High-density Cultivation Of Heterofermentive Lactobacillus And Methods To Improve The Freeze-drying Survival Rate

Heterofermentive Lactobacillus have a special metabolic pathway that can convert carbohydrates into lactic acid,acetic acid,ethanol,CO₂and other metabolites.They also have a variety of probiotic functions and have a wide range of applications in food,medicine and health care industries.At present,the main growth inhibitory factors for constant pH culture of heterofermentive Lactobacillus were still unclear,and the high-density culture strategy based on growth inhibitory factors had not yet been clarified.In this paper,Lactobacillus fermentum,Lactobacillus reuteri and Lactobacillus brevis were selected as the research objects to systematically study their growth inhibitory factors,and the optimum growth substrates were slected.Based on the growth inhibition factors,the substrate concentration,proportion and culture process conditions were optimized,and the high-density culture strategy of heterofermentive Lactobacillus was proposed.On this basis,the fermentation regulation methods to improve the freeze-drying survival rate of heterofermentive Lactobacillus were studied,and the mechanisms were explored.The main findings were as follows:(1)Substrates concentration,acid anions concentration and osmotic pressure in the fermentation broth at the point of completely inhibiting heterofermentive Lactobacillus in pH7.0-controlled and feed culture were determined.The results showed that there was no substrate inhibition for heterofermentive Lactobacillus in pH7.0-controlled and feed culture.The accumulation of organic acids and the increasing osmotic pressure were the main inhibitors.In addition,the minimum inhibitory concentrations of sodium lactate,sodium acetate and sodium chloride for strains at pH7.0 were determined.The results showed that acid anions had no toxic effect on heterofermentive Lactobacillus,and the increase in osmotic pressure caused by acid anions accumulation was the growth inhibitory factor in pH7.0-controlled and feed culture.(2)The proliferation of L.fermentum,L.reuteri and L.brevis using different carbon sources,nitrogen sources,inorganic salts and trace elements were determined.The results showed that glucose was the best carbon source for heterofermentive Lactobacillus.The different species of heterofermentive Lactobacillus had different preferences for nitrogen sources utilization.Among them,the peptone of the yeast powder composite macromolecular peptide was the best nitrogen source for L.fermentum,and the small molecular peptide yeast powder was the best nitrogen source for L.reuteri and L.brevis.Inorganic salt was not a proliferative substrate of heterofermentive Lactobacillus.Both Mn²⁺and Mg²⁺were the restrictive trace elements of heterofermentive Lactobacillus.The degree of utilization of different growth substrates by heterofermentive Lactobacillus showed a certain regularity.(3)On the basis of the most suitable substrates,representative strains of L.fermentum FXJCJ6-1,L.reuteri CCFM1040 and L.brevis 173-1-2 were selected to determine the carbon-nitrogen ratio when the growth rate of the strain was inhibited,concentration and ratio of trace elements.The optimum culture pH and culture process were also optimized.The results showed that the substrate carbon-nitrogen ratio was based on the carbon-nitrogen consumption ratio when the strain growth rate was inhibited,and the substrate concentration was based on the carbon-nitrogen consumption calculated based on the osmotic pressure when the strain growth rate was inhibited.Then the preparation of the medium was most conductive to the growth of heterofermentive Lactobacillus.The optimum concentration of Mg²⁺and Mn²⁺was 0.1 g/L-0.2 g/L.The optimal growth pH of heterofermentive Lactobacillus was 5.0-6.0.The constant pH batch culture method was more conducive to the proliferation of heterofermentive Lactobacillus.The optimum culture strategy of L.fermentum FXJCJ6-1 was as follows:nitrogen source:40 g/L(yeast extract powder FM528:8 g/L,bovine bone peptone FM326:16 g/L,fish bone peptone FM351:16 g/L),glucose:108 g/L,Mg SO₄:0.175 g/L,Mn SO₄:0.175 g/L,Tween 80:1 m L/L,cultured at constant pH6.0,the number of viable bacteria reaches(1.3±0.1)×10¹⁰CFU/m L,which was higher than the number of viable bacteria when cultured in MRS medium statically 3.1 times.The optimum culture strategy of L.reuteri CCFM1040 was as follows:nitrogen source:40 g/L(yeast extract FM528),glucose:112 g/L,Mg SO₄:0.175 g/L,Mn SO₄:0.175 g/L,Tween 80:1 m L/L,cultured at constant pH5.0,the number of viable cells was(8.0±0.4)×10⁹CFU/m L,which was higher than the number of viable cells when cultured in MRS medium statically 5.2 times.The optimum culture strategy of L.brevis 173-1-2 was as follows:nitrogen source:40 g/L(yeast extract FM528),glucose:108 g/L,Mg SO₄:0.125 g/L,Mn SO₄:0.125 g/L,Tween 80:1m L/L,cultured at constant pH5.0,the number of viable bacteria was(1.0±0.14)×10¹⁰CFU/m L,which was higher than the viable bacteria in the static culture of MRS medium statically 6.0 times.(4)Then on the basis of high-density culture technology,the freeze-drying survival rate was determined by collecting three strains of bacteria cultured at different harvest times,different culture pH and adding different compatible solutes.The results showed that the freeze-drying resistance of heterofermentive Lactobacillus in the late stable phase was stronger than that of other stages.A lower pH culture environment helped to improve the freeze-drying survival rate of heterofermentive Lactobacillus.For strains with a higher optimal growth pH,stopping alkali supplementation at the end of the logarithmic phase could also improve the freeze-drying survival rate.The addition of exogenous compatible solutes such as lactose,alanine and betaine could improve the freeze-drying survival rate of the three strains,while the addition of trehalose could improve the freeze-drying survival rate of L.fermentum FXJCJ6-1.The fermentation regulation methods to improve the freeze-drying survival rate of L.fermentum FXJCJ6-1 were as follows:trehalose was added,the optimal growth pH6.0 was first cultivated to the end of the logarithmic phase(10 h),then alkali supplementation was stopped,and finally the bacteria were harvested at the end of the stable phase(14 h).The freeze-drying survival rate increased by(27.53±1.36)%.The fermentation regulation methods to improve the freeze-drying survival rate of L.reuteri CCFM1040 were as follows:betaine was added and was directly cultivated at the optimal growth pH5.0 to the late stable phase(14 h).The freeze-drying survival rate increased by(16.17±3.11)%.The fermentation regulation methods to improve the freeze-drying survival rate of L.brevis173-1-2 were as follows:lactose was added and was directly cultivated at the optimal growth pH5.0 to the late stable phase(14 h).The freeze-drying survival rate increased by(19.16±0.43)%.(5)Trehalose and lactose were used to culture L.fermentum FXJCJ6-1,L.reuteri CCFM1040 and L.brevis 173-1-2,and by measuring the intracellular substance,changes in the morphology of the bacteria and the production of extracellular polysaccharides,the difference in the effect of trehalose and lactose on improving the freeze-drying survival rate of heterofermentive Lactobacillus was analyzed.The results showed that the accumulation of trehalose was detected in the cells of L.fermentum FXJCJ6-1 when the high-density culture reached the hypertonic environment,but there was no trehalose accumulation in the cells of the hypotonic environment.It was speculated that the strain induced hypertonic resistance to transport trehalose from the environment under hypertonic conditions,thereby enhancing its stress resistance.However,this result was not found in L.reuteri CCFM1040 and L.brevis173-1-2,which may also be related to the existence of a trehalose transport system in the bacteria.When using lactose as a carbon source to cultivate L.fermentum FXJCJ6-1 and L.reuteri CCFM1040,it could significantly reduce the output of their extracellular polysaccharides compared with glucose,thereby increasing the freeze-drying survival rate of the strains.