CN112006066A - Mixed fermentation liquor with improved antibacterial activity and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microbial fermentation, and in particular relates to a mixed fermentation broth with improved bacteriostatic activity and a preparation method and application thereof. The mixed fermentation liquid with improved bacteriostatic activity of the present invention is obtained by mixed fermentation of Lactobacillus plantarum and Propionibacterium freudenreii subsp. In the mixed fermentation process of the present invention, two kinds of bacteria are added to the fermentation tank in proportion to carry out anaerobic fermentation, and the growth of the two bacteria is controlled by adding glucose during the fermentation process. When the amount of bacteria in the fermentation liquid reaches the maximum, End fermentation. The supernatant obtained by centrifuging the fermentation broth is the fermentation extract, and the fermentation liquid contains various organic acids, which have a good inhibitory effect on spoilage microorganisms, such as Penicillium, Aspergillus, and Cladosporium.

Description

Mixed fermentation broth with improved antibacterial activity and preparation method and application thereof

technical field

The invention belongs to the technical field of microbial fermentation, and in particular relates to a mixed fermentation broth with improved bacteriostatic activity and a preparation method and application thereof.

Background technique

Food is highly susceptible to spoilage due to external mold contamination during processing, preservation, and transportation. As the most important additive, chemical preservatives are widely used in the food industry. However, chemically synthesized preservatives have potential carcinogenicity, teratogenicity, or food poisoning and other potential safety hazards to the human body. Therefore, finding and developing natural and safe antibacterial products with high bacteriostatic activity has become a research hotspot in the food field.

Lactic acid bacteria (LAB) are a class of bacteria that can ferment carbohydrates to produce large amounts of lactic acid. Probiotic lactic acid bacteria are recognized as safe microorganisms. Lactobacillus plantarum is an important probiotic bacteria, which can produce antibacterial substances such as organic acids, hydrogen peroxide and lactobacillus in the process of metabolism. It is currently recognized as a potential microbial source of development value. , The producing bacteria of natural biological food preservatives.

Propionibacterium is widely found in nature. Propionibacterium freudenreichii derived from dairy products can synthesize various organic acids, diacetyl and special peptides or proteins in the process of metabolism. These substances have a wide range of antibacterial effects, and The bacteriostatic effect is even better than that of common chemical preservatives such as sodium benzoate and potassium sorbate.

Due to technical reasons, when lactic acid bacteria or propionic acid bacteria are cultured alone, the bacteriostatic activity of the fermentation broth is often low, and the preservative products need to be purified, and the process is complicated and costly. Therefore, improving the bacteriostatic activity of the fermentation broth and reducing the purification operation are very important for the industrial production of natural preservatives.

Mixed fermentation refers to a new type of fermentation technology that uses the synergy of two or more microorganisms to complete a certain fermentation process, which can improve the fermentation efficiency and even form new products. However, when two or more different strains of bacteria are cultivated in the same system, a competitive relationship will form between them, and even the metabolites of different strains will have an antagonistic effect. Effect. According to the data, under the optimum culture conditions, lactic acid bacteria metabolize glucose 5 to 10 times faster than Propionibacterium. The invention adjusts the growth of the two strains and the ratio of metabolites by controlling the fermentation conditions to achieve a synergistic effect, thereby improving the bacteriostatic activity of the fermentation broth.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a mixed fermentation broth with improved bacteriostatic activity.

Another object of the present invention is to provide a method for preparing the above-mentioned mixed fermentation broth.

Another object of the present invention is to provide the application of the above mixed fermentation broth.

According to the mixed fermentation liquid with improved bacteriostatic activity according to the specific embodiment of the present invention, the mixed fermentation liquid is obtained by mixed fermentation of Lactobacillus plantarum and Propionibacterium freudenreichii subsp. Xie, wherein the preservation number of Lactobacillus plantarum is CGMCCNO .18027, Propionibacterium freudenreichii subsp. Shetner's strain number is ATCC 9614.

The preservation number of Lactobacillus plantarum of the present invention is CGMCCNO.18027, and it was deposited in the General Microorganism Center of China Microorganism Culture Collection Management Committee on June 26, 2019, and the preservation address is No. 1 Beichen West Road, Chaoyang District, Beijing No. 3 Institute of Microbiology, Chinese Academy of Sciences.

According to the mixed fermentation broth with improved bacteriostatic activity according to a specific embodiment of the present invention, the mixed fermentation process of the Lactobacillus plantarum and Propionibacterium freudenreichii subsp.

(1) respectively preparing the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreichii subsp.

(2) adding the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreii subsp. Xie to the liquid fermentation medium in a volume ratio of 1:1 to 10 for mixed fermentation;

(3) adding glucose to the fermentation broth in stages, and the final concentration of glucose is 0.5-1.5%;

(4) when the bacterial mass in the fermentation broth stopped growing, the fermentation was terminated;

(5) Centrifuging the fermentation broth to obtain a supernatant, which is a mixed fermentation broth.

The invention adopts the mixed fermentation of two strains to improve the antibacterial activity of the fermentation broth through the synergistic effect of the metabolites; meanwhile, the two strains are fully grown by controlling the inoculation ratio and the sugar supplementing process, and the metabolites of the two strains are further improved. In order to achieve better synergistic antibacterial effect.

According to the mixed fermentation broth with improved bacteriostatic activity according to the specific embodiment of the present invention, the inoculation volume ratio of Lactobacillus plantarum and Propionibacterium is preferably 1:2 to 1:5. The inoculation ratio of strains will affect the growth rate and metabolites of the two strains, and then affect the synergistic bacteriostatic effect of the fermentation broth.

According to the mixed fermentation broth with improved bacteriostatic activity according to the specific embodiment of the present invention, after 24 hours of mixed fermentation, 25% to 30% glucose solution is added to the fermentation broth in stages, until the final concentration of glucose in the fermentation broth is 0.5 to 1.5 %, wherein the time interval for adding glucose solution is 5-12 hours, and the final concentration of glucose is preferably 0.8-1.2%. By adjusting the amount of glucose supplemented and the supplementation time, the synergistic effect between the metabolites of the two strains was enhanced, thereby further improving the bacteriostatic effect of the fermentation broth.

According to the mixed fermentation broth with improved bacteriostatic activity according to the specific embodiment of the present invention, in step (2), the total inoculation amount of the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreii subsp. , the more preferred inoculum amount is 8-10%; the mixed fermentation conditions are anaerobic culture at a temperature of 30°C. The appropriate inoculum size has an important influence on the growth rate of the bacteria and the nutrient composition of the medium. 30 ℃ is the best temperature for the growth of propionic acid bacteria, and it will not affect the metabolism of lactic acid bacteria, which is conducive to the synergistic effect of the growth of the two strains.

According to the mixed fermentation broth with improved bacteriostatic activity according to the specific embodiment of the present invention, in step (2), the liquid fermentation medium contains 5-10 g/L of yeast powder, 15-25 g/L of peptone, 15-30 g/L of glucose, Calcium carbonate 10～25g/L, initial pH 7.0～7.2.

According to the method for the mixed fermentation broth with improved bacteriostatic activity according to the specific embodiment of the present invention, the method comprises the following steps:

(1) respectively preparing the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenrechtii subsp. siemens, wherein, the preservation number of Lactobacillus plantarum is CGMCC NO.18027, and the bacteria of P. The species number is ATCC9614;

(2) adding the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreii subsp. Xie to the liquid fermentation medium in a volume ratio of 1:1 to 10 for mixed fermentation;

(3) adding glucose to the fermentation broth in stages, and the final concentration of glucose is 0.5-1.5%;

(4) when the bacterial mass in the fermentation broth stopped growing, the fermentation was terminated;

(5) Centrifuging the fermentation broth to obtain a supernatant, which is a mixed fermentation broth.

According to the method for preparing a mixed fermentation broth with improved bacteriostatic activity according to a specific embodiment of the present invention, after 24 hours of mixed fermentation, 25%-30% glucose solution is added to the fermentation broth in stages, until the final concentration of glucose in the fermentation broth is 0.5-1.5%, wherein, the time interval of adding glucose solution is 5-12h.

According to the method for preparing the mixed fermentation broth with improved bacteriostatic activity according to the specific embodiment of the present invention, the total inoculation amount of the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreichii subsp. Xie is 5-20%; the mixed fermentation The conditions were anaerobic cultivation at a temperature of 30°C.

According to the method for preparing the mixed fermentation broth with improved bacteriostatic activity according to the specific embodiment of the present invention, in step (2), the liquid fermentation medium contains 5-10 g/L of yeast powder, 15-25 g/L of peptone, and 15-30 g of glucose. /L, calcium carbonate 10～25g/L, initial pH 7.0～7.2.

According to the method for mixing fermentation broth with improved bacteriostatic activity according to the specific embodiment of the present invention, in step (4), the fermentation broth is centrifuged at a temperature of 15 to 30° C., the centrifugal speed is 3000 to 5000 r/min, and the centrifugation time is 10 to 100 rpm. 20min to obtain the supernatant.

The present invention also provides the application of the above-mentioned mixed fermentation liquid, and its antibacterial effect on common spoilage microorganisms in bakery products, such as Penicillium, Aspergillus flavus, Cladosporium and the like, is better than the effect of single strain fermentation liquid.

Beneficial effects of the present invention:

The invention provides a mixed fermentation liquid obtained by mixed fermentation of Lactobacillus plantarum and Propionibacterium freudenreii subspecies Xie. During the fermentation process, the growth of the two bacteria is controlled by adding glucose, and the fermentation is ended when the amount of bacteria in the fermentation broth reaches the maximum. The supernatant obtained by centrifuging the fermentation broth is the fermentation extract, and the fermentation liquid contains various organic acids, which have a good inhibitory effect on spoilage microorganisms, such as Penicillium, Aspergillus, and Cladosporium.

Description of drawings

Fig. 1 shows the bacteriostatic effect on Aspergillus flavus and Penicillium of the mixed fermentation broth obtained by separate fermentation and mixed fermentation of Lactobacillus plantarum and Propionibacterium freudenii, wherein A is the fermentation liquid plate prepared by separate fermentation of Lactobacillus plantarum; B is The fermentation liquid plate containing Lactobacillus plantarum and Propionibacterium freudenreii mixed fermentation; C is the fermentation liquid plate containing Propionibacterium freudenreii fermentation alone; a ~ Aspergillus flavus colony, b ~ Penicillium colony.

Figure 2 shows the bacteriostatic effect of the obtained mixed fermentation broth on Penicillium and Aspergillus niger with different inoculation ratios of lactic acid bacteria and propionic acid bacteria under the same culture conditions. ①, ②, ③, ④, ⑤ represent Lactobacillus plantarum and Propionibacterium inoculation volume ratios of 1:4, 2:3, 1:1, 3:2 and 4:1, respectively

Figure 3 shows the bacteriostatic effect of the obtained mixed fermentation broth on Penicillium and Aspergillus niger under the same inoculation ratio with different sugar supplementing methods.

The preservation number of Lactobacillus plantarum L9 of the present invention is CGMCCNO.18027, and it was preserved in the General Microorganism Center of China Microorganism Culture Collection Administration Committee on June 26, 2019, and the preservation address is 1 Beichen West Road, Chaoyang District, Beijing. No. 3, Institute of Microbiology, Chinese Academy of Sciences.

Detailed ways

Example 1 Preparation of Lactobacillus plantarum and Propionibacterium freudenreii mixed fermentation extract

(1) Preparation of seed liquid:

Pick the activated Lactobacillus plantarum colony and inoculate it into MRS liquid medium, 30-37 ℃, static culture, culture time 12-20h, when the OD of the medium reaches 2-4, take a sample for microscopic examination without impurities, and reserve it for later use. . Among them, each liter of MRS medium contains peptone 3-7g, beef extract 5-9g, yeast powder 5-9g, glucose 10-15g, sodium acetate 1-2g, citrate diamine 1-2g, Tween 800.2-0.5g , 0.5-1 g of dipotassium hydrogen phosphate, 0.5-0.7 g of magnesium sulfate, and 0.03-0.05 g of manganese sulfate.

Pick a single colony of P. freudenreichii after activation and inoculate it into SLB liquid medium, and culture it statically at 25-32° C. for 30-50 h. When the OD of the medium reaches 1 to 2, take a sample for microscopic examination without impurities and reserve it for later use. Wherein, the SLB medium contains 1-5 g of yeast powder, 5-15 g of peptone, 25-30 mL of sodium lactate aqueous solution, 0.01-0.05 g of dipotassium hydrogen phosphate, and 0.01-0.05 g of manganese sulfate per liter.

(2) Liquid anaerobic fermentation: inoculate the prepared seed liquid with 12% of the total inoculation, wherein the volume ratio of Lactobacillus plantarum and Propionibacterium is 1:3; inoculate the seed liquid into a new liquid medium containing Fermentation culture was carried out in the liquid medium containing 10 g/L yeast powder, 20 g/L peptone, 20 g/L glucose, 20 g/L calcium carbonate, and the initial pH was 7.0-7.2. The fermentation temperature was 30°C.

After 24 hours of fermentation, glucose solution was added in batches to a final concentration of 1%, and the interval between each supplementation was 10 hours.

The fermentation was terminated when the biomass in the fermentation broth no longer increased for 80 hours.

Centrifuge at 4000 rpm for 20 min at room temperature, and the supernatant is the mixed fermentation broth.

Embodiment 2 detects the antibacterial activity of mixed fermentation broth

2.1 Preparation of indicator bacteria

For the common molds that cause food spoilage, Aspergillus flavus and Penicillium were selected as bacteriostatic indicator strains.

The species of Aspergillus flavus, Penicillium and Cladosporium preserved in 20% glycerol were transferred to PDA solid medium, respectively, and activated and cultured at 30°C. Then the activated mold spores were transferred to the PDA slant, cultured at 30°C for 7 days, and the ME medium containing 0.1% Tween 80 (malt extract 20g/L, sucrose 20g/L, peptone 1g/L, natural pH ) Wash the spores from the culture medium and count them with a hemocytometer to prepare Aspergillus flavus and Penicillium spores with a spore content of 1*10 ⁵ .

2.2 Preparation of Lactobacillus plantarum fermentation extract

(1) Preparation of seed solution: Pick a single colony of Lactobacillus plantarum after activation, inoculate it into MRS liquid medium, and culture it anaerobic at 25°C to 30°C for 12 to 20 hours respectively. Sampling for microscopic examination without impurities, spare.

(2) Liquid anaerobic fermentation: Inoculate the prepared seed liquid with 10% inoculum amount into a new one containing 10g/L yeast powder, 20g/L peptone, 20g/L glucose and 20g/L calcium carbonate. Fermentation culture was carried out in a liquid medium with pH 7.0 to 7.2 at a temperature of 30°C. When the biomass no longer increased after 48h, the fermentation was stopped. Centrifuge at 4000 rpm for 20 min at room temperature, and the supernatant is the Lactobacillus plantarum fermentation extract.

2.3 Preparation of Propionibacterium freudenreii fermentation extract

(1) Preparation of seed solution: Pick a single colony of Propionibacterium freudenreichii after activation, inoculate it into SLB liquid medium, and anaerobic culture at 30°C for 45h respectively. Sampling for microscopic examination without impurities, spare.

(2) Liquid anaerobic fermentation: Inoculate the prepared seed liquid with 10% of the total inoculation amount, and inoculate it into a new container containing 10g/L yeast powder, 20g/L peptone, 20g/L glucose, 20g/L calcium carbonate, Fermentation culture was carried out in a liquid medium with an initial pH of 7.0 to 7.2 at a temperature of 30°C. Fermentation was carried out for 96h, and the fermentation was completed when the biomass no longer increased. Centrifuge at 4000 rpm for 20 min at room temperature, and the supernatant is the fermented extract of P. freudenreichii.

2.4 Observe the antibacterial effect

Take 50 mL of distilled water and 50 mL of separately fermented Lactobacillus plantarum, Propionibacterium freudenii and the above mixed fermentation to prepare a mixed fermentation liquid, add an appropriate amount of potato dextrose agar to melt, adjust the pH of the medium to 6, sterilize with high pressure steam, and pour flat. After the plate was solidified, 10uL of Penicillium and Aspergillus flavus spore suspension with a concentration of 1*10 ⁵ was added dropwise, and cultured at 30°C. Watch for mold growth.

As can be seen from Figure 1, the Penicillium on the B plate containing the mixed fermented extract was almost completely inhibited, and the colony of Aspergillus flavus on the B plate was smaller than the single fermentation Lactobacillus plantarum A plate and the C plate of the fermentation broth of P. freudenreichii. , and the color of Aspergillus flavus on the B plate is lighter than that of the A and C plates, indicating that Aspergillus flavus is also significantly inhibited. Therefore, the mixed fermentation broth of the present invention has higher bacteriostatic activity than the single fermentation extract of Lactobacillus plantarum and Propionibacterium freudenreichii.

The effect of the inoculation ratio of embodiment 3 Lactobacillus plantarum and Propionibacterium on the bacteriostatic activity of fermentation broth

(1) Preparation of seed liquid:

Pick the activated Lactobacillus plantarum colonies and inoculate them into MRS liquid medium, culture at 30-37°C for 12-20 hours, and when the OD of the medium reaches 2-4, take a sample for microscopic examination to be free of impurities and reserve for use. Among them, each liter of MRS medium contains peptone 3-7g, beef extract 5-9g, yeast powder 5-9g, glucose 10-15g, sodium acetate 1-2g, citrate diamine 1-2g, Tween 800.2-0.5g , 0.5-1 g of dipotassium hydrogen phosphate, 0.5-0.7 g of magnesium sulfate, and 0.03-0.05 g of manganese sulfate.

Pick a single colony of P. freudenreichii after activation and inoculate it into SLB liquid medium, and culture it statically at 25-32° C. for 30-50 h. When the OD of the medium reaches 1 to 2, take a sample for microscopic examination without impurities and reserve it for later use. Wherein, the SLB medium contains 1-5 g of yeast powder, 5-15 g of peptone, 25-30 mL of sodium lactate aqueous solution, 0.01-0.05 g of dipotassium hydrogen phosphate, and 0.01-0.05 g of manganese sulfate per liter.

(2) Liquid anaerobic fermentation: Inoculate the prepared seed liquid with 12% of the total inoculation, wherein the volume ratios of Lactobacillus plantarum and Propionibacterium are ①1:4, ②2:3, ③1:1, ④3:2 , ⑤4:1; inoculate the seed liquid into a new liquid medium for fermentation culture, the liquid medium contains 10g/L yeast powder, 20g/L peptone, 20g/L glucose, 20g/L calcium carbonate, initial pH 7.0 to 7.2. The fermentation temperature was 30°C.

After 24 hours of fermentation, glucose was supplemented in batches to a final concentration of 1%, and the interval between each supplementation was 10 hours. The fermentation was terminated when the biomass in the fermentation broth no longer increased for 80 hours. The fermentation broth was centrifuged at 4000 rpm for 20 min, and the supernatant was the mixed fermentation broth.

According to the method of Example 2.4, the antibacterial activities of mixed fermentation broths with different inoculation ratios were compared. The results are shown in Figure 2. It can be seen that different inoculation ratios have a certain influence on the antibacterial activity of mixed fermentation broths. With the increase of the inoculation ratio of Lactobacillus plantarum, the antibacterial activity of the fermentation broth against Penicillium and Aspergillus flavus increased first and then decreased. When the inoculation ratio of Lactobacillus plantarum and Propionibacterium was 3:2, the antibacterial activity of the fermentation broth was the strongest.

Example 4 Effect of sugar supplementation on the antibacterial activity of metabolites

(1) Preparation of seed liquid:

Pick the activated Lactobacillus plantarum colony and inoculate it into MRS liquid medium, 30-37 ℃, static culture, culture time 12-20h, when the OD of the medium reaches 2-4, take a sample for microscopic examination without impurities, and reserve it for later use. . Among them, each liter of MRS medium contains peptone 3-7g, beef extract 5-9g, yeast powder 5-9g, glucose 10-15g, sodium acetate 1-2g, citrate diamine 1-2g, Tween 800.2-0.5g , 0.5-1 g of dipotassium hydrogen phosphate, 0.5-0.7 g of magnesium sulfate, and 0.03-0.05 g of manganese sulfate.

Pick a single colony of P. freudenreichii after activation and inoculate it into SLB liquid medium, and culture it statically at 25-32° C. for 30-50 h. When the OD of the medium reaches 1 to 2, take a sample for microscopic examination without impurities and reserve it for later use. Wherein, the SLB medium contains 1-5 g of yeast powder, 5-15 g of peptone, 25-30 mL of sodium lactate aqueous solution, 0.01-0.05 g of dipotassium hydrogen phosphate, and 0.01-0.05 g of manganese sulfate per liter.

(2) Liquid anaerobic fermentation: inoculate the prepared seed liquid with 12% of the total inoculation, wherein the volume ratio of Lactobacillus plantarum and Propionibacterium is 1:3; inoculate the seed liquid into a new liquid medium containing Fermentation culture was carried out in the liquid medium containing 10 g/L yeast powder, 20 g/L peptone, 20 g/L glucose, 20 g/L calcium carbonate, and the initial pH was 7.0-7.2. The fermentation temperature was 30°C. The fermentation process does the following four ways of supplementing sugar.

A. After 24 hours of fermentation, glucose was added in three batches to a final concentration of 1%, and the interval between each supplementation was 6 hours.

B. After 24 hours of fermentation, glucose was added in three batches to a final concentration of 1%, and the interval between each supplementation was 10 hours.

C. After 24 hours of fermentation, glucose was added in three batches to a final concentration of 1%, and the interval between each supplementation was 20 hours.

D. After 24 hours of fermentation, glucose was added in two batches to a final concentration of 1.5%, and the interval between each supplementation was 10 hours.

E. After 24 hours of fermentation, glucose was added in two batches to a final concentration of 1.5%, and the interval between each supplementation was 20 hours.

According to the method of Example 2.4, the mixed fermentation broths of different sugar supplementing methods were compared, and the results are shown in Figure 3. The mixed fermentation broth obtained by several different sugar supplementing methods has little difference in inhibitory effect on Penicillium, while on Aspergillus niger The inhibitory effect of group B showed obvious advantages. Therefore, after 24 hours of fermentation, glucose was supplemented in three batches to a final concentration of 1%, and the interval between each supplementation was 10h.

In this example, on the basis of finding that the two strains have synergistic antibacterial effects, the mixed fermentation inoculation ratio and the sugar supplementation method are optimized, which further improves the antibacterial activity of the fermentation broth, which will be an important contribution to the industry of this kind of natural antiseptic product. Chemical development has laid a good foundation.

Claims (10)

1. the mixed fermented liquid that antibacterial activity improves, it is characterized in that, described mixed fermented liquid is obtained through mixed fermentation by Lactobacillus plantarum and Propionibacterium freudenreichii subspecies Xie, wherein, the preservation number of Lactobacillus plantarum is CGMCC NO .18027, Propionibacterium freudenreichii subsp. Shetner's strain number is ATCC 9614. 2. the mixed fermentation liquid that antibacterial activity improves according to claim 1, it is characterised in that the mixed fermentation process of described Lactobacillus plantarum, Propionibacterium freudenreichii subsp. (1) respectively preparing the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreichii subsp. (2) the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreii subsp. Xie are inserted into the liquid fermentation medium in a volume ratio of 1:1 to 10 to carry out mixed fermentation; (3) adding glucose to the fermentation broth in stages, and the final concentration of glucose reaches 0.5-1.5%; (4) when the bacterial mass in the fermentation broth stopped growing, the fermentation was terminated; (5) After centrifuging the fermentation broth, take the supernatant, which is the mixed fermentation broth. 3. The mixed fermentation broth with improved bacteriostatic activity according to claim 2, characterized in that, after the mixed fermentation for 24h, 25%～30% glucose solution is added to the fermentation broth in stages, until the concentration of glucose in the fermentation broth is reached. The final concentration reaches 0.5-1.5%, wherein, the time interval for supplementing the glucose solution is 5-12 hours. 4. the mixed fermentation liquid that antibacterial activity according to claim 2 improves, it is characterised in that in step (2), the total inoculation of the liquid seed of Lactobacillus plantarum and the liquid seed of Propionibacterium freudenreichii subspecies The amount is 5-20%; the mixed fermentation conditions are anaerobic culture at a temperature of 30 °C. 5. The mixed fermentation broth with improved bacteriostatic activity according to claim 2, characterized in that, in step (2), the liquid fermentation medium comprises yeast powder 5～10g/L, peptone 15～25g/L, glucose 15～30g/L, calcium carbonate 10～25g/L, initial pH 7.0～7.2. 6. the method for preparing the mixed fermentation broth that the antibacterial activity of claim 1 improves, is characterized in that, described method comprises the following steps: (1) respectively preparing the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenrechtii subsp. siemens, wherein, the preservation number of Lactobacillus plantarum is CGMCC NO.18027, and the bacteria of P. The species number is ATCC 9614; (2) adding the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreii subsp. Xie to the liquid fermentation medium in a volume ratio of 1:1 to 10 for mixed fermentation; (3) adding glucose to the fermentation broth in stages, and the final concentration of glucose reaches 0.5-1.5%; (4) when the bacterial mass in the fermentation broth stopped growing, the fermentation was terminated; (5) Centrifuging the fermentation broth to obtain a supernatant, which is a mixed fermentation broth. 7. The method for preparing a mixed fermentation broth with improved bacteriostatic activity according to claim 6, characterized in that, after the mixed fermentation for 24 hours, 25%-30% glucose solution is added to the fermentation broth in stages, until the fermentation broth is The final concentration of glucose in the medium reaches 0.5-1.5%, and the time interval for adding the glucose solution is 5-12 hours. 8. the method for preparing the mixed fermentation broth that antibacterial activity improves according to claim 6, is characterized in that, the total inoculum of the liquid seed of Lactobacillus plantarum and the liquid seed of Propionibacterium freudenreichii subspecies is 5 ~20%; the mixed fermentation conditions are anaerobic culture at a temperature of 30°C. 9. The method for preparing the mixed fermentation broth with improved bacteriostatic activity according to claim 6, wherein in step (2), the liquid fermentation medium comprises yeast powder 5～10g/L, peptone 15～25g/L L, glucose 15～30g/L, calcium carbonate 10～25g/L, initial pH 7.0～7.2. 10. The application of the mixed fermentation broth with improved bacteriostatic activity according to claim 1 in inhibiting Penicillium, Aspergillus and Cladosporium.

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