| Lactobacillus bulgaricus is one of the main starter strains used to prepare fermented milk products,and its high biomass is the key to the formation of a high-efficiency Directed Vat Set.Current studies have suggested that the ability of proliferation decreases even at the late-exponential phase under the condition of adequate nutrition is closely related to the physiological states of bacterial cells.Therefore,for high-viable-cell starter culture preparation,it is important to resolve the bottleneck of cell proliferation and culture cells at higher density by exploring the dynamics of the physiological states and their effects on viable cell density to establish the mechanism of cell division and thereby promote bacterial cell division.To provide a basis of the industrial high-density culture of Lactobacillus,this study aimed to analyze the physiological states of L.bulgaricus sp1.1 in the culture process,obtain information about cell division and death,and regulate cell division and proliferation.Fluorescence quantitative polymerase chain reaction(qPCR)combined with plate counting method was used to detect differences in bacterial viability,cultivability,and membrane damage as well as to explore the main physiological state of L.bulgaricus sp1.1 during the culture process.The qPCR method possessed high efficiency and high accuracy in detecting the cell numbers,and the qPCR results revealed a linear relationship between the copy number of the 16S r RNA gene and cell density(R~2=0.9981),with a detection limit of 15.1 colony-forming units/m L/reaction.The efficiency of molecular amplification was determined to be98.42%.On introducing propidium bromide azide at a final concentration of 60μmol/L,qPCR could distinguish and quantify viable cells,and no significant difference was noted with the plate counting method(p>0.05).Detecting the physiological states of L.bulgaricus sp1.1 by using the above methods suggested that the culturable(33.71%–54.63%),viable but nonculturable(23.30%–39.72%),membrane-permeabilized(12.73%–30.59%),and lysed cells existed during the culture process.Among these,the culturable state was the main physiological state during the late-exponential phase and decline phase.Cell lysis occurred continuously during the culture process;however,it could not be accurately quantified.To explore the dynamic changes in bacterial cell division and cell death during the culture process,a fluorescent dye tracking assay was applied to monitor the cell division rate and death rate of L.bulgaricus sp1.1.The fluorescent dye tracking method was optimized and suggested that 10μmol/L carboxyfluorescein diacetate succinimidyl ester could not interfere with cell viability,proliferation,and acidification ability.The cell division number tracked by this method was consistent with that by the conventional methods.The threshold of cell division detection reached 6 in this case.The monitoring of cell division and cell death of L.bulgaricus sp1.1 suggested that the division rate of L.bulgaricus sp1.1 decreased rapidly after the late-exponential phase,and only 0.46 divisions were completed from the late-exponential phase to the decline phase.The cell death rate was higher than the division rate in the stationary phase.According to the precursor cohort model,the decrease in the division rate(regression coefficient=0.2386)had a greater impact on the change in the viable cell number than the death rate(regression coefficient=0.0140).Decreased pH is one of the main factors influencing the proliferation of lactic acid bacteria.To explore the mechanisms of pH affecting cell division of L.bulgaricus sp1.1,changes in the cell division number,nutrient metabolisms,and key cell cycle processes under the conditions of lower pH were analyzed.It was found that cell division was inhibited at pH 4.1 and the culturable cell number decreased at pH<4.1.Thus,pH 4.1 was the critical pH at which cell division was inhibited,but cell culturability was maintained.The consumption of main nutrients and changes in the intracellular energy at the critical pH were further analyzed,and the results revealed that the consumption rate of glucose was reduced from 22.6%(pH 6.5)to5.27%,while the consumption rate of protein was almost 0%.The intracellular adenosine triphosphate(ATP)was significantly decreased(p<0.05)and the activity of H~+-ATPase was significantly increased(p<0.05);moreover,the supplement of energy was affected by the critical pH.Furthermore,the influence of the critical pH on the cell cycle of L.bulgaricus sp1.1 was analyzed,and the results revealed that low pH inhibited cell division by inhibiting cell size increase,interfering with DNA replication,and inhibiting the expression of the key gene fts Z.Furthermore,the cell wall hydrolase and synthetase enzymes were investigated,and the results demonstrated that the activity and transcriptional expression of peptidoglycan hydrolase increased with a decrease in the pH,although the expression of peptidoglycan synthase was downregulated,which unbalanced the cell wall synthetase and hydrolase enzymes,causing cell injury and lysis.Based on the above research,to promote the proliferation of L.bulgaricus sp1.1,bacterial cell division was regulated with amino acids at the critical pH(pH 4.1)and the pH of the medium was adjusted by using ion exchange resin.The results showed that when 50 mmol/L histidines were added,the maximum cell division number at the critical pH reached 2.36,which was close to the division number at pH 6.5(3.61),and the cell area and cell length reached 1.38–1.92-times and 1.28–1.73-times greater than those at the critical pH,respectively.The maximum expression folds of the key cell cycle genes dna A and fts Z were upregulated to 3.76 and 7.04,respectively.In addition,when the ion exchange resin was applied to maintain the pH at 5.5–5.8,the number of culturable L.bulgaricus sp1.1 cells in the batch culture was approximately 1.75-times higher than that without resin,while the number of culturable cells in the fed-batch culture was approximately 10-times higher than that when no resin was added.The number of culturable cells in the resin-fed-batch reached 7.83×10~9 CFU/m L within 24 h,and we cultured L.bulgaricus sp1.1 at a higher cell density.The following conclusions can be drawn based on the study results:four physiological states occur during the culture process of L.bulgaricus sp1.1,as determined using the qPCR,PMA-qPCR and plate counting methods.Further analysis revealed that the division rate had a greater impact on the viable cell number than the death rate,and we noted that the low pH caused by lactic acid inhibited cell division by interfering with cell metabolism and downregulating the expression of key genes in the cell cycle.Finally,cell division of L.bulgaricus sp1.1 was promoted by the addition of amino acids,and the high-density culture of L.bulgaricus sp1.1was achieved using ion exchange resins to adjust the pH of the culture medium.These results together provide the basis for producing a starter culture with high activity of L.bulgaricus for industrial application. |
