CN108676830A - The method of lactobacillus plantarum ZJ316 high density fermentation bacteriocinogeny - Google Patents

Abstract

The invention discloses a method for preparing bacteriocin by high-density fermentation of plant lactobacillus ZJ316. In this method, Lactobacillus plantarum ZJ316 is inoculated into the medium for fermentation and culture to obtain bacteriocin, and the culture is fed by exponential feeding. The mass ratio of carbon source and nitrogen source in the substrate of exponential feeding is 1: 1.9-2.1; the pH of the culture medium is 5.8-6.2. This method regulates and controls the mass ratio of carbon source and nitrogen source in the substrate, as well as the pH of the medium, and carries out the high-density fermentation culture of Lactobacillus plantarum ZJ316 through the mode of exponential feeding, which greatly improves the production of bacteriocin, and at the same time The purity and activity of the bacteriocins are also guaranteed.

Description

Method for producing bacteriocin by high-density fermentation of Lactobacillus plantarum ZJ316

technical field

The invention relates to the field of biotechnology, in particular to a method for preparing bacteriocin by high-density fermentation of plantarum lactobacillus ZJ316.

Background technique

Bacteriocin is a short antibacterial peptide produced by Lactobacillus, which is high temperature resistant, acid resistant, degradable in the human body, safe and non-toxic, and has strong antibacterial activity. Bacteriocin is a kind of protein or low molecular weight polypeptide substance with antibacterial activity produced by microorganisms through the ribosome synthesis mechanism. The antibacterial range is not limited to homologous bacteria, and the producing bacteria are autoimmune to their bacteriocins.

As a special antibacterial and antibacterial protein, bacteriocin has the characteristics of strong antibacterial activity, good biocompatibility, stable performance, biodegradability, digestibility and high biological safety. It is used in the prevention and treatment of livestock and poultry diseases, The production of green biological food, feed additives and biological veterinary medicine has broad development and application prospects.

The growth of Lactobacillus plantarum ZJ316 has a strong correlation with the production of bacteriocins, and the production of bacteriocins can be increased by increasing the density of the bacteria. The bacterial cell density is related to the feeding method and the pH of the fermentation culture, but the relationship between the feeding method and the pH’s influence on the bacterial cell density is more complicated, and there is not a simple linear relationship between them, so there is still no good way to pass it. A method for increasing the bacteriocin production by increasing the cell density.

Contents of the invention

The object of the present invention is to propose a method for preparing bacteriocins by high-density fermentation of Lactobacillus plantarum ZJ316 in view of the deficiencies in the prior art above. The density of Lactobacillus plantarum ZJ316 was increased by using the mass ratio of the nitrogen source and the pH of the medium, thereby increasing the production of bacteriocin.

The present invention proposes a method for preparing bacteriocins by high-density fermentation of Lactobacillus plantarum ZJ316, inoculating Lactobacillus plantarum ZJ316 into a culture medium for fermentation and culture to obtain bacteriocins, and the cultivation adopts an exponential feeding method for feeding culture, and the The mass ratio of carbon source and nitrogen source in the exponential feeding substrate is 1:1.9-2.1; the pH of the culture medium is 5.8-6.2.

When Lactobacillus is cultured and fermented, the growth rate of the bacteria is slow at 0-2 hours, which is the lag phase. The body's stable growth period. In terms of consumption: in the lag phase, the nutrients in the fermentation broth are consumed slowly; after entering the logarithmic phase, the nutrient content in the fermentation broth decreases rapidly; in the stable phase, the consumption of nutrients slows down, and the nutrients are the lowest at the end of fermentation . For the production of bacteriocins, bacteriocins began to be produced at 2 h, and after 6 h, the production speed of bacteriocins began to accelerate, and the potency value of bacteriocins reached the maximum in the 24 h fermentation broth. As the fermentation continued, the activity of the bacteriocins began to decrease.

Therefore, the method of exponential feeding is most in line with the growth needs of Lactobacillus. With the growth of the bacteria, the required supply is given, which not only meets the growth needs of the bacteria, but also avoids the growth of the bacteria in the lagging period. When supplementing a large amount of nutrients, such as causing the glucose effect and accumulating a large amount of organic acids, it inhibits the growth of bacteria.

The ratio of carbon source and nitrogen source in the substrate will also have a greater impact on the growth of Lactobacillus and the production of bacteriocins. With the increase in the ratio of nitrogen sources in the substrate, the number of bacteria and the amount of bacteriocins produced has seen an increase. This may be due to the lack of nutritional factors such as vitamins and free amino acids that are provided by the nitrogen source in the fermentation broth to promote the growth and metabolism of the bacteria; however, when the ratio of the nitrogen source increases to a certain value, the biomass of the bacteria and the fermentation broth The potency value of the bacteriocins in the medium decreased, and the nitrogen source was transformed into a limiting factor at this time. Therefore, the ratio of the carbon source to the nitrogen source used in the present invention is 1:1.9-2.1, and the bacterial biomass, bacterial density and bacteriocin potency all reach the highest value.

The acidic environment is conducive to the growth of lactic acid bacteria and the production of bacteriocins, while the higher pH is not conducive to the growth of lactic acid bacteria and the production of bacteriocins. This is because high pH is conducive to the adsorption of bacteriocins to cells, which promotes the formation of some inactive bacteriocin aggregates, and also promotes the degradation of bacteriocins. The optimum pH for bacterial growth and bacteriocin production is not consistent, that is, there is not a complete positive correlation between the production of bacteriocin and bacterial growth. Because the required pH value of bacteriocin is lower than the required pH value of thalline growth, so the pH value in the culture medium is set at 5.8～6.2 in the present invention, like this in the initial stage of thalline growth, the pH value is partial The high environment is more suitable for the growth and reproduction of the bacteria. When the exponential feeding continues, the substrate will continue to increase, and the pH value will also decrease. In this way, when the bacteria enter the logarithmic phase, they will reach a higher density. , that is, during the important production stage of bacteriocins, the pH value is acidic, and the environment is more suitable for the production of bacteriocins, which increases the production of bacteriocins.

Further, the carbon source is one or more of glucose, molasses and lactose. More preferably, the molasses is one or both of beet molasses and sugarcane molasses. Since sugar is not only the most important nutrient for the bacteria, it also provides the necessary carbon source for the growth of the bacteria and the production of bacteriocins. Molasses contains vitamins, minerals, bacterial proteins, nucleic acids, surfactants, and growth-promoting factors (bioactive substances), which can better promote the growth and production of bacteria and bacteriocins; lactose is decomposed under the action of lactic acid bacteria into galactose and glucose, ensuring the slow-release effect of carbon source. At the same time, in order to meet more needs, glucose, molasses and lactose can also be used in combination to achieve the desired effect.

Further, the nitrogen source includes soybean peptone, yeast extract, ammonium citrate and silkworm chrysalis powder. Silkworm chrysalis powder is not only rich in fat, but also contains a lot of amino acids, calcium and phosphorus, which can promote the metabolism and reproduction of bacteria, increase the density of bacteria, and accelerate the production of bacteriocins.

Further, the substrate also contains one or both of arginine and methionine. Arginine and methionine can promote the metabolism of bacteria and accelerate the production of bacteriocins.

Further, the pH of the substrate is 5.3-5.7. In the process of exponential feeding, with the addition of the substrate, the environmental pH value will also decrease. At this time, the bacteria have obtained better growth and reproduction at the initial pH value of the medium. When the environmental pH value decreases , will be more suitable for the production of bacteriocins.

Further, the culture medium also contains a surfactant. The surfactant includes one or both of Tweens and Spans. The mass ratio of the Tweens and Spans is 9:0.5-1.5.

Surfactants can reduce the surface tension between the bacterial cells and the medium contact surface, thereby changing the permeability of the Lactobacillus cell membrane, promoting the entry of nutrients into the cells and the excretion of metabolites, thus promoting the production of bacteriocins and making bacteriocins The vitality enhancing network. In addition, Tweens can provide fatty acids for the growth of bacteriocin-producing strains and also act as a vitamin-like substance that contributes to bacteriocin production.

Further, the Lactobacillus plantarum ZJ316 was purified and cultured for 6-9 passages before inoculation. The purpose of this is to ensure that after the high-purification treatment of the bacteria, high-quality bacteria can be obtained for inoculation, which can better reproduce or produce bacteriocins during the fermentation process, and can also improve the activity of bacteriocins.

The method for preparing bacteriocin by high-density fermentation of Lactobacillus plantarum ZJ316 of the present invention, the method regulates and controls the mass ratio of carbon source and nitrogen source in the substrate, and the pH of the medium, and carries out Lactobacillus plantarum by means of exponential feeding The high-density fermentation culture of ZJ316 greatly increases the yield of bacteriocin, and also ensures the purity and activity of bacteriocin.

Detailed ways

For the following in conjunction with specific examples, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, various changes or modifications made by those skilled in the art to the present invention, and these equivalent forms also fall within the scope of protection claimed by the present application. The proportions in the embodiments of the present invention are all by weight.

Example 1

Lactobacillus plantarum ZJ316 is activated, and the seed liquid that has been passed down for 6 times is put into the fermenter with an inoculum of 3%. The medium components are peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, citric acid Triamine, sodium acetate, glucose, magnesium sulfate, manganese sulfate, Tween 80 and Span 20, the mass ratio of Tween 80 to Span 20 was 9:0.5, the pH value of the medium was 5.8; the stirring speed during fermentation was 100 r/min, the culture temperature was 30°C, feeding was carried out according to the exponential feeding method, and the feeding was carried out by calculating the outflow acceleration rate through the fed-batch equation. The carbon source of the base material was glucose; the nitrogen source was soybean peptone and yeast extract , ammonium citrate, silkworm chrysalis powder, arginine and methionine, the mass ratio of carbon source and nitrogen source in the substrate is 1:1.9, and the pH value of the substrate is 5.3; finally, bacteriocin is obtained.

Example 2

Lactobacillus plantarum ZJ316 is activated, and the seed liquid that has been passed down for 8 times is put into the fermenter with an inoculum of 3%. The medium components are peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, citric acid Triamine, sodium acetate, glucose, magnesium sulfate, manganese sulfate, Tween 80 and Span 20, the mass ratio of Tween 80 to Span 20 is 9:1, the pH value of the medium is 6; the stirring speed during fermentation is 100 r/min, the culture temperature was 30°C, feeding was carried out according to the exponential feeding method, and the feeding was carried out by calculating the flow acceleration rate through the fed-batch equation. The carbon source of the base material was glucose; the nitrogen source was soybean peptone and yeast extract , ammonium citrate, silkworm chrysalis powder, arginine and methionine, the mass ratio of carbon source and nitrogen source in the substrate is 1:2, and the pH value of the substrate is 5.5; finally, bacteriocin is obtained.

Example 3

Lactobacillus plantarum ZJ316 is activated, and the seed liquid that has been passaged 9 times is put into the fermenter with an inoculum of 3%. The medium components are peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, citric acid Triamine, sodium acetate, glucose, magnesium sulfate, manganese sulfate, Tween 80 and Span 20, the mass ratio of Tween 80 to Span 20 is 9:1.5, the pH value of the medium is 6.2; the stirring speed during fermentation is 100 r/min, the culture temperature was 30°C, feeding was carried out according to the exponential feeding method, and the feeding was carried out by calculating the flow acceleration rate through the fed-batch equation. The carbon source of the base material was glucose; the nitrogen source was soybean peptone and yeast extract , ammonium citrate, silkworm chrysalis powder, arginine and methionine, the mass ratio of carbon source and nitrogen source in the substrate is 1: 2.1, and the pH value of the substrate is 5.7; finally, bacteriocin is obtained.

Comparative example 1

Lactobacillus plantarum ZJ316 is activated, and the seed liquid that has been passed down for 6 times is put into the fermenter with an inoculum of 3%. The medium components are peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, citric acid Triamine, sodium acetate, glucose, magnesium sulfate, manganese sulfate, Tween 80 and Span 20, the mass ratio of Tween 80 to Span 20 is 9:5, the pH value of the medium is 5.8; the stirring speed during fermentation is 100 r/min, the culture temperature was 30°C, feeding was carried out according to the exponential feeding method, and the feeding was carried out by calculating the flow acceleration rate through the fed-batch equation. The carbon source of the base material was glucose; the nitrogen source was soybean peptone and yeast extract , ammonium citrate, silkworm chrysalis powder, arginine and methionine, the mass ratio of carbon source and nitrogen source in the substrate is 1:1.9, and the pH value of the substrate is 5.3; finally, bacteriocin is obtained.

Comparative example 2

Lactobacillus plantarum ZJ316 is activated, and the seed liquid that has been passaged 9 times is put into the fermenter with an inoculum of 3%. The medium components are peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, citric acid Triamine, sodium acetate, glucose, magnesium sulfate, manganese sulfate, Tween 80 and Span 20, the mass ratio of Tween 80 to Span 20 is 9:1.5, the pH value of the medium is 6.5; the stirring speed during fermentation is 100 r/min, the culture temperature was 30°C, feeding was carried out according to the exponential feeding method, and the feeding was carried out by calculating the flow acceleration rate through the fed-batch equation. The carbon source of the base material was glucose; the nitrogen source was soybean peptone and yeast extract , ammonium citrate, silkworm chrysalis powder, arginine and methionine, the mass ratio of carbon source and nitrogen source in the substrate is 1: 2.1, and the pH value of the substrate is 5.7; finally, bacteriocin is obtained.

Comparative example 3

Lactobacillus plantarum ZJ316 is activated, and the seed liquid that has been passaged 9 times is put into the fermenter with an inoculum of 3%. The medium components are peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, citric acid Triamine, sodium acetate, glucose, magnesium sulfate, manganese sulfate, Tween 80 and Span 20, the mass ratio of Tween 80 to Span 20 is 9:1.5, the pH value of the medium is 6.2; the stirring speed during fermentation is 100 r/min, the culture temperature was 30°C, feeding was carried out according to the exponential feeding method, and the feeding was carried out by calculating the flow acceleration rate through the fed-batch equation. The carbon source of the base material was glucose; the nitrogen source was soybean peptone and yeast extract , ammonium citrate, silkworm chrysalis powder, arginine and methionine, the mass ratio of carbon source and nitrogen source in the substrate is 1:4, and the pH value of the substrate is 5.7; finally, bacteriocin is obtained.

Comparative example 4

Lactobacillus plantarum ZJ316 is activated, and the seed liquid that has been passaged 9 times is put into the fermenter with an inoculum of 3%. The medium components are peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, citric acid Triamine, sodium acetate, glucose, magnesium sulfate, manganese sulfate, Tween 80 and Span 20, the mass ratio of Tween 80 to Span 20 is 9:1.5, the pH value of the medium is 6.2; the stirring speed during fermentation is 100 r/min, the culture temperature was 30°C, and the feeding was carried out according to the constant feeding method. The carbon source of the bottom material was glucose; the nitrogen source was soybean peptone, yeast extract, ammonium citrate, silkworm chrysalis powder, arginine and methionine , the mass ratio of carbon source and nitrogen source in the substrate is 1:2, and the pH value of the substrate is 5.7; the bacteriocin is finally obtained.

Comparative example 5

Lactobacillus plantarum ZJ316 is activated, and the seed liquid that has been passaged 9 times is put into the fermenter with an inoculum of 3%. The medium components are peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, citric acid Triamine, sodium acetate, glucose, magnesium sulfate, manganese sulfate, Tween 80 and Span 20, the mass ratio of Tween 80 to Span 20 is 9:1.5, the pH value of the medium is 6.2; the stirring speed during fermentation is 100 r/min, the culture temperature was 30°C, and the feeding was carried out according to the constant feeding method. The carbon source of the bottom material was glucose; the nitrogen source was soybean peptone, yeast extract, ammonium citrate, silkworm chrysalis powder, arginine and methionine , the mass ratio of carbon source and nitrogen source in the substrate is 1:2, and the pH value of the substrate is 5.7; the bacteriocin is finally obtained.

Evaluation:

The bacteriocins obtained in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3 were collected, and the yield and specific activity were detected. The detection results are shown in Table 1.

By comparing the data in Table 1, it can be seen that the yield and specific activity of the bacteriocins obtained in Example 1, Example 2, and Example 3 are significantly better than those of Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. It is fully illustrated that the feeding mode adopted by the present invention, the ratio of carbon source and nitrogen source, and the selection of medium pH have obvious improvement on the output of bacteriocins, and the mass ratio of surfactant Tweens and Spans in comparative example 1 It also has a certain impact on the production of bacteriocin.

The embodiments of the present invention have been described above as examples, but the content described is only a preferred embodiment of the present invention, and cannot be considered as limiting the implementation scope of the present invention. All equal changes and improvements based on the scope of the application of the present invention shall belong to the scope covered by the patent of the present invention.

Claims (10)

1. lactobacillus plantarum ZJ316 is seeded to culture medium by the method that lactobacillus plantarum ZJ316 high density fermentations prepare bacteriocin Interior carry out fermented and cultured, obtains bacteriocin, it is characterised in that：The culture carries out feed-batch culture by the way of index feed supplement, The mass ratio of carbon source and nitrogen source is 1: 1.9~2.1 in the substrate of the index feed supplement；The pH of the culture medium is 5.8~6.2.

2. the method that lactobacillus plantarum ZJ316 high density fermentations as described in claim 1 prepare bacteriocin, it is characterised in that： The carbon source is one or more of glucose, molasses and lactose.

3. the method that lactobacillus plantarum ZJ316 high density fermentations as claimed in claim 2 prepare bacteriocin, it is characterised in that： The molasses are one or both of beet molasses and cane molasses.

4. the method that lactobacillus plantarum ZJ316 high density fermentations as described in claim 1 prepare bacteriocin, it is characterised in that： The nitrogen source includes soy peptone, yeast extract, ammonium citrate and dried silkworm chrysalis meal.

5. the method that lactobacillus plantarum ZJ316 high density fermentations as described in claim 1 prepare bacteriocin, it is characterised in that： Also include one or two kinds of in arginine and methionine in the substrate.

6. the method that lactobacillus plantarum ZJ316 high density fermentations as described in claim 1 prepare bacteriocin, it is characterised in that： The pH of the substrate is 5.3~5.7.

7. the method that lactobacillus plantarum ZJ316 high density fermentations as described in claim 1 prepare bacteriocin, it is characterised in that： The culture medium also includes surfactant.

8. the method that lactobacillus plantarum ZJ316 high density fermentations as claimed in claim 7 prepare bacteriocin, it is characterised in that： The surfactant includes one or two kinds of in Tweens and spans.

9. the method that lactobacillus plantarum ZJ316 high density fermentations as claimed in claim 8 prepare bacteriocin, it is characterised in that： The mass ratio of the Tweens and spans is 9: 0.5~1.5.

10. the method that lactobacillus plantarum ZJ316 high density fermentations as described in claim 1 prepare bacteriocin, it is characterised in that： The lactobacillus plantarum ZJ316 carries out the purifying culture of 6~9 passages before inoculation.