CN112961797A - Lactobacillus acidophilus high-density fermentation medium and application thereof - Google Patents
Lactobacillus acidophilus high-density fermentation medium and application thereof Download PDFInfo
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Abstract
The invention discloses a lactobacillus acidophilus high-density fermentation medium and application thereof, belonging to the field of microbial fermentation. The culture medium comprises the following components in percentage by weight: 40-60g/L of lactose, 20-30g/L of bovine bone peptone, 15-30g/L of yeast extract powder, 10-20g/L of beef extract powder, 5-10g/L of sodium acetate, 3-6g/L of sodium citrate, 2-4g/L of dipotassium hydrogen phosphate, 1-2g/L of L-cysteine hydrochloride, 0.5-1g/L of magnesium sulfate, 0.1-0.5g/L of manganese sulfate, 1-1 g/L of Tween-800.5, 3-5g/L of tomato extract powder, 0.05-0.1g/L of methionine, 0.1-0.2g/L of histidine and 0.05-0.1g/L of serine. The culture medium can be used for culturing lactobacillus acidophilus, so that the viable bacteria concentration in the obtained fermentation liquor is greatly improved.
Description
Technical Field
The invention belongs to the field of microbial fermentation, relates to a culture medium, and particularly relates to a lactobacillus acidophilus high-density culture medium and application thereof.
Background
Lactobacillus acidophilus is a lactic acid bacterium with good probiotic function, but the main factors restricting the large-scale application of the lactobacillus at present are high-density culture of bacterial strains and preparation of high-activity bacterial powder. At present, the research on high-density culture of lactic acid bacteria mainly comprises the optimization of a culture medium and the optimization of a culture process.
The main components of the culture medium comprise: carbon source, nitrogen source, inorganic salt, growth factor and water. The carbon source and the nitrogen source provide energy for the microorganisms and form carbon and nitrogen elements required by the microorganisms; inorganic salts can maintain the osmotic pressure required for cell survival and provide other chemical elements required by microorganisms; the growth factor provides essential enzyme cofactor and active substance for the microorganism; the water provides a stable growth environment for the microorganisms. These factors can influence the growth rate of the microorganisms in the medium and the ultimate maximum density and biomass that can be achieved. Different lactic acid bacteria have different requirements for these conditions. The existing reported lactobacillus acidophilus high-density fermentation culture medium has the problems of high raw material cost and difficult acquisition. For example, in the high-density fermentation culture medium or culture method disclosed in chinese patents CN201710499446.7 and CN201910140769.6, dandelion extract, organic pea protein, and fish collagen peptide are added.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a lactobacillus acidophilus high-density fermentation medium and application thereof.
The purpose of the invention is realized by the following technical scheme:
a lactobacillus acidophilus high-density fermentation medium consists of a carbon source, a nitrogen source, buffer salt, trace elements, growth factors, Tween-80 and water, and comprises the following components in percentage by weight: 40-60g/L of lactose, 20-30g/L of bovine bone peptone, 15-30g/L of yeast extract powder, 10-20g/L of beef extract powder, 5-10g/L of sodium acetate, 3-6g/L of sodium citrate, 2-4g/L of dipotassium hydrogen phosphate, 1-2g/L of L-cysteine hydrochloride, 0.5-1g/L of magnesium sulfate, 0.1-0.5g/L of manganese sulfate, 1-1 g/L of Tween-800.5, 3-5g/L of tomato extract powder, 0.05-0.1g/L of methionine, 0.1-0.2g/L of histidine and 0.05-0.1g/L of serine.
Further, the lactobacillus acidophilus high-density fermentation medium comprises the following components in percentage by weight: 50g/L of lactose, 20g/L of bovine bone peptone, 15g/L of yeast extract powder, 15g/L of beef extract powder, 8g/L of sodium acetate, 4g/L of sodium citrate, 3g/L of dipotassium phosphate, 1g/L of L-cysteine hydrochloride, 0.8g/L of magnesium sulfate, 0.2g/L of manganese sulfate, 801 g/L of tween-801, 5g/L of tomato extract powder, 0.1g/L of methionine, 0.2g/L of histidine and 0.1g/L of serine.
The lactobacillus acidophilus high-density fermentation medium can be used for culturing lactobacillus acidophilus, so that the viable bacteria concentration in the obtained fermentation liquid is greatly improved.
A method for culturing lactobacillus acidophilus at high density comprises the following steps: inoculating Lactobacillus acidophilus seed solution into the high-density fermentation culture medium, adjusting initial fermentation pH to 6.4-6.8, adjusting fermentation rotation speed to 80-100rpm, fermenting for 6-8h, controlling fermentation broth pH to 5.4-5.7 with 16-23% NaOH solution, and culturing at 35-37 deg.C for 10-12 h.
Further, the lactobacillus acidophilus seed liquid is prepared by a method comprising the following steps: the lactobacillus acidophilus which is frozen and preserved is streaked on an MRS solid culture medium plate for culture, and the culture is carried out for 48h-60h at the temperature of 35-37 ℃. And selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and culturing at 37 ℃ for 10-12h for primary activation. Inoculating the primary activation solution to MRS liquid culture medium, culturing at 37 deg.C for 4-6h, and activating again to obtain seed solution. Wherein, the MRS liquid culture medium comprises the following components in percentage by weight: 5g/L beef extract, 4g/L yeast powder, 10g/L peptone, 20g/L glucose, 2g/L dipotassium hydrogen phosphate, 5g/L sodium acetate, 2g/L ammonium citrate, 0.2g/L magnesium sulfate and 0.05g/L manganese sulfate; MRS solid culture medium is added with 15g/L agar based on MRS liquid culture medium.
The lactobacillus acidophilus is preferably lactobacillus acidophilus LA 85.
The lactobacillus acidophilus high-density fermentation medium has the following characteristics:
(1) the carbon source and the nitrogen source are main components for forming the culture medium, and provide energy for the microorganisms and form carbon and nitrogen elements required by the microorganisms. The invention selects carbon source lactose and nitrogen source bovine bone peptone, yeast extract powder and beef extract powder which are suitable for lactobacillus acidophilus fermentation through research, and determines the mass ratio of the total amount of the carbon source and the nitrogen source to the mass ratio as follows: 50g/L carbon source +50g/L nitrogen source. If the nitrogen source is too much, the thalli grow excessively vigorously, and the thalli are easy to age and autolyze; too much carbon source tends to form a lower pH, which is not favorable for growth of the cells.
(2) The inhibition of low pH value on the bacteria caused by lactic acid can be relieved by adding a certain amount of buffer salt in the culture medium, the growth and the propagation of the bacteria are promoted, and the high-density culture of the lactobacillus acidophilus is realized. The buffer salt consists of sodium citrate, sodium acetate, dipotassium hydrogen phosphate and L-cysteine hydrochloride.
(3) The trace elements are used as enzyme activator or bioactive substance, and are essential for growth and propagation of microbe. The trace elements are required to different degrees by different microorganisms or different strains. Through research, the magnesium sulfate in the culture medium of Lactobacillus acidophilus LA85 is 0.5-1g/L, and the manganese sulfate is 0.1-0.5 g/L. Manganese ions are a composition component of lactate dehydrogenase, are one of key growth factors for growth of lactobacillus acidophilus, have promotion effect on microbial growth and product synthesis at low concentration, and often show obvious inhibition effect at high concentration.
(4) Lactobacillus acidophilus has high nutritional requirement, and needs abundant peptide, amino acid and vitamin nutrient substances for growth, so that certain growth factors are added into a culture medium to promote the rapid proliferation of the lactobacillus acidophilus, and the optimized screening shows that the amino acid and tomato extract powder have good proliferation promoting effects. Therefore, 3-5g/L of tomato extract powder and 0.2-0.4g/L of compound amino acid are added into the culture medium to promote the rapid proliferation of Lactobacillus acidophilus LA 85.
Compared with the existing culture medium and culture method, the lactobacillus acidophilus high-density fermentation culture medium and the culture method thereof have the advantages of simple components, simple and convenient preparation and low cost, and save materials and labor cost. The viable bacteria concentration in the fermentation liquid obtained by culturing Lactobacillus acidophilus LA85 with the culture medium can reach 9 × 109CFU/mL。
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
EXAMPLE 1 Effect of fermentation Medium Nitrogen Source proportioning
(1) Activation of a glycerol tube: lactobacillus acidophilus LA85 preserved in glycerol tubes was streaked on MRS solid medium plates and cultured at 37 ℃ for 48 h. Each 1L of MRS solid culture medium consists of the following components in percentage by weight: 1000mL of MRS liquid medium and 15g of agar. Each 1L of MRS liquid culture medium comprises the following components by weight: 5g of beef extract, 4g of yeast powder, 10g of peptone, 20g of glucose, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of ammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 1000mL of water.
(2) Liquid activation: and selecting a single colony, inoculating the single colony in a test tube containing 10mL of MRS liquid culture medium for static culture, performing primary activation at 37 ℃ for 10-12h, inoculating the strain suspension liquid in an infusion bottle containing 200mL of MRS liquid culture medium in an inoculation amount of 2% (V/V), and performing secondary activation at 37 ℃ for 4-6h to obtain a seed solution.
(3) Fermentation: inoculating the seed solution into a 15L fermentation tank filled with fermentation culture media with different formulas shown in the following table 1 in an inoculation amount of 2% (V/V), wherein the liquid loading amount is 10L, the initial pH value is adjusted to 6.8, the rotating speed is 100rpm/min, after fermentation for 6h, the pH value of the fermentation liquid is controlled to 5.4 by 23% NaOH solution, and the fermentation liquid is cultured for 12h at 37 ℃. The viable cell count of the resulting fermentation broth is shown in table 2 below.
TABLE 1 fermentation media with different ratios of nitrogen sources
TABLE 2 number of viable bacteria in fermentation broth
| Fermentation medium | Formulation 1 | Formulation 2 | Formulation 3 | Formulation 4 |
| Viable count (. times.10)9CFU/mL) | 9 | 8.4 | 8.1 | 7.4 |
Example 2 Effect of carbon Source species in fermentation Medium
The culture was carried out in a fermentation medium according to the method of example 1, containing the following composition: 50g/L carbon source, 20g/L beef bone peptone, 15g/L yeast extract powder, 15g/L beef extract powder, 8g/L sodium acetate, 4g/L sodium citrate, 3g/L dipotassium phosphate, 1 g/L-cysteine hydrochloride, 0.8/L magnesium sulfate, 0.2g/L manganese sulfate, 801 g/L tween-801 g/L, 5g/L tomato extract powder, 0.1g/L methionine, 0.2g/L histidine and 0.1g/L serine. Wherein the carbon source is lactose, glucose, sucrose and maltose.
The number of viable bacteria in the fermentation broth is shown in table 3 below.
TABLE 3 number of viable bacteria in fermentation broth
| Kind of carbon source | Lactose | Glucose | Sucrose | Maltose |
| Viable count (. times.10)9CFU/mL) | 9 | 5 | 7.3 | 6 |
Example 3 influence of the type and amount of amino acid addition to the fermentation Medium
The culture was carried out in a fermentation medium according to the method of example 1, containing the following composition: 50g/L of lactose, 20g/L of bovine bone peptone, 15g/L of yeast extract powder, 15g/L of beef extract powder, 8g/L of sodium acetate, 4g/L of sodium citrate, 3g/L of dipotassium phosphate, 1g/L of L-cysteine hydrochloride, 0.8/L of magnesium sulfate, 0.2g/L of manganese sulfate, 801 g/L of tween-801, 5g/L of tomato extract powder and amino acids with different types and contents, wherein the types and contents of the compound amino acids are shown in Table 4.
TABLE 4 amino acid types and amounts
The number of viable bacteria in the fermentation broth is shown in table 5 below.
TABLE 5 number of viable bacteria in fermentation broth
Example 4 Effect of tomato extract powder addition in fermentation Medium
The culture was carried out in a fermentation medium according to the method of example 1, containing the following composition: 50g/L of lactose, 20g/L of bovine bone peptone, 15g/L of yeast extract powder, 15g/L of beef extract powder, 8g/L of sodium acetate, 4g/L of sodium citrate, 3g/L of dipotassium phosphate, 1g/L of L-cysteine hydrochloride, 0.8/L of magnesium sulfate, 0.2g/L of manganese sulfate, 801 g/L of Tween-E, 0.1g/L of methionine, 0.2g/L of histidine, 0.1g/L of serine and tomato extract powder with different contents, wherein the content of the tomato extract powder is shown in Table 6, and the number of colonies in fermentation liquid is shown in Table 7.
TABLE 6 tomato extract content
| Content 1 | Content 2 | Content 3 | Content 4 | Content 5 | Content 6 | Content 7 | |
| Tomato extract content (g/L) | 5 | 1 | 2 | 3 | 4 | 6 | 7 |
TABLE 7 viable cell count in fermentation broth
| Tomato extract content (g/L) | Content 1 | Content 2 | Content 3 | Content 4 | Content 5 | Content 6 | Content 7 |
| Viable count (. times.10)9CFU/mL) | 9 | 2.1 | 3.2 | 5.8 | 7.1 | 9.1 | 9.1 |
Comparative example 1MRS liquid Medium As fermentation Medium
The culture was carried out in MRS liquid medium according to the method of example 1, the composition of MRS liquid medium was the same as above. The number of colonies in the fermentation broth was 0.48X 109CFU/mL。
Comparative example 2 fermentation Medium for culturing other species of Lactobacillus
Other lactobacilli were cultured in the medium according to the method of example 1, and the number of colonies in the fermentation broth is shown in table 8 below.
TABLE 8 number of viable bacteria in fermentation broth
From the above results of examples and comparative examples, it can be seen that: lactose and a nitrogen source are selected as sufficient composite nitrogen sources for the carbon source fermented by the lactobacillus acidophilus LA85, and when the composite amino acid and sufficient tomato extract powder are added, the high-density culture of the lactobacillus acidophilus LA85 is facilitated, the lactobacillus acidophilus LA85 is cultured by using the culture medium, and the viable count is higher than that of other lactobacillus. By using the high-density fermentation culture medium, the concentration of the viable bacteria in the lactobacillus acidophilus LA85 fermentation liquid can reach 9.1 multiplied by 109cfu/mL is about 20 times higher than MRS culture medium, and compared with other strains, the culture medium is more suitable for culturing Lactobacillus acidophilus LA 85.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A lactobacillus acidophilus high-density fermentation medium is characterized in that: comprises the following components in percentage by weight: 40-60g/L of lactose, 20-30g/L of bovine bone peptone, 15-30g/L of yeast extract powder, 10-20g/L of beef extract powder, 5-10g/L of sodium acetate, 3-6g/L of sodium citrate, 2-4g/L of dipotassium hydrogen phosphate, 1-2g/L of L-cysteine hydrochloride, 0.5-1g/L of magnesium sulfate, 0.1-0.5g/L of manganese sulfate, 1-1 g/L of Tween-800.5, 3-5g/L of tomato extract powder, 0.05-0.1g/L of methionine, 0.1-0.2g/L of histidine and 0.05-0.1g/L of serine.
2. The lactobacillus acidophilus high-density fermentation medium according to claim 1, characterized in that: comprises the following components in percentage by weight: 50g/L of lactose, 20g/L of bovine bone peptone, 15g/L of yeast extract powder, 15g/L of beef extract powder, 8g/L of sodium acetate, 4g/L of sodium citrate, 3g/L of dipotassium phosphate, 1g/L of L-cysteine hydrochloride, 0.8g/L of magnesium sulfate, 0.2g/L of manganese sulfate, 801 g/L of tween-801, 5g/L of tomato extract powder, 0.1g/L of methionine, 0.2g/L of histidine and 0.1g/L of serine.
3. A lactobacillus acidophilus high-density fermentation medium according to claim 1 or 2, characterized in that: the lactobacillus acidophilus is lactobacillus acidophilus LA 85.
4. Use of a high-density fermentation medium of lactobacillus acidophilus according to claim 1 or 2 for the culture of lactobacillus acidophilus.
5. A method for culturing lactobacillus acidophilus at high density is characterized in that: the method comprises the following steps: inoculating lactobacillus acidophilus seed liquid into a lactobacillus acidophilus high-density fermentation culture medium according to claim 1 or 2, adjusting the initial fermentation pH value to be 6.4-6.8, adjusting the fermentation rotation speed to be 80-100rpm, controlling the fermentation liquid pH value to be 5.4-5.7 by using 16-23% NaOH solution after fermenting for 6-8h, and culturing for 10-12h at 35-37 ℃.
6. The method for high-density culture of Lactobacillus acidophilus according to claim 5, characterized in that: the lactobacillus acidophilus seed liquid is prepared by the method comprising the following steps: the lactobacillus acidophilus which is frozen and preserved is streaked on an MRS solid culture medium plate for culture, and the culture is carried out for 48h-60h at the temperature of 35-37 ℃. Selecting single colony, inoculating in MRS liquid culture medium, culturing at 37 deg.C for 10-12 hr, and activating; inoculating the primary activation solution to MRS liquid culture medium, culturing at 37 deg.C for 4-6h, and activating again to obtain seed solution.
7. The method for high-density culture of Lactobacillus acidophilus according to claim 5 or 6, characterized in that: the lactobacillus acidophilus is lactobacillus acidophilus LA 85.
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