| The world's largest functional food market is occupied by probiotic foods. One of the developments of probiotics is Lactobacillus acidophilus whose powder is widely used in food and pharmaceutical industry. The study of high density culture and protective agent of freeze-dried of Lactobacillus acidophilus was to increase the number of production living cells, achieve industrial production and reduce the production of cost. In the work, MRS (De Man Rogosa and Sharpe) medium was optimized, culture methods of Lactobacillus acidophilus were discussed, the most bacterial count powder was obtained by optimizing cryoprotectant and adding anti-freezing factors and the storage effect of powder was discussed in different environments.The effect of amino acids, sugars, reducing substances and natural extracts on the growth of Lactobacillus acidophilus was studied by single-factor test. The research shows that glutamic acid, arginine, leucine, lysine, methionine, proline, phenylalanine, ascorbic acid, cysteine hydrochloride, carrot juice, tomato juice and mushroom sauce can promote the growth of Lactobacillus acidophilus.Modified MRS medium which is peptone 10 g/L, beef extract 8 g/L, yeast extract 6g/L, glucose 21 g/L-22g/L, KH2PO43.3g/L, Ammonium citrate dibasic 2g/L, Sodium acetate trihydrate 6 g/L-6.25g/L, MgSO42g/L, Tween-80 lml/L and MnSO40.05g/L, was optimized by response surface method. The optimized culture condition was 5% inoculum, incubation time 18h, the culture temperature 37℃and anaerobic static culture. Bacterial Count of Lactobacillus acidophilus of Medium was up to 5.3×109cfu/mL by adding alkali, compared with the control increased by 2 times.The results show that Cell-Recycle culture could make the number of cells in the medium reach to 3.5 X 1010 cfu/mL-3.6×1010 cfu/mL. The accumulation of metabolites in the medium is the primary factor inhibited growth of Lactobacillus acidophilus.Freeze-drying process conditions were discussed by Single-factor test. The results showed 6000r/min×10min collecting cells, standing 30min,-35℃prefreezing 24h.Using factorial experiment, the steepest ascending method and two-factor experiment screened and optimized to cryoprotectant of Lactobacillus acidophilus. The optimum compounding of cryoprotectant is skimmed milk powder 100g/L, glycerol 50mL/L, trehalose 50g/L, ascorbic acid lg/L, NaCl 3g/L, gelatin 1g/L, the survival rate of freeze-drying cells is up to 98% and the number of viable cells of the powder is up to 1.2×1011cfu/g. If the composite anti-freeze factors were added medium to culture Lactobacillus acidophilus, the survival rate of freeze-drying cells was increased. Research shows that 2 groups of anti-freeze compound factors:①glycerol 4mL/L, trehalose 4g/L, sodium chloride 4g/L and alanine 40mg/L,②inositol 0.1g/L, NaCl 3g/L, bovine serum albumin 70 mg/L and alanine 30mg/LBy comparing the color found that the powder color of FD1, FD2 and FD3 (freeze-drying process of the powder) were ideal, and by comparing scanning electron microscopy images found that medium(water) of FD1, FD2 and FD3 did not form large ice crystals, and cells, cryoprotectant and media (water) did not occur to Separation in the process of pre-freeze. Study proves that the effect environmental and human factors on the quality of the powder were very significant, while the controlled situation of FD1 and FD3 was better than FD2 in the overall consideration. The probiotic powder and the goat milk added to probiotic powder cold storage were much better, the storage of FD1 and FD2 powder was better than FD3, while the storage of the goat milk added to FD3 powder was best than FD1 and FD2 at 4℃.The high density culture of Lactobacillus acidophilus way, that could effectively reduce the cost of freeze-dried powder preparation, was studied in the work. The number of prepared powder living cells has reached the requirements of industrial production. The application technology of anti-freezing factors lay the technical support to improve anti-freezing ability of Lactobacillus acidophilus. |
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