The Impact Factors And Rules Of Growth Inhibition And Freeze-drying Survival For Lactic Acid Bacteria

Lactic acid bacteria?LAB? have been highly appreciated because of its good fermentation performance and superior probiotic properties. In order to improve the culture density of LAB and preparation efficiency of probiotics, the key factors inhibiting the growth of LAB and the effect of which on preparing probiotics powder would be understood. This paper systematically investigated the main growth inbititors of different homofermentative Lactobacilli and Bifidobacteria, and analyzed the law of the highest biomass. Then, based on the law of growth inhibition, a fed-batch culture coupled with a lactic acid removal system was develepped to study the effect of inhibiting factor removed on the growth of LAB. Finally, acid production from bacteria mixed with protective agent before freezing was determined and its effect on the survive of LAB during freeze-driying was studied. The main results were shown as follows:The accumulation of the end products and the depletion of nutrients by various homofermentative Lactobacilli were systematically evaluated at the point of the highest biomass in both batch culture and fed batch culture. The results showed that there was no substrate inhibition for two kinds of culture ways. Low pH value and organic acid accumulation were the main factors inhibiting the growth of Lactobacillus in batch culture. In addition, the minimum inhibitory concentrations?MICs? of sodium lactate, sodium acetate and sodium chloride for various strains at pH 7.0 were examined. The MICs of acid anions were the same as one of sodium chloride for Lactobacillus plantarum, L. casei, L. bulgaricus, L. acidophilus and L. helveticus except L. rhamnosus. The lactate concentration at the point of complete inhibition was not significantly different from the MIC of lactate for all of the strains, although the inhibition mechanism of lactate and acetate on Lactobacillus rhamnosus was different from the other strains which were inhibited by the osmotic pressure caused by acid anions at pH 7.0. When the lactate concentration accumulated to the MIC, the strains stopped growing. The maximum biomass was closely related to the biomass yield per unit of lactate produced(Y_X/P) and the MIC?C? of lactate for different homofermentative Lactobacillus. The rule of the maximum biomass of different homofermentative Lactobacillus was obtained as follows: X_max- X_O =?0.59 ± 0.02?·Y_X/P ·C.In the same way, the main inhibitory factors of Bifidobacterium bifidum, B. longum, B. longum subsp. Infantis, B. adolescentis and B. animal in both batch culture and fed batch culture were analyzed. It was found that the main inhibitors of Bifidobacteria during batch culture was the low pH value and the accumulation of organic acids. Similarly, MICs of acid anions on a range of strains were examined at pH 7.0. The inhibition of acid anions, which had the same MIC as sodium chloride, was due to the osmotic pressure under pH 7.0 conditions. Moreover, the concentration of total acid-anions completely inhibiting each strain in the fed-batch culture at pH 7.0-controlled had no significant differences with the MIC of acid anions for the corresponding strains. The osmotic pressures under two conditions were not significantly different. Finally, the maximum biomass concentration of Bifidobacteria was found to be closely related to biomass yield per unit of acid anion produced(Y_X/P) and MIC?C? which were needed for the prediction, and different strains exhibited marked correlation?p<0.01, R=0.985?. The rule of the maximum biomass of different Bifidobacteria was developed as follows: X_max- X_O =?0.71 ± 0.03? ·Y_X/P·C.The rate of glucose converted to acids by different homofermentative Lactobacillus and Bifidobacterium was determined during culture. About 90% glucoses were metablized to acids by all tested strains. This suggestd that the amount of glucose consumed was calculated according to the quantity of alkali fed and the conversion rate of glucose to alkali, since the amount of alkali fed up represented the acids producd. Feeding automaticly k mL mediums was designed following 1 m L NaOH solution. The concentration of NaOH and glucose could be prepared as the following formula: C_?/40×2×90%=C_?/180?k?homofermentative Lactobacillus?;C_?/40×5×90%=C_?/180×2?k?Bifidobacterium?. This feeding method followed the law of substrate consumption, and ensured the stability of the substrate of the culture system.By fitting the osmotic pressure as the only inhibiting factor, the growth kinetics equation of Lactobacillus plantarum CCFM 8610 in pH 7.0-controlled and fed-batch culture was obtained:?=?0.96±0.09?×(1- (O-?336±1?/?2416±97?-?336±1? ^0.25±0.04). The kinetics implied that the strain was sensitive to osmotic pressure, and the growth rate decreased rapidly when the osmotic pressure rised, and the growth rate of the strain decreased with the increase of osmotic pressure. With the osmotic pressure continues to rise, the growth rate of the strain decreased slowly.By measuring the adsorption capacity for lactic acid and the selectivity for glucose and amino acids of different anion exchange resins at 37?, the optimal anion exchange resin? D319? was obtained. After converted to the OH^-, it was used to form a lactic acid removal system. Then the effect of acid anions selectively removed on the growth was studied. The results showed that the bacteria could continue to proliferate after the acid anions removed, and the growth dynamics of the bacteria were in line with the the growth kinetics equation above. It was confirmed that the osmotic pressure caused by total acid-anions accumulation was the main inhibitory factor of L. plantarum in pH 7.0-controlled and fed-batch culture. This acid removal system with anion-exchange resins provides a new method for improving the growth of LAB. In this cultivation system, a cell concentration of 34.5 gL_-1 was obtained after 12 h, being about 13.3 and 2.3 times higher respectively than that of a batch culture and a fed-batch and pH-controlled culture without the addition of resins. The proposed culture system was effective in increasing the biomass of lactic acid bacteria.Vacuum freeze drying was the key technology of the preparation of LAB powder, and the strains should be mixed with the protective agent before freeze drying. Acids production of the strains in the process of being mixed with the protective agent, and the effect of the pH decrease caused by acids produced on the survival rate of strains for pre-freezing and freeze-drying were systematically studied. The acid production rate was determined at room temperature and refrigerated temperature. The effect of different pH values during pre-freezing and freeze-drying on ATPase, ?-galactosidase, lactate dehydrogenase?LDH?, cell membrane integrity and flow conditions were analyzed. The results showed that the rate of acid production at the room temperature was very fast, and the pH value of the bacterial suspension could be reduced to less than 4 in a short time. Moreover, the survival of the strain was significantly reduced at pH lower than 4.0 for pre-freezing while the survival rate for freeze-drying decreased dramaticlly at pH below 5.0. The acids caused the loss in viability of Lactobacillus due to the damages aggravated to ATPase, ?-galactosidase and cell membrane fluidity, but not LDH and cell membrane integrity.The amount of cryoprotectants?M? and cell pastes?m?, total cell count?N?, total surface area of strains? cytomembrane?S_t?, total volumn?V_t? of bacteria for 100% survival rate was respectively linear-regression analyzed. The total areas of microorganism?s cytomembrane were found to be highly correlated with the amount of proposed cryoprotectant. For 100% survival rate of freeze-dried Lactobacillus, the amount of cryoprotectant?M? and the total areas of strains? cytomembrane?S_t? are in accordance with the following rule: N ·?4?r² + 2?l?=?0.66 ± 0.03? · M. The optimum amount of protective agent could be calculated according to this rule.