CN103834602A - High density fermentation medium for lactic acid bacteria - Google Patents

Abstract

The invention relates to a culture technology for microorganism, and particularly relates to a high density fermentation medium for lactic acid bacteria. The minimal medium of the high density fermentation medium for the lactic acid bacterial comprises following components and contents: 25g/L of glucose, 10g/L of soy peptone, 8g/L of yeast powder, 2g/L of K2HPO4, 7g/L of CH3COONa, 3g/L of sodium citrate, 0.2 g/L of MgSO4, 0.1g/L of FeCl2, and 0.05g/L of MnSO4, the minimal medium comprises following additive and additive amount: 25mL/L of fermented supernate of sterile saccharomyces cerevisiae. According to the high density fermentation medium, the fermented supernate of sterile saccharomyces cerevisiae is firstly prepared, then the fermented supernate is added into the sterilized and cooled fermentation minimal medium, so that the operation is simple, the cost is low; what is more important is that the density of the active lactic acid bacteria reaches 1012CFU/g, which is about 100 times that (1010CFU/g) of common zymophyte.

Description

A kind of milk-acid bacteria high density fermentation culture medium

Technical field

The present invention relates to the culture technique of a kind of microorganism, especially relate to the high density fermentation culture medium of a kind of milk-acid bacteria.

Background technology

Milk-acid bacteria, as the putative safe microorganism of a class, for the processing of multiple fermented product, has been eaten more than one thousand years by the mankind for a long time.The regulating effect of milk-acid bacteria to HUMAN HEALTH, as regulated gastrointestinal bacterial flora composition, prevent and treat various diarrhoea, immunomodulatory, hypertension, oxidation-resistance, reduction serum cholesterol etc. and obtained broad research and discussion.

China's " prebiotic mushroom protective foods is declared and evaluated regulation " regulation: viable bacteria class probiotic health food viable bacteria number within its quality guaranteed period must not be less than 10 ⁶cfu/mL (g).Can find out, the activity of milk-acid bacteria microbial inoculum and its quantity are closely related, and therefore, the high-density culture technology of milk-acid bacteria just becomes the key that milk-acid bacteria is applied.In the fermented liquid that traditional standing for fermentation method obtains, the fermentation density of thalline is lower, in order to obtain more viable count, just need to increase the volume of reactor or increase fermentation times etc., thereby increase the separation costs of biomass, extend manufacture cycle, thereby improve production cost, reduction production efficiency.

At present, both at home and abroad all in the high-density culture technology of inquiring into milk-acid bacteria, the method adopting has carries out controlled atmosphere cultivation, has and adds the somatomedins such as amino acid, also has to adopt to add that the material of polypeptide class, oligosaccharides or tea polyphenols cultivates.Utilize above-mentioned technology to cultivate, have that complicated operation, cost are large, product component complicated with separate the problems such as difficult, be difficult for applying.

Summary of the invention

The technical problem to be solved in the present invention is to overcome the deficiencies in the prior art, provides a kind of with low cost, simple to operate, and the lactobacillus-fermented substratum that milk-acid bacteria thalline composition is single, activity is high, concentration is high obtaining.

For solving the problems of the technologies described above, the technical scheme that the present invention proposes is as follows:

A kind of milk-acid bacteria high density fermentation culture medium, minimum medium component and content: glucose 25g/L, soy peptone 10g/L, yeast powder 8g/L, K ₂hPO ₄2g/L, CH ₃cOONa7g/L, Trisodium Citrate 3g/L, MgSO ₄0.2g/L, FeCl ₂0.1g/L, MnSO ₄0.05g/L; Minimum medium additive and addition thereof: aseptic fermentation by saccharomyces cerevisiae supernatant liquor 25mL/L.

A kind of milk-acid bacteria high density fermentation culture medium, while it is characterized in that configuring substratum until 121 DEG C of autoclaving 15min of minimum medium and cooling after, by aseptic technique, aseptic fermentation by saccharomyces cerevisiae supernatant liquor is added, and stirs.

A kind of milk-acid bacteria high density fermentation culture medium, is characterized in that the preparation method of the aseptic fermentation by saccharomyces cerevisiae supernatant liquor of minimum medium additive is as follows:

(1) preparation of yeast saccharomyces cerevisiae seed liquor: inoculate after test tube slant activation being kept at the yeast saccharomyces cerevisiae in dormant state in lyophilize pipe, pass through again shake-flask culture, 30 DEG C, under 200rpm condition, cultivate 24h, obtain seed liquor, by measuring OD value under 560nm wavelength, to guarantee that in every a collection of seed liquor, yeast saccharomyces cerevisiae thalline number is consistent;

(2) configuration fermentation by saccharomyces cerevisiae substratum: peptone 20g/L, yeast extract 10g/L, glucose 2g/L, appropriate distilled water dissolves, then adds 15mL/L0.2% adenine solution, and 120 DEG C of autoclaving 15min are cooling for subsequent use;

(3) inoculation fermentation: the inoculum size with 5% is inoculated into the yeast saccharomyces cerevisiae seed liquor of preparation in (1) in (2) described substratum, at 30 DEG C, cultivates 36h under 200rpm condition;

(4) aseptic fermentation by saccharomyces cerevisiae supernatant liquor preparation: shift to an earlier date sterilized centrifuge tube and collect the fermentation by saccharomyces cerevisiae liquid of preparation in (3), 3000rpm is centrifugal, and 10min obtains supernatant liquor, concentrated 100 times of rotary evaporation, through 0.45 μ m millipore filtration suction filtration (sterilizing in advance such as filter flask is for subsequent use), finally obtain aseptic supernatant liquor again.

First the present invention prepares aseptic fermentation by saccharomyces cerevisiae supernatant liquor, is then joined in fermentation minimum medium, simple to operate, with low cost; The more important thing is and make viable lactic acid bacteria cell density reach 10 ¹²cFU/g is 100 times of left and right of common fermentation cell density.

Embodiment

Embodiment 1

1. be divided into five groups of experimental group A, experimental group B, experimental group C, experimental group D, control groups according to the add-on of aseptic fermentation by saccharomyces cerevisiae supernatant liquor in minimum medium, as follows:

2. specific experiment step is as follows:

(1) bacterial classification and seed liquor preparation thereof

Enterococcus faecalis (Enterococcus faecalis), the enterococcus faecalis of freezing preservation is activated 3 times at MRS agar plate, picking list colony inoculation is in deep layer MRS liquid tube, cultivate after 8h for 37 DEG C, transfer in MRS liquid nutrient medium with 1% inoculum size, cultivate after 8-10h as seed liquor.

(2) substratum configuration

Minimum medium component and content: glucose 25g/L, soy peptone 10g/L, yeast powder 8g/L, K ₂hPO ₄2g/L, CH ₃cOONa7g/L, Trisodium Citrate 3g/L, MgSO ₄0.2g/L, FeCl ₂0.1g/L, MnSO ₄0.05g/L; Minimum medium additive and addition thereof: aseptic fermentation by saccharomyces cerevisiae supernatant liquor X mL/L.

According to ordinary method configuration minimum medium, 121 DEG C of autoclaving 15min, cooling, then add aseptic fermentation by saccharomyces cerevisiae supernatant liquor, stir.

(4) inoculation

The seed liquor of with 5% inoculum size being prepared by (1) is inoculated in the substratum of (2) configuration.

(5) fermentation

In fermenting process, maintain pH value for 6.0-6.8, when pH is during lower than set(ting)value, auto-feeding 30%NaCO ₃, any in 20%KOH, 20%NaOH or ammoniacal liquor; 37 DEG C of temperature, rotating speed 50rpm condition bottom fermentation 12h, passes into N in fermenting process ₂, maintain dissolved oxygen amount and be less than 1%.

3. interpretation of result

Experimental result is as shown in the table:

Above experimental data illustrates that aseptic fermentation by saccharomyces cerevisiae supernatant liquor can promote enterococcus faecalis breeding, and in the time that aseptic fermentation by saccharomyces cerevisiae supernatant liquor add-on is 25mL/L, enterococcus faecalis thalline sum is the highest, compares and has improved nearly 100 times with control group.

Embodiment 2

1. be divided into experimental group and control group according to the addition sequence of aseptic fermentation by saccharomyces cerevisiae supernatant liquor in minimum medium, as follows:

2. specific experiment step is as follows:

(1) bacterial classification and seed liquor preparation thereof

Enterococcus faecalis (Enterococcus faecalis), the enterococcus faecalis of freezing preservation is activated 3 times at MRS agar plate, picking list colony inoculation is in deep layer MRS liquid tube, cultivate after 8h for 37 DEG C, transfer in MRS liquid nutrient medium with 1% inoculum size, cultivate after 8-10h as seed liquor.

(2) substratum configuration

Minimum medium component and content: glucose 25g/L, soy peptone 10g/L, yeast powder 8g/L, K ₂hPO ₄2g/L, CH ₃cOONa7g/L, Trisodium Citrate 3g/L, MgSO ₄0.2g/L, FeCl ₂0.1g/L, MnSO ₄0.05g/L; Minimum medium additive and addition thereof: aseptic fermentation by saccharomyces cerevisiae supernatant liquor 25mL/L.

According to ordinary method configuration minimum medium, require to add aseptic fermentation by saccharomyces cerevisiae supernatant liquor according to experimental group in 1 and control group.

(4) inoculation

The seed liquor of with 5% inoculum size being prepared by (1) is inoculated in the substratum of (2) configuration.

(5) fermentation

In fermenting process, maintain pH value for 6.0-6.8, when pH is during lower than set(ting)value, auto-feeding 30%NaCO ₃, any in 20%KOH, 20%NaOH or ammoniacal liquor; 37 DEG C of temperature, rotating speed 50rpm condition bottom fermentation 12h, passes into N in fermenting process ₂, maintain dissolved oxygen amount and be less than 1%.

3. interpretation of result.

Experimental result is as shown in the table:

Group number	Experimental group	Control group
			Gained enterococcus faecalis thalline sum (CFU/g)	5.6×10 12	4.8×10 10

Above experimental data explanation, in aseptic fermentation by saccharomyces cerevisiae supernatant liquor some can promote the factor of enterococcus faecalis breeding can be in 121 DEG C of high-pressure sterilizing courses inactivation, so aseptic fermentation by saccharomyces cerevisiae supernatant liquor need to 121 DEG C of autoclavings of minimum medium and cooling after add.

Claims (3)

1. a milk-acid bacteria high density fermentation culture medium, minimum medium component and content: glucose 25g/L, soy peptone 10g/L, yeast powder 8g/L, K ₂hPO ₄2g/L, CH ₃cOONa7g/L, Trisodium Citrate 3g/L, MgSO ₄0.2g/L, FeCl ₂0.1g/L, MnSO ₄0.05g/L; Minimum medium additive and addition thereof: aseptic fermentation by saccharomyces cerevisiae supernatant liquor 25mL/L.

2. a kind of milk-acid bacteria high density fermentation culture medium according to claim 1, while it is characterized in that configuring substratum until 121 DEG C of autoclaving 15min of minimum medium and cooling after, by aseptic technique, aseptic fermentation by saccharomyces cerevisiae supernatant liquor is added, and stir.

3. a kind of milk-acid bacteria high density fermentation culture medium according to claim 1, is characterized in that the preparation method of the aseptic fermentation by saccharomyces cerevisiae supernatant liquor of minimum medium additive is as follows:

(1) preparation of yeast saccharomyces cerevisiae seed liquor: inoculate after test tube slant activation being kept at the yeast saccharomyces cerevisiae in dormant state in lyophilize pipe, pass through again shake-flask culture, 30 DEG C, under 200rpm condition, cultivate 24h, obtain seed liquor, by measuring OD value under 560nm wavelength, to guarantee that in every a collection of seed liquor, yeast saccharomyces cerevisiae thalline number is consistent;

(2) configuration fermentation by saccharomyces cerevisiae substratum: peptone 20g/L, yeast extract 10g/L, glucose 2g/L, appropriate distilled water dissolves, then adds 15mL/L0.2% adenine solution, and 120 DEG C of autoclaving 15min are cooling for subsequent use;

(3) inoculation fermentation: the yeast saccharomyces cerevisiae seed liquor that the inoculum size with 5% is prepared (1) is inoculated in (2) described substratum, at 30 DEG C, cultivates 36h under 200rpm condition;

(4) aseptic fermentation by saccharomyces cerevisiae supernatant liquor preparation: shift to an earlier date sterilized centrifuge tube and collect the fermentation by saccharomyces cerevisiae liquid of preparation in (3), 3000rpm is centrifugal, and 10min obtains supernatant liquor, concentrated 100 times of rotary evaporation, through 0.45 μ m millipore filtration suction filtration (sterilizing in advance such as filter flask is for subsequent use), finally obtain aseptic supernatant liquor again.