected strains DY-2was a favourable inoculants for JA pickle under low temperature. Temperature seemed to be a key factor that influences the organic acids and volatile compounds production, both for production rate and final concentration in pickling juice, which will affected the flavour of final product. (3) Jerusalem artichoke powder by hot-air drying can be used as a good substrate for LAB fermentation. The maximum cell density could be obtained in10%JA juice with addition of8.0g/L of glucose,40.0g/L of soya peptone and0.45g/L of MnSO4, respectively. By using this optimal medium, the maximum cell concentration in the fermented broth could reach3.3×109CFU/mL.(4) Batch fermentation of L. plantarum DY-2to get high quality starter culture was developed in5-L fermentor. The effects of pH, fermentation temperature, fed-batch strategy on the growth and fermentative activity were investigated. Cell density of LAB could be highly improved when the cultivation was performed with constant pH value, and a maximum cell concentration in the fermented broth could reach9.6×109CFU/mL when the pH value was controlled at5.0. Growth rate could be changed under different fermentation temperature, but the cell concentration at the end of fermentation was similar. Cultivation temperature of34℃was more suitable to cell growth and higher temperature could make a fermentative activity lose of the LAB. A lower nutrient concentration will make the fermentation slower and the cell density in the fermentation broth was remarkable declined. Feeding glucose in the fermentation process had major positive impact on the rate of acid production, and then promotes the decline of LAB in the later phage of fermentation.(5) The effects of medium components on the quality of L. plantarum DY-2starter culture was investigated, and the results showed that the addition of glucose and soya peptone had a positive promotion on the viable number of LAB in the fermentation process, and manganese was proved to be an key factor in LAB culture medium for its subsequent fermentative activity. Lack of manganese would cause the subsequent fermentation significantly slower, whether the cell density in starter culture was higher or lower.(6) The influence of manganese on LAB metabolism was further elucidated by the time course analysis of the specific activities of metabolism key enzymes during the culture processes of L plantarum DY-2. Compared to the fermentation processes without manganese addition, it was found that manganese addition would enhance the lactate dehydrogenase (LDH) activity but reduce the activities of pyruvate dehydrogenase (PDH) and ATPase activity. Therefore, it could be concluded that the improvement of L. plantarum starter fermentative activity was probably a consequence of manganese acting as "metabolic switch", which regulated the metabolic flux from pyruvic acid to lactic acid and other metabolism pathway.(7) The relationship of manganese and intracellular metabolite in L. plantarum was exploiting in metabolism level by using GC-MS. Glycolysis, tricarboxylic acid cycle, organic acids and amino acids were both response to the lack (addition) of manganese, and the effect of the amino acids changes was most marked. After fermentation for6hours, concentrations of intracellular amino acids were improved by the lack of manganese, but this tendency was reversed after12hours.(8) The technique of isobaric tags for relative and absolute quantitation (iTRAQ) was used to analyze the influence of manganese on protein expression in L. plantarum based on mass spectrometry. Fifty-six intracellular proteins were identified having differential expression after6hours with lack of manganese, and these56proteins include31up-regulated proteins and25down-regulated proteins. Seventy-eigth intracellular proteins were identified having differential expression after9hours with lack of manganese, while the expression level of40proteins was up-regulated and38proteins was down-regulated. Sixteen proteins were found to have significant differences at both time points. Proteins involved in the construction of ribosom were down-regulated with lack of manganese, while the up-regulated proteins were involved in DNA repair and stress response, and UspA family proteins did show significant difference in the fermentation process. GO analysis and KEGG metabolic pathway analysis revealed that manganese have a significant impact on metabolism pathway in L. plantarum, such as pyruvate and amino acid metabolism, which demonstrate that manganese was one of the most important factors for maintain the normal metabolism in L. plantarum. |
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