CN106434465B - Method for realizing high-density fermentation of lactic acid bacteria by adopting amorphous solid attachments - Google Patents

Abstract

The invention discloses a technology for realizing high-density fermentation of lactic acid bacteria by using amorphous solid attachments. (2) Fermentation culture is carried out at 37°C, and after the culture is completed, the cells are harvested. The fermentation technology provided by the invention does not require feeding, and can complete the fermentation process at one time; a general fermenter can be used, and no special fermenter needs to be invested; the invention can obtain lactic acid bacteria cells, and all production processes using lactic acid bacteria biomass as the purpose of fermentation are It can be applied; after the fermentation, the total number of bacterial colonies can reach 10 ^10-12 cfu/mL; the obtained bacterial cells are resistant to high temperature and stress, and the survival rate is increased by 2-3 times in the simulated gastric acid and bile salt environment.

Description

A kind of method that adopts amorphous solid attachment to realize lactic acid bacteria high-density fermentation

technical field

The invention relates to the field of food microorganism fermentation engineering, in particular to a method for realizing high-density fermentation of lactic acid bacteria by using amorphous solid attachments.

Background technique

Lactic acid bacteria is a general term for a group of Gram-positive bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. Lactic acid bacteria are a kind of microbial flora that are beneficial to the human body and are widely distributed in nature. The fermentation of lactic acid bacteria can produce a large amount of metabolites such as organic acids, alcohols and various amino acids, which have physiological effects such as inhibiting spoilage, improving digestibility, and preventing cancer. Lactic acid bacteria have a long history of application, and are now widely used in the fermented dairy industry, food processing industry, feed preparation, medicine and health, and the prevention and control of livestock diseases.

With the rapid development of my country's fermented dairy product industry, active research and vigorous development of high-efficiency concentrated yogurt starter is of great significance for promoting the industrialization of lactic acid bacteria starter in my country and the development of my country's fermented dairy product industry. The key to the preparation of lactic acid bacteria super-concentrated starter is to realize its high activity and high density culture. Therefore, the research on high density culture engineering technology of lactic acid bacteria has important practical significance and broad prospects.

High-density culture of lactic acid bacteria can be used to produce starter, probiotic preparations, lactic acid, bacteriocins, and other products, but the acidification of the fermentation broth and metabolites of lactic acid bacteria during the culture process will inhibit the growth of lactic acid bacteria, resulting in cultured bacteria. Continuity of density and biomass accumulation is severely hindered.

In the process of bacterial culture, the product inhibition coefficient affects the growth rate and product conversion rate. Controlling changes in pH only partially mitigated this inhibition. In the prior art, in order to achieve high-density fermentation of lactic acid bacteria, (1) feeding and adding a membrane filtration device can be used to dilute or remove metabolic waste to relieve growth inhibition; (2) immobilization or microencapsulation can also be used to fix cells, preventing metabolites from affecting the growth of lactic acid bacteria. In addition, (3) biofilm induction method can also be used to make lactic acid bacteria form biofilms through exogenous stimulation to achieve high-density culture; finally, some scholars observe the cell growth cycle through (4) mathematical model and calculate the change trend of cell concentration , biosynthetic activity, and substrate consumption rate, etc., and finally obtain the best target production model or high-density culture.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the object of the present invention is to provide a method for realizing high-density fermentation of lactic acid bacteria by adopting amorphous solid attachments, and adopting amorphous solid suspensions to induce lactic acid bacteria to produce biofilm, thereby realizing the high-density cultivation of lactic acid bacteria.

To achieve the above object, the present invention provides the following technical solutions:

A method for realizing high-density fermentation of lactic acid bacteria using amorphous solid attachments, comprising the following steps:

(1) Select the excellent lactic acid bacteria strains that grow vigorously and ensure that they can reach the logarithmic growth phase within 4-8 hours after inoculation for activation, and inoculate the activated lactic acid bacteria strains in the lactic acid bacteria medium;

(2) Fermentation culture is carried out at 37°C, and after the culture is completed, the cells are harvested.

In the above-mentioned method for realizing high-density fermentation of lactic acid bacteria by using unshaped solid attachments, a preferred solution is that the lactic acid bacteria strain in step (1) is one of Lactobacillus salivarius, Lactobacillus fermentum and Pediococcus lactis.

In the above-mentioned method for realizing high-density fermentation of lactic acid bacteria by using unshaped solid attachments, a preferred solution is that the inoculum of the lactic acid bacteria strain described in step (1) is 0.5-1%.

In the above-mentioned method for realizing high-density fermentation of lactic acid bacteria by using amorphous solid attachments, a preferred solution is that the lactic acid bacteria medium in step (1) is a PY medium.

Above-mentioned a kind of method that adopts amorphous solid attachment to realize lactic acid bacteria high-density fermentation, preferred scheme is, described PY medium is made up of the raw material of following weight components: unhydrolyzed macromolecular protein 50g, peptone 5g, tryptone 5g, Yeast powder 10g, anhydrous sodium acetate 20g, KH ₂ PO ₄ 6g, MgSO ₄ ∙7H ₂ O 0.6g, MnSO ₄ ∙4H ₂ O 0.2g, water 1000g.

In the above-mentioned method for realizing high-density fermentation of lactic acid bacteria by using amorphous solid attachments, a preferred solution is that the pH of the PY medium is 6.8.

In the above-mentioned method for realizing high-density fermentation of lactic acid bacteria by using amorphous solid attachments, a preferred solution is that the fermentation time in step (2) is 36-48 hours.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The fermentation method provided by the present invention does not require feeding materials, and can complete the fermentation process at one time; a general fermentation tank can be used, and there is no need to invest in a special fermentation tank;

(2) The present invention can obtain lactic acid bacteria cells, and all production processes with lactic acid bacteria biomass as the purpose of fermentation can be applied, and the high-density culture method of immobilization (biofilm) cannot obtain lactic acid bacteria cells, only lactic acid bacteria metabolites can be used. as the target product;

(3) In the fermentation method provided by the present invention, after the fermentation, the total number of bacterial colonies can reach 10 ^10-12 cfu/mL; the obtained starter is wrapped by acid curdling protein, and most of the bacterial cells are embedded;

(4) The bacteria obtained by the fermentation method of the present invention have high temperature resistance and strong stress resistance, and the survival rate is increased by 2-3 times in the simulated gastric acid and bile salt environment.

Description of drawings

Figures 1-3 show the comparison of the total number of colonies obtained by the fermentation method of the present invention and the control group.

Figures 4-6 show a comparison chart of the high temperature resistance of the fermentation products of the present invention and the control group.

Figures 7-9 are graphs showing the comparison of gastric acid tolerance of the fermentation products of the present invention and the control group.

Figures 10-12 are graphs showing the comparison of bile salt tolerance of the fermentation products of the present invention and the control group.

Detailed ways

The technical solutions of the present invention will be described in detail below with reference to the embodiments and experimental examples, but the protection scope is not limited thereto. The raw materials used in the present invention can be purchased from the market.

Embodiment 1 A method for realizing high-density fermentation of lactic acid bacteria by using unshaped solid attachments, comprising the following steps:

(1) Select excellent Lactobacillus salivarius strains that grow vigorously and ensure that they can reach the logarithmic growth phase within 4-8 hours after inoculation for activation, and inoculate the activated Lactobacillus salivarius strains in PY medium with a pH of 6.8 In, the inoculation amount of described Lactobacillus salivarius strain is 0.5～1%, described PY medium is made up of the raw material of following weight components: unhydrolyzed macromolecular protein 50g, peptone 5g, tryptone 5g, yeast powder 10g, Anhydrous sodium acetate 20g, KH ₂ PO ₄ 6g, MgSO ₄ ∙7H ₂ O 0.6g, MnSO ₄ ∙4H ₂ O 0.2g, water 1000g;

(2) Fermentation culture is carried out at 37°C for 36-48 hours. After the culture is completed, the cells are harvested.

Embodiment 2 A method for realizing high-density fermentation of lactic acid bacteria by using unshaped solid attachments, comprising the following steps:

(1) Select the excellent Lactobacillus fermentum strains that grow vigorously and ensure that they can reach the logarithmic growth phase within 4-8 hours after inoculation for activation, and inoculate the activated Lactobacillus fermentum strains in PY medium with a pH of 6.8 Among them, the inoculum of the described Lactobacillus fermentum strain is 0.5~1%, and the PY medium is composed of the raw materials of the following weight components: unhydrolyzed macromolecular protein 50g, peptone 5g, tryptone 5g, yeast powder 10g, Anhydrous sodium acetate 20g, KH ₂ PO ₄ 6g, MgSO ₄ ∙7H ₂ O 0.6g, MnSO ₄ ∙4H ₂ O 0.2g, water 1000g;

(2) Fermentation culture is carried out at 37°C for 36-48 hours. After the culture is completed, the cells are harvested.

Embodiment 3 A method for realizing high-density fermentation of lactic acid bacteria by using unshaped solid attachments, comprising the following steps:

(1) Select the excellent Pediococcus lactis strains that grow vigorously and ensure that they can reach the logarithmic growth phase within 4-8 hours after inoculation for activation, and inoculate the activated Pediococcus lactis strains in PY medium with a pH of 6.8 In, the inoculation amount of described Pediococcus lactis strain is 0.5～1%, and described PY medium is made up of the raw material of following weight components: unhydrolyzed macromolecular protein 50g, peptone 5g, tryptone 5g, yeast powder 10g, Anhydrous sodium acetate 20g, KH ₂ PO ₄ 6g, MgSO ₄ ∙7H ₂ O 0.6g, MnSO ₄ ∙4H ₂ O 0.2g, water 1000g;

(2) Fermentation culture is carried out at 37°C for 36-48 hours. After the culture is completed, the cells are harvested.

Test Example The present invention is a kind of experimental research on the method for realizing high-density fermentation of lactic acid bacteria by using unshaped solid attachments:

1. Test subjects

Experimental group: Lactobacillus salivarius, Lactobacillus fermentum and Pediococcus lactis were respectively cultured at high density with the fermentation method of the present invention adding the unhydrolyzed protein culture medium.

Control group: Lactobacillus salivarius, Lactobacillus fermentum and Pediococcus lactis were cultured at high density by the fermentation method of basal medium without adding unhydrolyzed protein.

2. Test method

2.1 Measurement of the total number of colonies

The total number of colonies was determined by plate count method. Take 1 mL of sample diluted 10-fold with sterile water, then pipette 200 μL into a petri dish, and then pour it into MRS agar medium with a temperature not exceeding 70 °C. After 48 h incubation at 37°C, the colonies visible to the naked eye were counted. The number of colonies is required to be between 30 and 300 as an effective dilution gradient, and the number of colonies in the original sample is calculated according to the dilution multiple. It can be seen from Figure 1-3 that after the basal medium (control group) is cultured (24 h), the total number of colonies is 10 ^8-9 cfu/mL, while the total number of colonies obtained by the method of the present invention (experimental group) after fermentation is 10 ^{10- 12} cfu/mL, equivalent to 100-1000 times that of the control group.

2.2 High temperature resistance test

Take the lactic acid bacteria liquids of the control group and the experimental group that have been cultured for 12 h under aseptic conditions, transfer 1 mL to a sterile centrifuge tube, and after 30 min in a water bath at 50 °C, 60 °C, and 70 °C, the ice water is rapidly cooled to room temperature. The total number of colonies was then determined initially and after the high temperature water bath. According to the bacterial survival rate, the high temperature resistance ability of lactic acid bacteria in different treatment groups was judged. As can be seen from Figures 4-6, when the lactic acid bacteria are cultivated by the method of the present invention, the high temperature resistance of the bacteria body is significantly enhanced, especially when treated at 60°C for 30 min, all the lactic acid bacteria in the control group died, while the experimental groups had varying degrees of survival. However, when treated at 50 °C, the survival rate of the control group was 73-86%, and the survival rate of the experimental group was 85-95%. However, when treated at 70°C for 30 min, all the cells died and the survival rate was 0.

2.3 Preparation and experimental methods of simulated gastric juice and bile salts

The simulated gastric juice refers to the formula of JJ Ahire et al. (2011): peptone 8.3, glucose 3.5, NaCL 2.05, KH ₂ PO ₄ 0.6, CaCl ₂ 0.11, KCL 0.37, bile salt 0.05, lysozyme 0.1, pepsin 13.3 (unit: mg /mL), and the final pH was adjusted to 2.50 with 1 mol/L HCL. Dilute 10 times when using.

When measuring the survival rate of bacteria in simulated gastric juice, centrifuge the lactic acid bacteria liquid at 10,000 g for 5 min, discard the supernatant, replace it with the same amount of simulated gastric juice (10-fold dilution), and incubate at 37°C for 30-180 min. , to measure the total number of colonies. Figures 7-9 show that the lactic acid bacteria in the experimental group and the control group have a certain tolerance to simulated gastric acid. The survival rate of the experimental group was significantly higher than that of the control group (P < 0.05).

The preparation of bile salts is to prepare a corresponding bile salt solution according to the mass fraction of 0.5-2%. Figures 10-12 show that the tested lactic acid bacteria showed strong tolerance to bile salts. With the increase of bile salt concentration, the bacterial survival rate decreased. Overall, the bacterial survival rate in the experimental group was significantly higher than that in the control group (P < 0.05).

It will be apparent to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, but that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments are to be regarded in all respects as illustrative and not restrictive, and the scope of the invention is to be defined by the appended claims rather than the foregoing description, which are therefore intended to fall within the scope of the claims. All changes within the meaning and scope of the equivalents of , are included in the present invention. Any reference signs in the claims shall not be construed as limiting the involved claim.

Claims (5)

1. a kind of method that adopts amorphous solid attachment to realize lactic acid bacteria high density fermentation, is characterized in that, comprises the following steps: (1) Select the excellent lactic acid bacteria strains that grow vigorously and ensure that they can reach the logarithmic growth phase within 4-8 hours after inoculation for activation, and inoculate the activated lactic acid bacteria strains in the lactic acid bacteria medium; (2) Fermentation culture is carried out at 37°C, and after the culture is completed, the cells are harvested; Wherein, the lactic acid bacteria culture medium is PY culture medium; The PY medium is composed of the following raw materials by weight: 50 g of unhydrolyzed macromolecular protein, 5 g of peptone, 5 g of tryptone, 10 g of yeast powder, 20 g of anhydrous sodium acetate, 6 g of KH ₂ PO ₄ , MgSO ₄ ∙7H ₂ O 0.6g, MnSO ₄ ∙4H ₂ O 0.2g, water 1000g. 2 . The method according to claim 1 , wherein the lactic acid bacteria strain in step (1) is one of Lactobacillus salivarius, Lactobacillus fermentum and Pediococcus lactis. 3 . 3 . The method according to claim 1 , wherein the inoculation amount of the lactic acid bacteria strain in step (1) is 0.5-1%. 4 . 4. The method according to claim 1, wherein the pH of the PY medium is 6.8. 5 . The method according to claim 1 , wherein the fermentation time of step (2) is 36-48 h. 6 .

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