CN101606552B - Bifidobacterium deep freezing direct vat starter culture and composite cryoprotectant thereof - Google Patents

Abstract

The invention discloses a bifidobacterium submerged freezing fermentation agent and a cryoprotectant thereof. The bifidobacterium cells were obtained through high-density culture, and the sludge was collected by centrifugation, and the cells were resuspended with the optimal compound protective agent, and stored in deep freeze at -80°C, effectively maintaining the number and functional characteristics of the bifidobacteria. The bifidobacterium deep-layer frozen starter of the present invention is used as an auxiliary starter, and has a remarkable effect on the effective application of probiotics to dairy product fermentation.

Description

A bifidobacterium deep-frozen direct-throwing starter and its composite cryoprotectant

technical field

The invention relates to a bifidobacterium deep-layer freezing direct-throwing starter and a composite cryoprotectant thereof.

Background technique

As a classic probiotic, the probiotic function of bifidobacteria has been widely recognized, and its application in functional foods has also received extensive attention, among which the application in dairy products is the most representative. However, bifidobacteria have various disadvantages in fermentation performance and production performance. First of all, Bifidobacteria are extremely sensitive to the external environment, and the survival rate of Bifidobacteria is low when they experience low temperature, dryness, and other adversity stimuli during the preparation of the starter; secondly, Bifidobacteria are obligate anaerobic bacteria and have high requirements for fermentation conditions. The rate of acid production by aerobic fermentation is low, and the flavor is poor; it is difficult to control the ratio of strains in the preparation of mixed strains of starter starter, and the number of viable bacteria in the final product is unstable. Bifidobacterium is prepared into a direct-injection starter, and used as an auxiliary starter and mixed with traditional lactic acid bacteria in-line starter, which can not only ensure the number of probiotics in the product, but also ensure the texture and taste of yogurt, and control the curdling time. It has broad application prospects in functional dairy products.

The effective application of bifidobacteria in dairy fermentation mainly depends on the preparation and preservation technology of the starter. At present, the development of bifidobacterium fermentation agents is mainly concentrated in the fields of freeze-drying and spray-drying, but these technologies will cause a series of side effects: first, the survival rate in the drying process is only about 30%-40%, due to the impact of bifidobacteria on nutrition and growth The condition requirements are high, and the production cost of the bacteria is high. Improving the survival rate during the preparation of the starter is of great significance for production and application; secondly, the continuous low water activity during the preparation and storage of the starter will cause cell wall, cell membrane, DNA, etc. Many surviving cells will lose their physiological functions, thus losing the value of probiotics; third, due to cell damage, the ability of the bacteria to withstand adversity and the ability to tolerate oxygen, acid, bile salts, etc. in the product and the intestine It will also decrease, so that the number of bacteria reaching the intestinal tract and colonizing the intestinal tract cannot be guaranteed, which is also the bottleneck in the application process of bifidobacteria.

Deep freezing technology is usually used for the preservation of tissues, cells, strains, etc. It has a good effect on the maintenance of biological activity, and the preservation effect is good for a long storage time. Utilizing the effective protective agent, the survival rate of deep freezing technology used in the preparation of starter can be close to 100%, which plays an important role in reducing the cost of bifidobacterium cultivation. Commonly used cryoprotectants include dimethyl sulfoxide, polyethylene glycol, serum, etc., which are expensive and unsuitable for food, which limits their application in the production of starter cultures. The invention relates to a food-derived bifidobacterium submerged frozen starter, which is used for the preparation of the bifidobacterium direct input starter, so as to ensure the bacterial count and functional characteristics of the bifidobacteria in the product.

According to the different mechanism of action of protective agents, protective agents can be divided into penetrating protective agents, semi-permeable protective agents and non-permeable protective agents. The osmotic protectant can penetrate into the inside of the cell, fully hydrate the cell, prevent excessive dehydration of the cell, reduce the toxicity of high salt to the cell, and prevent the formation of ice crystals in the cell. The semi-permeable protective agent can prevent the separation of the plasmodium of the cell before freezing, and can be concentrated between the cell wall and the cytoplasm as a buffer layer to prevent the growth of ice crystals and chemically protect the cell membrane. The non-permeable protective agent adsorbs on the surface of microbial cells to form a protective layer with high viscosity, which prevents the growth of ice crystals by increasing the viscosity of the solution, and forms an amorphous structure near the cells. The combined use of cryoprotectants with various mechanisms has a significant effect on the maintenance of the number of viable bacteria and strain functions, and is the key to the preparation of high-efficiency cryogenic Bifidobacteria starter.

Skim milk is a good buffer system, which contains a lot of protein, sugar, etc. It is an ideal protective medium, which can prevent cell damage by protecting the stability of the cell membrane, and the protein in it can also be used as a membrane to protect cells To play a protective role, adding different protective agents to skim milk can also enhance its protective effect to varying degrees.

As a semi-permeable protective agent, the function of sugar is to protect the protein by forming a high-viscosity glassy substance during the freezing process; it combines with the protein as a water substitute to prevent the denaturation of the protein. In addition to protecting the structure and function of the protein, trehalose and other carbohydrates can also reduce the phase transition temperature of the cell membrane by replacing the hydrophilic head of the lipid, thereby preventing the phase transition of the cell membrane and the resulting leakage of the cell membrane .

The cryoprotective effect of glutamic acid is that the amino group of the protective agent combines with the carboxyl group of microbial protein to stabilize the protein, and glutamic acid may also improve the fixed water in the cell.

Bifidobacterium is a strict anaerobic fermentation agent. Oxygen stress during the preparation process will affect the survival and vitality of the bacteria. Adding antioxidants to the composition of the fermentation agent will have a significant effect on the number and vitality of Bifidobacteria.

Contents of the invention

The purpose of the present invention is to prepare a bifidobacterium submerged freezing starter and an effective cryoprotectant thereof. The method solves the problems of low survival rate and decreased strain viability in the preparation process of conventional bifidobacteria starter.

The present invention is used for the protective agent of bifidobacterium deep freezing and direct-throwing fermentation agent into weight groups: 6%-16% of skim milk; 2.5%-7.5% of trehalose; 2.5%-7.5% of fructose; 1%-4% of glycerin; Vc0.01%-0.1%; L-Glu0.01%-0.1%.

The bifidobacteria submerged deep-frozen direct-throwing starter is preferably composed of the following weight components: 8% skim milk; 5% trehalose; 5% fructose; 3% glycerin; Vc0.05%; L-Glu0.05%.

The bifidobacterium deep-layer frozen directly-introduced starter prepared by the invention is obtained by adding the above-mentioned protective agent to the bifidobacterium thallus and then freezing at -80°C.

The above protective agent is added according to 20/1-1/5 of the volume of the original bifidobacteria solution, mixed thoroughly and quickly frozen at -80°C.

The bifidobacterium cells are obtained by using corn steep liquor dry powder medium or NPNL medium or MRS culture based on 37°C, pH6.5-pH7.0 constant pH anaerobic culture for 15-24h.

The bacterium is obtained by centrifuging at 4000rpm-8000rpm for 8-20min.

The corn steep liquor dry powder culture medium is made of following components: corn steep liquor dry powder 55g/L, glucose 10g/L, L cysteine hydrochloride 0.05g/L, A liquid 10mL/L, B liquid 5ml/L L.

The liquid A is composed of the following components: KH ₂ PO ₄ 100g/L, K ₂ HPO ₄ 100g/L.

The liquid B is composed of the following components: MgSO ₄ .7H ₂ O 2g/L, FeSO ₄ .7H ₂ O 2g/L, Nacl 2g/L, MnSO ₄ 1.3g/L.

The NPNL medium is composed of the following components: beef extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, yeast extract juice 5g/L, beef liver extract powder 10g/L , glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, Tween 80 1g/L, cysteine hydrochloride 0.5g/L.

The liquid A is composed of the following components: KH ₂ PO ₄ 100g/L, K ₂ HPO ₄ 100g/L.

The liquid B is composed of the following components: MgSO ₄ .7H ₂ O 2g/L, FeSO ₄ .7H ₂ O 2g/L, NaCl 2g/L, MnSO ₄ 1.3g/L.

The MRS medium is composed of the following components: peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 20g/L, sodium acetate 5g/L, diammonium citrate 2g/L, KH ₂ PO ₄ 2g/L, Tween 80 1g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.28g/L, cysteine hydrochloride 0.5g/L.

Experiments show that bifidobacteria prepare deep-layer cryoprotectant under the action of protecting agent of the present invention, the number of bacteria remains stable after freezing, the viability of the bacterial strain determined by titration acidity remains good, the stability is good during storage, and the number of viable bacteria used in yogurt fermentation products reaches 10 ⁷ cfu/mL or more, meeting the requirements for direct-use starter cultures.

Description of drawings

Figure 1: Changes in the logarithmic value of viable Bifidobacterium BBMN68 during yogurt fermentation and storage.

Figure 2: Changes in the logarithmic value of viable Bifidobacterium BBMN23 during yogurt fermentation and storage.

Figure 3: Changes in the logarithmic value of viable Bifidobacterium BBMN01 during yogurt fermentation and storage.

Detailed ways

Examples 1-24

(1) Strain cultivation: the preserved Bifidobacterium longum BBMN68 (preserved in China Agricultural University) was subcultured twice to rejuvenate, and the strain morphology of the culture solution was examined under a microscope, and the activated strain was inoculated in corn steep liquor dry powder medium at 10 ⁷ cfu/mL , constant pH7.0 anaerobic culture for 24h.

Corn steep liquor dry powder medium is prepared from the following components: corn steep liquor dry powder 55g/L, glucose 10g/L, L-cysteine hydrochloride 0.05g/L, A liquid 10mL/L, B liquid 5ml/L.

The liquid A is composed of the following components: KH ₂ PO ₄ 100g/L, K ₂ HPO ₄ 100g/L.

The liquid B is composed of the following components: MgSO ₄ .7H ₂ O 2g/L, FeSO ₄ .7H ₂ O 2g/L, Nacl 2g/L, MnSO ₄ 1.3g/L.

(2) Collect the bacteria: centrifuge the bacteria liquid obtained in (1) at 4000 rpm for 10 minutes to obtain the bacteria, and collect the bacteria sludge.

(3) Add protective agent:

Bifidobacterium cryoprotectant consists of:

Table 1

Add 1/10 of the original bacterial liquid volume of the protective agent described in Table 1 to the collected bacterial cells, stir and mix well to obtain a concentrated bacterial cell suspension, and immediately freeze and store at -80°C.

(4) Determination of strain viability

The frozen concentrate was inoculated in 12% skim milk with an inoculum size of 1% (V/V), cultured at 37°C for 7h, and the titrated acidity (°T) was measured.

The method for determining the titration acidity is as follows: take 10 mL of the above-mentioned fermented milk, dilute it with 20 mL of distilled water, add 2 drops of 1% phenolphthalein, titrate with 0.1mol/L NaOH, multiply the number of milliliters of NaOH consumed by 10, which is the amount of acidity needed to neutralize 100 mL of fermented milk. The number of milliliters of 0.1mol/L NaOH needed is 1°T when 1 mL is consumed. Can reflect the vitality of the strain.

The results listed in the above table 1 show that the strain viability remains good after freezing under the action of the protective agent, and the optimal result is skim milk with a mass fraction of 8%; trehalose with a mass fraction of 5%; fructose with a mass fraction of 5%; The fraction is 3% glycerol; the mass fraction is 0.05% Vc; the mass fraction is 0.05% L-Glu.

Preparation and Application of Example 25 Bifidobacterium Deep Freezing Fermentation Agent

(1) Strain cultivation: the preserved Bifidobacterium longum BBMN68 (preserved in China Agricultural University) was subcultured twice to rejuvenate, and the strain morphology of the culture solution was examined under a microscope, and the activated strain was inoculated in corn steep liquor dry powder medium at 10 ⁷ cfu/mL , constant pH7.0 anaerobic culture for 24h.

Corn steep liquor dry powder medium is prepared from the following components: corn steep liquor dry powder 55g/L, glucose 10g/L, L-cysteine hydrochloride 0.05g/L, A liquid 10mL/L, B liquid 5ml/L.

The liquid A is composed of the following components: KH ₂ PO ₄ 100g/L, K ₂ HPO ₄ 100g/L.

The liquid B is composed of the following components: MgSO ₄ .7H ₂ O 2g/L, FeSO ₄ .7H ₂ O 2g/L, Nacl 2g/L, MnSO ₄ 1.3g/L.

(2) Collect the bacteria: centrifuge the bacteria liquid obtained in (1) at 4000 rpm for 10 minutes to obtain the bacteria, and collect the bacteria sludge.

(3) Add protective agent:

Bifidobacteria cryoprotectant composition is as described in Example 24.

Add 1/10 of the original bacterial liquid volume of the above protective agent to the collected bacterial cells, stir and mix well to obtain a concentrated bacterial cell suspension, and immediately freeze and store at -80°C.

(4) starter for yogurt fermentation

The above-prepared starter was inoculated with 12% skim milk at the same time as the traditional starter, fermented at 42°C until the yoghurt coagulates, stored at 4°C, and the number of bifidobacteria was measured during the storage period. Figure 1 shows the changes in the number of viable bacteria in yogurt during storage.

Bifidobacteria selective enumeration medium was used for the enumeration of viable bifidobacteria in the above-mentioned yogurt.

Bifidobacteria selective counting medium consists of: beef extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, yeast extract juice 5g/L, beef liver extract powder 10g/L, Glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, solution C 40ml/L, Tween 80 1g/L, cysteine hydrochloride 0.5/L, agar 15g/L L.

The liquid A is composed of the following components: KH ₂ PO ₄ 100g/L, K ₂ HPO ₄ 100g/L.

The liquid B is composed of the following components: MgSO ₄ .7H ₂ O 2g/L, FeSO ₄ .7H ₂ O 2g/L, Nacl 2g/L, MnSO ₄ 1.3g/L.

The solution C consists of: 100 mg of paromomycin sulfate, 400 mg of neomycin sulfate, 15 g of sodium propionate, and 3 g of lithium chloride, placed in 40 ml of sterile water, filtered through a 0.45 μm microporous membrane, ready for immediate use.

Preparation and Application of Example 26 Bifidobacterium Deep Freezing Fermentation Agent

(1) Strain culture: the preserved Bifidobacterium adolescentis BBMN23 (preserved in China Agricultural University) was subcultured twice to restore its vitality, and the strain morphology of the culture solution was examined under a microscope, and the activated strain was inoculated in MRS medium at 10 ⁷ cfu/mL, and kept pH 6.5 anaerobic culture 18.

MRS medium consists of the following components: peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 20g/L sodium acetate 5g/L, diammonium citrate 2g/L, KH ₂ PO ₄ 2g /, Tween 80 1g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.28g/L, cysteine hydrochloride 0.5g/L.

(2) Collect the bacteria: centrifuge the bacteria liquid obtained in (1) at 4500 rpm for 8 minutes to obtain the bacteria, and collect the bacteria sludge.

(3) Add protective agent:

Bifidobacterium cryoprotectant composition is as described in Example 10.

Add 1/20 of the original bacterial liquid volume of the above protective agent to the collected bacteria, stir and mix to obtain a concentrated bacteria suspension, which is quickly stored at -80°C.

(4) starter for yogurt fermentation

The above-prepared starter was inoculated with 12% skim milk at the same time as the traditional starter, fermented at 42°C until the yoghurt coagulates, stored at 4°C, and the number of bifidobacteria was measured during the storage period. Figure 2 shows the changes in the number of viable bacteria in yogurt during storage.

Bifidobacteria selective enumeration medium was used for the enumeration of viable bifidobacteria in the above-mentioned yogurt.

Bifidobacteria selective counting medium consists of: beef extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, yeast extract juice 5g/L, beef liver extract powder 10g/L, Glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, solution C 40ml/L, Tween 80 1g/L, cysteine hydrochloride 0.5/L, agar 15g/L L.

The liquid A is composed of the following components: KH ₂ PO ₄ 100g/L, K ₂ HPO ₄ 100g/L.

The liquid B is composed of the following components: MgSO ₄ .7H ₂ O 2g/L, FeSO ₄ .7H ₂ O 2g/L, Nacl 2g/L, MnSO ₄ 1.3g/L.

The solution C consists of: 100 mg of paromomycin sulfate, 400 mg of neomycin sulfate, 15 g of sodium propionate, and 3 g of lithium chloride, placed in 40 ml of sterile water, filtered through a 0.45 μm microporous membrane, ready for immediate use.

Preparation and Application of Example 27 Bifidobacterium Deep Freezing Fermentation Agent

(1) Strain cultivation: the preserved Bifidobacterium animalis BBMN01 (preserved in China Agricultural University) was subcultured twice to restore its vitality, and the strain morphology of the culture solution was examined under a microscope. The activated strain was inoculated in NPNL medium at 10 ⁷ cfu/mL, and kept pH 6.8 anaerobic culture 12.

The NPNL medium is composed of the following components: beef extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, yeast extract juice 5g/L, beef liver extract powder 10g/L , glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, Tween 80 1g/L, cysteine hydrochloride 0.5g/L.

The liquid A is composed of the following components: KH ₂ PO ₄ 100g/L, K ₂ HPO ₄ 100g/L.

The liquid B is composed of the following components: MgSO ₄ .7H ₂ O 2g/L, FeSO ₄ .7H ₂ O 2g/L, Nacl 2g/L MnSO ₄ 1.3g/L.

(2) Collect bacteria: Centrifuge the bacteria liquid obtained in (1) at 5000 rpm for 15 minutes to obtain bacteria, and collect the bacteria sludge.

(3) Add protective agent:

Bifidobacterium cryoprotectant composition is as described in Example 18.

Add 1/5 of the original bacterial liquid volume of the above protective agent to the collected cells, stir and mix to obtain a concentrated cell suspension, which is quickly stored at -80°C.

(4) starter for yogurt fermentation

The above-prepared starter was inoculated with 12% skim milk at the same time as the traditional starter, fermented at 42°C until the yoghurt coagulates, stored at 4°C, and the number of bifidobacteria was measured during the storage period. Figure 3 shows the changes in the number of viable bacteria in yogurt during storage.

Bifidobacteria selective enumeration medium was used for the enumeration of viable bifidobacteria in the above-mentioned yogurt.

Bifidobacteria selective counting medium consists of: beef extract powder 3g/L, peptone 10g/L, tryptone 5g/L, plant peptone 3g/L, yeast extract juice 5g/L, beef liver extract powder 10g/L, Glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, solution C 40ml/L, Tween 80 1g/L, cysteine hydrochloride 0.5/L, agar 15g/L L.

The liquid A is composed of the following components: KH ₂ PO ₄ 100g/L, K ₂ HPO ₄ 100g/L.

The liquid B is composed of the following components: MgSO ₄ .7H ₂ O 2g/L, FeSO ₄ .7H ₂ O 2g/L, Nacl 2g/L, MnSO ₄ 1.3g/L.

The solution C consists of: 100 mg of paromomycin sulfate, 400 mg of neomycin sulfate, 15 g of sodium propionate, and 3 g of lithium chloride, placed in 40 ml of sterile water, filtered through a 0.45 μm microporous membrane, ready for immediate use.

Claims (7)

1. composite protectant for the preparation of the freezing direct vat inoculation of bifidobacterium deep is characterized in that this protective agent is made up of following components by part by weight: skimmed milk 6%-16%; Trehalose 2.5%-7.5%; Fructose 2.5%-7.5%; Glycerine 1%-4%; Vc0.01%-0.1%; L-Glu0.01%-0.1%; All the other are water.

2. the composite protectant for the preparation of the freezing direct vat inoculation of bifidobacterium deep according to claim 1 is characterized in that this protective agent is made up of following components by part by weight: skimmed milk 8%; Trehalose 5%; Fructose 5%; Glycerine 3%; Vc0.05%; L-Glu0.05%; All the other are water.

3. freezing direct vat inoculation of bifidobacterium deep is characterized in that adding as behind arbitrary described composite protectant among the claim 1-2 in the Bifidobacterium thalline, obtains through deep layer is freezing.

4. the freezing direct vat inoculation of bifidobacterium deep according to claim 3 is characterized in that, its preparation method is:

(1) Bifidobacterium is inoculated in Dried Corn Steep Liquor Powder culture medium or NPNL culture medium or the MRS culture medium, in 37 ℃, anaerobism is cultivated 15-24h under the pH6.5-pH7.0, and is centrifugal, 4000rpm-8000rpm, and 8-20min collects thalline;

(2) thalline of collecting is added the described composite protectant of 1/20-1/5 original bacteria liquid volume, stir and evenly mix, obtain concentrating thallus suspension liquid, rapidly in-80 ℃ of following freezings; Wherein, described Dried Corn Steep Liquor Powder culture medium is made by following component: Dried Corn Steep Liquor Powder 55g/L, glucose 10g/L, L cysteine hydrochloride 0.05g/L, A liquid 10mL/L, B liquid 5ml/L;

Described A liquid is made up of following component: KH ₂PO ₄100g/L, K ₂HPO ₄100g/L;

Described B liquid is made up of following component: MgSO ₄7H ₂O 2g/L, FeSO ₄7H ₂O 2g/L, NaCl 2g/L, MnSO ₄1.3g/L.

5. the freezing direct vat inoculation of bifidobacterium deep according to claim 4, it is characterized in that: described NPNL culture medium is made up of following component: ox meat extract powder 3g/L, peptone 10g/L, tryptone 5g/L plants peptone 3g/L, Yeast diffusion juice 5g/L, beef liver soaks powder 10g/L, glucose 10g/L, soluble starch 0.5g/L, solution A 10ml/L, solution B 5ml/L, Tween 80 1g/L, CYSTEAMINE HCL hydrochlorate 0.5g/L, wherein

Described solution A is made up of following component: KH ₂PO ₄100g/L, K ₂HPO ₄100g/L;

Described solution B is made up of following component: MgSO ₄7H ₂O 2g/L, FeSO ₄7H ₂O 2g/L, NaCl 2g/L, MnSO ₄1.3g/L.

6. the freezing direct vat inoculation of bifidobacterium deep according to claim 4, it is characterized in that: described MRS culture medium is made up of following component: peptone 10g/L, beef extract 10g/L, dusty yeast 5g/L, glucose 20g/L, sodium acetate 5g/L, dibasic ammonium citrate 2g/L, KH ₂PO ₄2g/L, Tween 80 lg/L, magnesium sulfate 0.58g/L, manganese sulfate 0.28g/L, CYSTEAMINE HCL hydrochlorate 0.5g/L.

7. as the application of the claim 3-6 freezing direct vat inoculation of each described bifidobacterium deep in fermented yoghourt.

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