| Starter culture is the chip of fermented milk is also the key problem.The low activity,poor performance and weak competitiveness of domestic bacteria powder have become the problems of the development of Chinese probiotics industry.In order to solve the current situation that excellent probiotics powder basically depends on imports.In this paper,Streptococcus thermophilus used in starter culture was taken as an example to study the optimal solution of lyophilized protective agent of probiotics powder,the cooperative optimization strategy of protective agent combined lyophilized process and the protective mechanism of protective agent lyophilized.Based on the strain reserve resources and technical invention obtained from previous studies,the development and application of highly active probiotics anti-stress powder was the main line of technology,combined with the process requirements and production costs.The survival rate of bacteria and the stress resistance of bacteria powder are the main evaluation indexes to carry out research,so as to promote the high quality and sustainable development of probiotic industry in our country.The main research is as follows:(1)Screening and optimization of lyophilized protective agents for Streptococcus thermophilus.The frequency counting method was used to analyze the information of different protective agent combinations and their concentrations extracted from 106 relevant literatures.On the basis that the final system of fixed protectant was 1%sodium glutamate+5%skim milk+4%sugar protectant,the types of lyophilized protectant were further screened.Through single-factor screening of species and storage stability test of sugar protectant,the combination of 1%sodium glutamate+5%skim milk+2%sucrose and 1%sodium glutamate+5%skim milk+2%sorbitol was determined for the subsequent strain high-density lyophilization.The optimal combination of S.thermophilus 937 was found to be 4%glutamate+3%sucrose+3%sorbitol+8%skim milk by high-density lyophilization.The number of viable bacteria was only 109 CFU/g before high-density lyophilization but significantly increased to 1.78×1011CFU/g after high-density lyophilization,and the survival rate increased to 90.59%after optimization of protective agent concentration.It was proved that 4%sodium glutamate+3%sucrose+3%sorbitol+8%skim milk had a good protective effect on S.thermophilus Grx02 and Enterococcus faecium218,with survival rates of 95.68%and 92.11%,respectively.But for two strains of bacillus Lactobacillus plantarum S7 and Lactobacillus fermentium DL02 freeze-dried survival rates were 60.3%and 67.7%respectively,and did not show a good protection effect.The survival rate of S.thermophilus 937 powder was 42.17%after storage at 4℃ for 12 months.(2)Based on the structure of casein micelles,the protective effect and mechanism of salt lyophilized protectors on S.thermophilus 937 were investigated.Turbidity analysis,particle size distribution,transmission electron microscopy and other tests were used,as well as lyophilized survival rate and other indicators,the particle size changes of casein nanoparticles under the influence of sodium/potassium ions were determined to affect the protective effect of freeze-drying.The results showed that the particle size of casein nanoparticles corresponding to 150 mmol/L Glu-Na was 261 nm,and the freeze-drying survival rate was 87.56%.The effect of salt ions on the structure of casein nanoparticles on the lyophilized survival rate of S.thermophilus 937 was investigated by Zeta potential,hydrophobicity of protective agent,surface hydrophobicity,synchronous fluorescence spectrum,circular dichroism,and Fourier infrared spectroscopy.The absolute value of Zeta potential decreased to 21.56 mV when 150 mmol/L Glu-Na was added into the protectant,the hydrophobicity of the protectant solution and the bacteria surface did not change significantly,the β-folding content decreased to 35.3%,and the contents of α-helix,β-angle,and random curl fluctuated.Finally,it was verified that the size and structure regulation of protein protectants could improve the protective effect of lyophilization.After the adjustment of protein particle size and structure,the survival rate after freezing was increased by 40.35%(soy protein)and 29.38%(casein),respectively.After ultrasonic treatment,the β-fold content of soybean protein increased significantly to 38.0%,while the rest of the casein showed a decreasing trend After ultrasonic treatment,the β-angle content of casein increased significantly,and the rest of the casein fluctuated slightly.The reduction of α-helix content can correspond to the partial expansion of the α-helix region caused by ultrasonic cavitation.The increased beta-fold content also helps expose the hydrophobic regions of the protein.(3)The protective effect of salt protectors on S.thermophilus 937 was investigated based on the sodium/potassium balance of the cell membrane.The effects of different potassium salts on S.thermophilus 937 lyophilized survival rate were studied with different concentrations and types of potassium salts.The effect sequence of four potassium salts on S.thermophilus 937 lyophilized survival rate was KH2PO4～potassium glutamate>KCl>potassium citrate.The effects of different concentrations and types of sodium salts on the lyophilized survival rate of S.thermophilus 937 were studied.It was found that the lyophilized survival rate of sodium salts was higher than that of potassium salts at the same concentration of acid ions.The ratio of sodium/potassium solution in the protector solution affected the cell membrane ion balance and the lyophilized survival rate of S.thermophilus 937.In glutamate,when sodium/potassium ions are 8:2,cell membrane potential and ion channel balance can be better stabilized.In addition,under this state,Na+/K+-ATPase activity is 9.59 U/mgprot.It can be concluded that sodium glutamate can better inhibit the reduction of enzyme activity and thus reduce the damage to cells during freezing. |
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