| Carbon dioxide is one of the main products of the yeast (Saccharomyces cerevisiae) fermentation. The inhibition of CO2 could contribute to the stuck or sluggish of the fermentation, and cause the variation of the flavor of the fermentation food. Knowledge about the inhibition mechanism(s) of CO2 on yeast is valuable in optimizing and controlling the fermentation process to improve the productivity and quality. However, there is no answer to what will happen and when will the fermentation stop caused by the CO2 accumulated in a sealed fermentor, and no quantity research about the effect of CO2 on the metabolic pathway of yeast has been reported.This study aimed to investigate the influences of CO2, endogenously produced and accumulated in a sealed fermentor by fermenting grape juice, on the performance of Saccharomyces cerevisiae. Firstly, the model of metabolic flux analysis (MFA) was built to guide the design of experiments. Then, according to the MFA model, germ-free grape juice were fermented by Saccharomyces cerevisiae, and the performance of atmospheric anaerobic fermentation(AAF) and sealed fermentation(SF) were compared from the ferment rate, the aroma components and the producing rates and yields of ethanol, glycerol, acetic acid and succinic acid. Finally, by using the uptake rate of glucose and the producing rates of ethanol, glycerol, acetic acid and succinic acid, the metabolic flux analysis were applied, and the flux distribution of those two kinds of fermentation were compared in order to find the action position of CO2. Following results were obtained:1. In sealed fermentor, CO2 generated from the fermenting of 5.73g L-1 glucose could contribute 1 bar of pressure, and the fermentation stopped when 82.0g L-1 glucose were consumed and the endogenously-produced CO2 pressure reached at 14.3 bar, and at that time the residual sugar content was 83.0 g L-1 and the viable yeast concentration was 5.5×107cfu mL-1, while the atmospheric anaerobic fermentation almost used up the sugar in the grape juice. The total yeast concentration in the atmospheric anaerobic fermented juice(AAFJ) was about twice of the sealed fermented juice(SFJ).2. The yield of ethanol under AAF and SF differed a little, but the glycerol yield of SF was 79.1% of AAF, and the yield of succinic acid and acetic acid of SF were 24.7 and 26 times of AAF, respectively. 3. 36 kinds of the detected aroma components were shared by AAFJ and SFJ, 6 kinds were found in SFJ only, and 2 kinds were peculiars of AAFJ. Compared with AAF, SF inhibited the production of esters, stimulated the production of higher alcohols, and the concentration of acetoin in SFJ was 23.45 time of that in AAFJ.4. Metabolic flux analysis showed that there were difference on the fluxes of metabolic pathways of acetaldehyde producing acetic acid, pyruvic acid producing oxaloacetic acid, dihydroxyacetone- phosphate producing glycerol and pentose phosphate pathway(PPP) under AAF and SF. The fluxes of PPP and dihydroxyacetone- phosphate producing glycerol pathway under SF were respectively 35.6% and 1.89 times of those under AAF.Synthesized the results, following conclusions were drew: CO2 could have a serious inhibition on the growth of Saccharomyces cerevisiae; the fermentation and growth of Saccharomyces cerevisia could totally inhibited by CO2 when the CO2 concentration reach a certain leve; CO2 could decrease the production of glycerol and esters, stimulate the production of higher alcohol, and increase the production of acetic acid, succinic acid and acetoin considerably; reactions catalyzed by phosphogluconate dehydrogenase and pyruvate carboxylase were influenced by CO2, and those carboxylation and decarboxylation reactions seem to be acting positions of CO2 on Saccharomyces cerevisiae, however, the decarboxylation reaction catalyzed by pyruvate decarboxylase was insensitive to CO2.
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