A kind of Lactobacillus casei proliferation culture medium and culture method

Abstract

The invention provides a lactobacillus casei proliferation culture medium and a culture method, comprising a culture medium A and a culture medium B, wherein the culture medium A comprises 10-30 g / L of a carbon source, 15-25 g / L of a nitrogen source, 1-2 g / L of black corn juice, 0.5-1.5 g / L of Tween, 0.1-0.5 μg / mL of Xanthate, and 0.1-0.5 g / L of ascorbic acid, and the culture medium B comprises 10-30 g / L of a carbon source, 20-40 g / L of a nitrogen source, 1.5-7 g / L of an inorganic salt, 0.5-1.5 g / L of Tween, 0.5-1 μg / mL of Xanthate, 5-10 μg / mL of sodium naphthaleneacetic acid, and 0.1-0.5 g / L of ascorbic acid. The lactobacillus casei proliferation culture medium can improve the growth and proliferation of lactobacillus casei and improve the bacterial cell concentration by adding xanthate, sodium naphthaleneacetic acid, and ascorbic acid, thereby obtaining high-density and high-activity lactobacillus casei.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a lactobacillus casei proliferation culture medium and a culture method. Background Art

[0002] Probiotics are a type of active microbial food raw material that is beneficial to human health. Commonly used probiotics include Lactobacillus acidophilus and Bifidobacterium, which are called "three healthy bacteria" together with Lactobacillus casei. Lactobacillus casei has good acid and bile resistance, can reduce plasma cholesterol, enhance the host's nonspecific resistance to microbial pathogens, accelerate the removal of pathogens in the intestine, treat intestinal flora disorders and enhance intestinal permeability, thereby preventing food allergies and acute diarrhea. In addition, Lactobacillus casei can also increase anti-low-density oxidized lipid antibodies and lymphocytes, significantly enhance the phagocytic effect of granulocytes, regulate the host immune system, and prevent the occurrence of tumors. Due to its ability to survive stably in the human intestine and its numerous medical and health care functions, Lactobacillus casei is widely used as a probiotic in the development of functional foods, especially dairy products, and has gradually attracted people's attention. At present, Lactobacillus casei, as one of the probiotics, is used as a starter and auxiliary starter for dairy products such as milk, yogurt, soy milk, cream and cheese, especially in cheese. The growth of Lactobacillus casei is affected by the composition of the culture medium. Therefore, the premise for obtaining high-density and high-activity Lactobacillus casei for industrial application is to find a proliferation culture medium suitable for the bacteria. Summary of the invention

[0003] In view of this, the present invention aims to provide a Lactobacillus casei proliferation culture medium and a culture method to solve the above problems.

[0004] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0005] A lactobacillus casei proliferation culture medium comprises a culture medium A and a culture medium B, wherein the culture medium A comprises 10-30 g / L of a carbon source, 15-25 g / L of a nitrogen source, 1-2 g / L of black corn juice, 0.5-1.5 g / L of Tween, 0.1-0.5 μg / mL of xanthate, and 0.1-0.5 g / L of ascorbic acid, and the culture medium B comprises 10-30 g / L of a carbon source, 20-40 g / L of a nitrogen source, 1.5-7 g / L of an inorganic salt, 0.5-1.5 g / L of Tween, 0.5-1 μg / mL of xanthate, 5-10 μg / mL of sodium naphthaleneacetic acid, and 0.1-0.5 g / L of ascorbic acid.

[0006] Furthermore, in culture medium A, the carbon source includes 15-20 g / L glucose and 5-15 g / L fructose; the nitrogen source includes 5-10 g / L tryptone, 10-15 g / L beef extract and 1-5 g / L yeast extract.

[0007] Furthermore, in culture medium B, the carbon source includes 15-20 g / L glucose and 5-15 g / L fructose; the nitrogen source includes 8-15 g / L tryptone, 15-25 g / L beef extract and 3-9 g / L yeast extract; the inorganic salts include 1-5 g / L diammonium hydrogen citrate, 0.05-0.15 g / L calcium sulfate, 0.2-0.8 g / L sodium acetate, 0.5-1 g / L magnesium sulfate, 0.03-0.8 g / L manganese sulfate, and 1-5 g / L dipotassium hydrogen phosphate.

[0008] Furthermore, in culture medium A, the ratio of xanthate to ascorbic acid was 1:1000.

[0009] Furthermore, the concentration of ascorbic acid added to medium A and medium B was kept consistent.

[0010] The present invention also provides a method for culturing Lactobacillus casei in the Lactobacillus casei proliferation culture medium as described above, comprising the steps of:

[0011] 1) Inoculate the activated Lactobacillus casei into culture medium A and culture at 35-37°C for 10-18 hours;

[0012] 2) Inoculate the bacterial suspension cultured in step 1) into medium B and continue culturing at 35-37°C for 12-48 hours.

[0013] Furthermore, the inoculation amount in step 1) is 4%-8%.

[0014] The inoculum size refers to the ratio of the volume of the seed solution transferred to the volume of the culture solution after inoculation. For example, the inoculum size of the seed solution can be 4% (v / v), 4.5% (v / v), 5% (v / v), 5.5% (v / v), 6% (v / v), 6.5% (v / v), 7% (v / v), 7.5% (v / v), 8% (v / v), etc.

[0015] Furthermore, the total culturing time of step 1) and step 2) is greater than 36 hours.

[0016] Compared with the prior art, the Lactobacillus casei proliferation culture medium and culture method of the present invention have the following advantages:

[0017] The lactobacillus casei proliferation culture medium of the invention comprises two culture mediums, and the growth and proliferation of the lactobacillus casei and the bacterial cell concentration can be improved by adding xanthate, sodium naphthaleneacetate and ascorbic acid into the culture medium, thereby obtaining high-density and high-activity lactobacillus casei. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 This is a comparison of the number of viable bacteria after the concentration of Xanthin was changed in Schemes 1-9;

[0020] Figure 2 This is a comparison of the number of viable bacteria after the concentration of Xanthin was changed in Schemes 10-19;

[0021] Figure 3 This is a comparison of the number of viable bacteria after the concentration of sodium naphthylacetic acid in schemes I-X was changed. DETAILED DESCRIPTION

[0022] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1 Cultivation of Lactobacillus casei

[0025] The cultivation method comprises the following steps:

[0026] 1) The activated Lactobacillus casei was inoculated into medium A with an inoculum size of 5% and cultured at 35°C for 12 hours; medium A included 15 g / L glucose, 10 g / L fructose, 8 g / L tryptone, 13 g / L beef extract, 2 g / L yeast extract powder, 1 g / L black corn juice, 0.8 g / L Tween, 0.1 μg / mL Xanthium sibiricum, and 0.1 g / L ascorbic acid;

[0027] 2) The bacterial suspension cultured in step 1) was inoculated into medium B and cultured at 35°C for 30 hours;

[0028] Culture medium B includes 18 g / L glucose, 10 g / L fructose, 10 g / L tryptone, 22 g / L beef extract, 6 g / L yeast extract, 1 g / L diammonium hydrogen citrate, 0.1 g / L calcium sulfate, 0.5 g / L sodium acetate, 0.6 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 3 g / L dipotassium hydrogen phosphate, 0.8 g / L Tween, 0.6 μg / mL xanthate, 5 μg / mL sodium naphthaleneacetic acid, and 0.1 g / L ascorbic acid.

[0029] After cultivation, the number of viable bacteria detected was 8.97×10 10 cfu / ml.

[0030] Example 2 Cultivation of Lactobacillus casei

[0031] The cultivation method comprises the following steps:

[0032] 1) The activated Lactobacillus casei was inoculated into medium A with an inoculum size of 5% and cultured at 36°C for 10 hours; medium A included 18 g / L glucose, 10 g / L fructose, 10 g / L tryptone, 10 g / L beef extract, 1 g / L yeast extract powder, 1.5 g / L black corn juice, 1.0 g / L Tween, 0.3 μg / mL Xanthium sibiricum, and 0.3 g / L ascorbic acid;

[0033] 2) The bacterial suspension cultured in step 1) was inoculated into medium B and cultured at 36°C for 32 hours;

[0034] Culture medium B includes 15 g / L glucose, 10 g / L fructose, 13 g / L tryptone, 20 g / L beef extract, 3 g / L yeast extract, 2 g / L diammonium hydrogen citrate, 0.13 g / L calcium sulfate, 0.2 g / L sodium acetate, 0.5 g / L magnesium sulfate, 0.15 g / L manganese sulfate, 3 g / L dipotassium hydrogen phosphate, 1 g / L Tween, 0.8 μg / mL xanthate, 8 μg / mL sodium naphthaleneacetate, and 0.3 g / L ascorbic acid.

[0035] After cultivation, the number of viable bacteria detected was 9.21×10 10 cfu / ml.

[0036] Example 3 Cultivation of Lactobacillus casei

[0037] The cultivation method comprises the following steps:

[0038] 1) The activated Lactobacillus casei was inoculated into medium A with an inoculum size of 5% and cultured at 37°C for 15 hours; medium A included 20 g / L glucose, 8 g / L fructose, 6 g / L tryptone, 14 g / L beef extract, 3 g / L yeast extract powder, 2 g / L black corn juice, 1.5 g / L Tween, 0.5 μg / mL Xanthium sibiricum, and 0.5 g / L ascorbic acid;

[0039] 2) The bacterial suspension cultured in step 1) was inoculated into medium B and cultured at 37°C for 28 hours;

[0040] Culture medium B includes 20 g / L glucose, 8 g / L fructose, 15 g / L tryptone, 18 g / L beef extract, 5 g / L yeast extract, 3 g / L diammonium hydrogen citrate, 0.08 g / L calcium sulfate, 0.3 g / L sodium acetate, 0.7 g / L magnesium sulfate, 0.2 g / L manganese sulfate, 2 g / L dipotassium hydrogen phosphate, 1.5 g / L Tween, 1 μg / mL xanthate, 10 μg / mL sodium naphthaleneacetate, and 0.5 g / L ascorbic acid.

[0041] After cultivation, the number of viable bacteria detected was 9.14×10 10 cfu / ml.

[0042] Experiment 1: Optimization of culture medium

[0043] Based on Example 1, the culture conditions of Lactobacillus casei were changed according to Table 1, and the final viable count was detected.

[0044] Table 1 Schemes of different Lactobacillus casei culture conditions

[0045]

[0046] It can be found by Table 1 and the embodiments that the addition of xanthate, sodium naphthaleneacetate, and ascorbic acid in culture medium A and culture medium B can improve the growth and proliferation of Lactobacillus casei, and the cell concentration is higher. It can be seen from schemes a, b, and n that culture medium A and culture medium B need to add xanthate simultaneously to better promote the growth and proliferation of Lactobacillus casei. It can be seen from schemes c and f that culture medium B needs to add sodium naphthaleneacetate, and can only be added in culture medium B, and adding in culture medium A is not conducive to the growth of Lactobacillus casei. It can be seen from schemes d, e, j, and k that ascorbic acid is indispensable in culture medium A and B, and the concentration of ascorbic acid in culture medium A and B should be kept consistent to promote the growth of Lactobacillus casei; in addition, in scheme j, xanthate is 0.1 μg / mL, and ascorbic acid is ascorbic acid 0.2g / L, and the addition ratio of xanthate and ascorbic acid not meeting is 1:1000, and its effect deteriorates. It can be seen from schemes g, h, and i that the amount of Xanthate in culture medium A should be less than that in culture medium B. Otherwise, it will be unfavorable for the growth of Lactobacillus casei and reduce the bacterial concentration. It can be seen from schemes l and m that the number of days of culture in culture medium A should be less than that in culture medium B. Culture in culture medium A is equivalent to activating Lactobacillus casei first, and then proliferating in culture medium B in large quantities, and the culture time in culture medium A cannot be too long.

[0047] Experiment 2 Effect of Xanthium sibiricum concentration

[0048] Based on Example 1, the concentration of Xanthate in culture medium A was changed to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 1, and 10 μg / mL, respectively, and they were named as Schemes 1-9, respectively, wherein Scheme 1 is Example 1; the concentration of Xanthate in culture medium B was changed to 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, and 10 μg / mL, respectively, and they were named as Schemes 10-19, wherein Scheme 12 is Example 1; and the viable counts at the end of each scheme were detected, and the results are shown in Tables 2-3, Figure 1-2 shown.

[0049] Table 2 Xanthium sibiricum concentration table for schemes 1-9

[0050]

[0051] Table 3 Xanthium sibiricum concentration table for scheme 10-19

[0052]

[0053] Through Table 2-3, Figure 1-2 It can be seen that when the concentration of Xanthate in culture medium A is 0.1-0.5 μg / mL and the concentration of Xanthate in culture medium B is 0.5-1 μg / mL, the number of viable bacteria is higher; and the effect is best when the concentration of Xanthate in culture medium A is 0.3 μg / mL and the concentration of Xanthate in culture medium B is 0.8 μg / mL.

[0054] Experiment 3 Effect of the concentration of sodium naphthaleneacetic acid

[0055] Based on Example 1, the concentration of sodium naphthaleneacetic acid in culture medium B was changed to 4, 5, 6, 7, 8, 9, 10, 11, 12, and 50 μg / mL, respectively, and named as Schemes I-X, wherein Scheme II is Example 1; and the viable counts at the end of each scheme were detected, and the results are shown in Table 4. Figure 3 shown.

[0056] Table 4 Naphthylacetic acid sodium salt concentration table for schemes I-X

[0057]

[0058] Through Table 4, Figure 3 It can be seen that the effect is best when the concentration of sodium naphthylacetic acid is 5-10 μg / mL.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Lactobacillus casei proliferation culture medium, characterized in that: The method comprises a culture medium A and a culture medium B. The culture medium A comprises 10-30 g / L of a carbon source, 15-25 g / L of a nitrogen source, 1-2 g / L of black corn juice, 0.5-1.5 g / L of Tween, 0.1-0.5 μg / mL of Xanthate, and 0.1-0.5 g / L of ascorbic acid. The culture medium B comprises 10-30 g / L of a carbon source, 20-40 g / L of a nitrogen source, 1.5-7 g / L of an inorganic salt, 0.5-1.5 g / L of Tween, 0.5-1 μg / mL of Xanthate, 5-10 μg / mL of sodium naphthaleneacetic acid, and 0.1-0.5 g / L of ascorbic acid.

2. Lactobacillus casei proliferation culture medium according to claim 1, is characterized in that: In culture medium A, the ratio of Xanthate to ascorbic acid was 1:1000.

3. Lactobacillus casei proliferation culture medium according to claim 1, is characterized in that: The concentration of ascorbic acid added to medium A and medium B was kept the same.

4. the method for culturing Lactobacillus casei in a Lactobacillus casei proliferation medium as claimed in any one of claims 1 to 3, is characterized in that: The steps include: 1) Inoculate the activated Lactobacillus casei into culture medium A and culture at 35-37°C for 10-18 hours; 2) Inoculate the bacterial suspension cultured in step 1) into medium B and continue culturing at 35-37°C for 12-48 hours.

5. the method for cultivating Lactobacillus casei in a lactobacillus casei proliferation medium according to claim 4 is characterized in that: The total culturing time of step 1) and step 2) is greater than 36 hours.

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