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CN-122012273-A - High-density culture method of lactobacillus amylophilus

CN122012273ACN 122012273 ACN122012273 ACN 122012273ACN-122012273-A

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a high-density culture method of lactobacillus amylophilus. The invention improves the fermentation raw materials and the fermentation process of the lactobacillus amylophilus, changes the traditional continuous culture into fed-batch culture, optimizes proper feeding time and feeding volume by feeding carbon and nitrogen sources, improves the fermentation bacterial load by about one time, and in addition, adopts trehalose, skimmed milk powder, vitamin C and other components as a spray drying protective agent, thereby obviously improving the stability of bacterial powder products.

Inventors

  • WU CHUANJU
  • ZHAO PENGBO
  • SHI XINHUI
  • ZHU RUIXIANG
  • WU HONGYAN

Assignees

  • 山东天润和生物工程有限公司

Dates

Publication Date
20260512
Application Date
20260119

Claims (10)

  1. 1. A high-density culture method of lactobacillus amylophilus, which is characterized by comprising the following steps: s1, activating and culturing strains; s2, culturing in a first-stage seed tank, namely inoculating the bacterial suspension cultured in the S1 into the seed tank containing a seed culture medium according to an inoculum size of 0.3% -1% (v/v), controlling the temperature to be 35-40 ℃, and culturing to obtain a first-stage seed liquid, wherein the initial rotating speed is 50-100 rpm, and the ventilation ratio is 0.03-0.05 vvm; S3, culturing in a fermentation tank, namely transferring the primary seed liquid in the step S2 into a fermentation tank containing a fermentation medium, and starting feeding when the fermentation culture is carried out until the net OD 600 is more than or equal to 3.0, wherein the feeding is carried out for 4-6 hours, and the fermentation period is 16-20 hours, so as to obtain the high-density fermentation liquid of the lactobacillus amylophilus; The formula of the fermentation medium comprises 15-40 g/L corn steep liquor/soybean protein powder, 5-30 g/L yeast extract, 10-30 g/L glucose, 10-20 g/L starch, 1-8 g/L monopotassium phosphate, 1-5 g/L dipotassium phosphate, 1-5 g/L sodium acetate, 1-8 g/L magnesium sulfate, 1-7 g/L urea, 0.5-5 g/L ammonium sulfate and 0.5-1.5 g/L manganese sulfate; the feed formula comprises 300-600 g/L of starch, 30-60 g/L of ammonium sulfate and 50-150 g/L of glucose.
  2. 2. The method according to claim 1, wherein the strain activation culture in S1 is specifically that the preserved strain of lactobacillus amylophilus is activated and cultured by a plate culture medium until a third-generation lactobacillus amylophilus strain suspension is obtained; The formula of the plate culture medium comprises 5-15 g/L peptone, 5-15 g/L beef extract powder, 5-8 g/L yeast extract powder, 15-25 g/L glucose, 0.5-1.5 g/L tween-80, 1.5-3.5 g/L dipotassium hydrogen phosphate, 2.5-5 g/L sodium acetate, 2.5-3.5 g/L ammonium citrate, 0.5-1 g/L magnesium sulfate, 0.1-0.2 g/L manganese sulfate, 0.1-0.5 g/L-cysteine hydrochloride and 15-20 g/L agar powder.
  3. 3. The culture method according to claim 1, wherein in S2, the seed culture medium comprises 10-20 g/L of peptone, 10-30 g/L of yeast extract, 10-30 g/L of glucose, 10-20 g/L of beef extract powder, 1-8 g/L of potassium dihydrogen phosphate, 1-8 g/L of magnesium sulfate, 3-5 g/L of sodium acetate, 0.5-1.5 g/L of manganese sulfate and 0.5-1 g/L of tween-80.
  4. 4. The method according to claim 1, wherein in S2, the first seed liquid is obtained when the pH and OD 600 are periodically detected and the microscopic examination is carried out, and when the pH is 4.5-5.0 and the OD 600 is not less than 3.5,100 times of the total of 100 per field of view in the microscopic examination is carried out.
  5. 5. The culture method according to claim 1, wherein in S3, the fermentation condition is that the inoculation amount is 5% -10% (v/v), sterile air is introduced, stirring is started at 60-100 rpm, the ventilation ratio is 0.03-0.05 vvm, the temperature is set at 36-37 ℃, fermentation is started, the initial pH is 6.3-6.5, the calibrated dissolved oxygen amount is 100%, the clicking pH is self-controlled, and the pH is controlled at 6.3-6.5.
  6. 6. The culture method according to claim 1, wherein in S3, when the pH is automatically controlled by clicking, pH is adjusted by using 20% -30% sodium hydroxide sterilized in advance.
  7. 7. The culture method according to claim 1, wherein in S3, when the foam reaches the top view mirror of the fermenter during the culture, the polyether defoamer is added in an amount of 20wt% to 30 wt%.
  8. 8. The method according to claim 1, wherein the lactobacillus amylophilus in the high-density fermentation broth has a bacterial count of 3X 10 10 cfu/mL or more.
  9. 9. The probiotics powder containing the lactobacillus amylophilus is characterized by comprising the lactobacillus amylophilus obtained by the culture method according to any one of claims 1-8 and a protective agent, wherein the protective agent comprises, by mass of lactobacillus amylophilus thalli, 1% -5% of skimmed milk powder, 1% -3% of maltodextrin, 1% -2% of trehalose, 2% -5% of microcrystalline cellulose sodium, 0.5% -1% of vitamin C and 1% -2% of sorbitol.
  10. 10. The probiotic bacterial powder according to claim 9, wherein the bacterial powder is obtained by spray drying, and the spray drying condition is that the air inlet temperature is 125-135 ℃ and the air outlet temperature is 65-75 ℃.

Description

High-density culture method of lactobacillus amylophilus Technical Field The invention belongs to the technical field of microbial fermentation, and particularly relates to a high-density culture method of lactobacillus amylophilus. Background The lactobacillus amylophilus is a gram-positive, facultative anaerobic and spore-free lactobacillus, has unique starch hydrolysis capability, can directly utilize raw starch or gelatinized starch to produce acid, and therefore has important application potential in the fields of feed, food, environmental protection, biological medicine and the like. At present, the liquid fermentation of the lactobacillus amylophilus mostly adopts a traditional MRS culture medium or a simple optimized basic culture medium, and has the following core technical bottlenecks: First, it is difficult to combine the fermentation yield and the bacterial activity. The viable count of the lactobacillus amyloliquefaciens obtained by adopting the traditional conventional liquid fermentation process as disclosed in the patent CN104212743A is only 30-50 hundred million CFU/mL, although the literature records that the bacterial count of the lactobacillus amylophilus can reach 8×10 9 CFU/mL during high-density culture, the lactobacillus amylovorus is extremely easy to cause the reduction of the integrity of cell membranes of the bacteria due to the accumulation of lactic acid, the increase of the concentration of metabolites and the superposition of triple stress with overhigh cell density, the breakage rate is increased to 35%, the activity of starch hydrolase is reduced by 50%, and the final viable count can reach a certain level, but the effective cell proportion with the starch hydrolysis function is only 60-70%, and the requirements (Zhang Jun and the like) of industrial high-activity products cannot be met, the stress response of the lactic acid bacteria and the influence on carbohydrate metabolism [ J ]. Chinese food science report, 2017,17 (6): 1-9.); Secondly, the cost of the culture medium is high and the suitability is poor. The traditional culture relies on nitrogen sources such as high-valence yeast extract, peptone and the like, the cost ratio of raw materials is high, and although industrial byproducts (such as bean curd yellow serofluid and sweet potato juice) can replace part of raw materials, the fluctuation of components is large, and the large difference of bacteria among batches is easily caused. The invention adopts low-cost raw materials as main fermentation raw materials, reduces the cost of the raw materials, and improves the fermentation bacteria amount to 300 hundred million by adjusting the carbon-nitrogen ratio in a fermentation formula and adopting a fed-batch process. In addition, the preparation of the probiotics into dry powder provides support and guarantee for the wide application of the probiotics, but the protective agents adopted in the probiotic spraying and drying process in the prior art are mainly maltodextrin, microcrystalline cellulose sodium, sorbitol and other components, the total yield of the spraying powder is only about 20%, and the loss rate of the probiotics is high. How to increase the viable count of the probiotics fermentation liquor and the spray drying survival rate becomes the research focus of the industrial production of the probiotics dry powder. Therefore, developing a culture medium suitable for lactobacillus amylophilus and preparing a set of specific fermentation process and spray drying protective agent are important to obtaining high-concentration fermentation liquor and high-yield bacterial powder. Disclosure of Invention In order to solve the technical problems, the invention provides a high-density culture method of lactobacillus amylophilus. Compared with the traditional method, the method has the greatest characteristics that in the fermentation culture process of the lactobacillus amylophilus, an optimized formula is adopted, on one hand, corn steep liquor/soybean protein powder with low price is adopted to replace raw materials such as peptone, beef extract powder and the like, so that the raw material cost of a fermentation culture medium is greatly saved, on the other hand, a feeding process is adopted, and components such as starch, glucose, ammonium sulfate and the like are fed in batches, so that a carbon source and an inorganic nitrogen source are provided for the propagation of the lactobacillus amylophilus, and finally, the fermentation bacteria amount of the lactobacillus amylophilus is remarkably improved. In addition, the formula of the spray drying protective agent is further optimized, and finally, the lactobacillus amylophilus obtained through fermentation culture by the process is improved from 50 hundred million to 250-300 hundred million before improvement, 13000 hundred million of spray drying protective agent and 85% -90% of spray powder yield. The technical scheme provided by the invention is as follows: a