CN112006066A - Mixed fermentation liquor with improved antibacterial activity and preparation method and application thereof - Google Patents
Mixed fermentation liquor with improved antibacterial activity and preparation method and application thereof Download PDFInfo
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- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 55
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- 238000012258 culturing Methods 0.000 description 6
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- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D15/00—Preserving finished, partly finished or par-baked bakery products; Improving
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to mixed fermentation liquor with improved antibacterial activity, and a preparation method and application thereof. The mixed fermentation liquor with improved antibacterial activity is obtained by mixing and fermenting lactobacillus plantarum and propionibacterium freudenreichii subspecies schizenii. In the mixed fermentation process, two kinds of bacteria are added into a fermentation tank in proportion for anaerobic fermentation, the growth of the two kinds of bacteria is controlled by adding glucose in the adding fermentation process, and the fermentation is finished when the bacteria amount in the fermentation liquid reaches the maximum. The supernatant fluid obtained by centrifuging the fermentation liquor is the fermentation extract, and the fermentation liquid contains various organic acids and has good inhibition effect on putrefying microorganisms such as penicillium, aspergillus and cladosporium.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to mixed fermentation liquor with improved antibacterial activity, and a preparation method and application thereof.
Background
The food is very easy to be polluted by external mould to be rotten and deteriorated in the processes of processing, preservation, transportation and the like, and the chemical preservative is widely applied to the food industry as the most important additive. However, the chemically synthesized preservative has potential carcinogenicity, teratogenicity or potential safety hazards such as food poisoning, so that the search and development of natural and safe antibacterial products with high bacteriostatic activity become a research hotspot in the field of food.
Lactic Acid Bacteria (LAB) are a class of bacteria that can utilize carbohydrate fermentation to produce large amounts of lactic acid. The probiotic lactic acid bacteria are generally recognized as safe microorganisms, the lactobacillus plantarum is an important probiotic bacterium, and can generate antibacterial substances such as organic acid, hydrogen peroxide, lactobacillin and the like in a metabolic process, so that the probiotic lactic acid bacteria are currently recognized as a microbe source with potential development value and a generation bacterium of a natural biological food preservative.
The propionibacterium exists widely in nature, the propionibacterium freudenreichii from milk products can be combined into various organic acids, diacetyl and special peptides or proteins in the metabolic process, the substances have wide bacteriostatic action, the bacteriostatic effect is even better than common chemical preservatives such as sodium benzoate, potassium sorbate and the like, and the propionibacterium freudenreichii has good application prospect in food preservation and fresh keeping.
For technical reasons, when lactobacillus or propionibacterium is cultured independently, the bacteriostatic activity of fermentation liquor is often low, and antiseptic products need to be purified, so that the process is complex and the cost is high. Therefore, the improvement of the bacteriostatic activity of the fermentation liquor and the reduction of the purification operation are very important for the industrial production of the natural preservative preparation.
The mixed fermentation is a novel fermentation technology which adopts the synergistic action of two or more microorganisms to jointly complete a certain fermentation process, can improve the fermentation efficiency and even can form a new product. However, two or more different strains are cultured in the same system, a competitive relationship is formed between the two or more strains, and even metabolites of the different strains generate antagonism, so that a good effect cannot be achieved by simply performing mixed culture on the two or more strains. According to data, the metabolism speed of the lactobacillus to glucose is 5-10 times that of the propionibacterium under the optimal culture condition, so that the propionibacterium rarely produces metabolites by simply mixing and culturing the lactobacillus and the propionibacterium. The invention adjusts the growth and metabolite proportion of the two strains by controlling the fermentation conditions, so as to achieve the synergistic effect, thereby improving the bacteriostatic activity of the fermentation liquor.
Disclosure of Invention
The invention aims to provide a mixed fermentation broth with improved antibacterial activity.
The invention also aims to provide a preparation method of the mixed fermentation liquor.
The invention also aims to provide application of the mixed fermentation liquor.
According to the mixed fermentation liquid with improved antibacterial activity, the mixed fermentation liquid is obtained by mixing and fermenting the lactobacillus plantarum and the propionibacterium freudenreichii subspecies schwerer, wherein the preservation number of the lactobacillus plantarum is CGMCC NO.18027, and the strain number of the propionibacterium freudenreichii subspecies schwerer is ATCC 9614.
The Lactobacillus plantarum (Lactobacillus plantarum) has a preservation number of CGMCC NO.18027, is preserved in China general microbiological culture Collection center (CCTCC) in 26 months and 06 months in 2019, and has a preservation address of China academy of sciences (China institute of sciences) No. 3, North West Lu 1 institute of south China, Inward, Beijing.
According to the mixed fermentation liquid with improved bacteriostatic activity, the mixed fermentation process of the lactobacillus plantarum and the propionibacterium freudenreichii subspecies comprises the following steps:
(1) respectively preparing liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schleriensler;
(2) liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schilder were mixed according to the ratio of 1: inoculating the mixture into a liquid fermentation culture medium in a volume ratio of 1-10 for mixed fermentation;
(3) adding glucose into the fermentation liquor in a grading manner, wherein the final concentration of the glucose is 0.5-1.5%;
(4) when the bacterial mass in the fermentation liquor stops increasing, the fermentation is finished;
(5) and centrifuging the fermentation liquor to obtain supernatant, namely the mixed fermentation liquor.
The invention adopts two strains for mixed fermentation, and improves the bacteriostatic activity of the fermentation liquor through the synergistic effect of metabolites; meanwhile, the two strains grow fully by controlling the inoculation proportion and the sugar supplement process, and the metabolites of the two strains are further improved, so that a better synergistic bacteriostatic effect is achieved.
According to the mixed fermentation liquid with improved antibacterial activity, the inoculation volume ratio of the lactobacillus plantarum to the propionibacterium is preferably 1: 2-1: 5. The inoculation ratio of the strains can affect the growth speed and metabolic products of the two strains, and further affect the synergistic bacteriostatic effect of the fermentation liquor.
According to the mixed fermentation liquor with improved antibacterial activity, after 24 hours of mixed fermentation, 25% -30% of glucose solution is added into the fermentation liquor in a divided manner until the final concentration of glucose in the fermentation liquor is 0.5-1.5%, wherein the time interval for supplementing the glucose solution is 5-12 hours, and the final concentration of glucose is preferably 0.8-1.2%. By adjusting the glucose supplementation amount and the glucose supplementation time, the synergistic effect between metabolites of the two strains is improved, so that the bacteriostatic effect of the fermentation liquor is further improved.
According to the mixed fermentation liquid with improved antibacterial activity, in the step (2), the total inoculation amount of the liquid seeds of the lactobacillus plantarum and the liquid seeds of the propionibacterium freudenreichii subspecies schilder is 5-20%, and the more preferable inoculation amount is 8-10%; the mixed fermentation condition is anaerobic culture at the temperature of 30 ℃. The proper inoculation amount has important influence on the growth rate of the thallus and the nutrient components of the culture medium. The temperature of 30 ℃ is the optimal temperature for the growth of the propionibacterium, and meanwhile, the metabolism of the lactobacillus cannot be influenced, thereby being beneficial to the growth synergistic effect of the two strains.
According to the mixed fermentation liquid with improved antibacterial activity, in the step (2), the liquid fermentation culture medium comprises 5-10 g/L of yeast powder, 15-25 g/L of peptone, 15-30 g/L of glucose and 10-25 g/L of calcium carbonate, and the initial pH is 7.0-7.2.
A method of mixing fermentation broth with improved bacteriostatic activity according to an embodiment of the present invention, the method comprising the steps of:
(1) respectively preparing liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schleri, wherein the preservation number of the lactobacillus plantarum is CGMCC NO.18027, and the strain number of the propionibacterium freudenreichii subspecies schleri is ATCC 9614;
(2) liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schilder were mixed according to the ratio of 1: inoculating the mixture into a liquid fermentation culture medium in a volume ratio of 1-10 for mixed fermentation;
(3) adding glucose into the fermentation liquor in a grading manner, wherein the final concentration of the glucose is 0.5-1.5%;
(4) when the bacterial mass in the fermentation liquor stops increasing, the fermentation is finished;
(5) and centrifuging the fermentation liquor to obtain supernatant, namely the mixed fermentation liquor.
According to the method for preparing the mixed fermentation liquor with improved antibacterial activity, after 24 hours of mixed fermentation, 25% -30% of glucose solution is added into the fermentation liquor in a grading manner until the final concentration of glucose in the fermentation liquor is 0.5-1.5%, wherein the time interval for supplementing the glucose solution is 5-12 hours.
According to the method for preparing the mixed fermentation liquor with improved antibacterial activity, the total inoculation amount of liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schildersha is 5-20%; the mixed fermentation condition is anaerobic culture at the temperature of 30 ℃.
According to the method for preparing the mixed fermentation liquor with improved antibacterial activity, in the step (2), the liquid fermentation culture medium comprises 5-10 g/L of yeast powder, 15-25 g/L of peptone, 15-30 g/L of glucose, 10-25 g/L of calcium carbonate, and the initial pH is 7.0-7.2.
According to the method for mixing the fermentation liquor with improved antibacterial activity, disclosed by the embodiment of the invention, in the step (4), the fermentation liquor is centrifuged at the centrifugal rotation speed of 3000-5000 r/min for 10-20 min at the temperature of 15-30 ℃, so that a supernatant is obtained.
The invention also provides application of the mixed fermentation liquor, and the bacteriostatic effect of the mixed fermentation liquor on common spoilage microorganisms in baked products, such as penicillium, aspergillus flavus, cladosporium and the like, is superior to that of single-strain fermentation liquor.
The invention has the beneficial effects that:
the invention provides a mixed fermentation liquid prepared by mixing and fermenting lactobacillus plantarum and propionibacterium freudenreichii subspecies Sheheli, wherein in the process of mixed fermentation, two strains are added into a fermentation tank in proportion for anaerobic fermentation, in the process of adding fermentation, the growth of the two strains is controlled by supplementing glucose, and when the amount of the strains in the fermentation liquid reaches the maximum, the fermentation is finished. The supernatant fluid obtained by centrifuging the fermentation liquor is the fermentation extract, and the fermentation liquid contains various organic acids and has good inhibition effect on putrefying microorganisms such as penicillium, aspergillus and cladosporium.
Drawings
FIG. 1 shows the bacteriostatic effect of mixed fermentation broth obtained by separately fermenting and mixed fermenting Lactobacillus plantarum and Propionibacterium freudenreichii on Aspergillus flavus and Penicillium, wherein A is a fermentation broth plate prepared by separately fermenting Lactobacillus plantarum; b is a fermentation liquid flat plate prepared by mixed fermentation of lactobacillus plantarum and propionibacterium freudenreichii; c is a fermentation liquid flat plate prepared by single fermentation of propionibacterium freudenreichii; a to Aspergillus flavus bacterial colony and b to Penicillium bacterial colony.
FIG. 2 shows the bacteriostatic effect of the mixed fermentation liquid on Penicillium and Aspergillus niger obtained by different inoculation ratios of lactic acid bacteria and Propionibacterium under the same culture conditions. The inoculation volume ratios of lactobacillus plantarum and propionibacterium are respectively 1:4, 2:3, 1:1, 3:2 and 4:1
FIG. 3 shows the bacteriostatic effect of the mixed fermentation liquid on Penicillium and Aspergillus niger obtained by different sugar-supplementing methods under the same inoculation ratio of lactobacillus and Propionibacterium.
The Lactobacillus plantarum (Lactobacillus plantarum) L9 has the preservation number of CGMCC NO.18027, is preserved in China general microbiological culture Collection center (CCCCCCCES) in 26.06.2019, and has the preservation address of China academy of sciences (China institute of sciences, No. 3, West Lu 1, North Cheng, Inward, Beijing city.
Detailed Description
Example 1 preparation of a Mixed fermentation extract of Lactobacillus plantarum and Propionibacterium freudenreichii
(1) Preparing a seed solution:
and (3) selecting the activated lactobacillus plantarum colony, inoculating the lactobacillus plantarum colony into an MRS liquid culture medium, carrying out static culture at the temperature of 30-37 ℃, carrying out culture for 12-20 h, and sampling to carry out microscopic examination when the OD of the culture medium reaches 2-4 so as to obtain a sterile lactobacillus plantarum strain for later use. The MRS culture medium contains 3-7 g of peptone, 5-9 g of beef extract, 5-9 g of yeast powder, 10-15 g of glucose, 1-2 g of sodium acetate, 1-2 g of diamine citrate, 800.2-0.5 g of tween, 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 per liter.
And (3) selecting the activated single bacterial colony of the propionibacterium freudenreichii, inoculating the single bacterial colony into an SLB liquid culture medium, and statically culturing at the temperature of 25-32 ℃ for 30-50 h. When the OD of the culture medium reaches 1-2, sampling and performing microscopic examination to remove foreign bacteria for later use. Wherein each liter of the SLB culture 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.
(2) Liquid anaerobic fermentation: inoculating the prepared seed liquid by 12 percent of total inoculation amount, wherein the volume ratio of the lactobacillus plantarum to the propionibacterium is 1: 3; inoculating the seed liquid into a new liquid culture medium for fermentation culture, wherein the liquid culture medium contains 10g/L yeast powder, 20g/L peptone, 20g/L glucose and 20g/L calcium carbonate, and the initial pH is 7.0-7.2. The fermentation temperature was 30 ℃.
After fermenting for 24h, glucose solution is supplemented in batches until the final concentration is 1%, and the sugar supplementing interval is 10h each time.
When the biomass in the fermentation liquor does not increase any more after the fermentation is carried out for 80 hours, the fermentation is finished.
Centrifuging at 4000rpm for 20min at room temperature to obtain supernatant as mixed fermentation liquid.
Example 2 detection of bacteriostatic Activity of Mixed fermentation broth
2.1 preparation of indicator bacteria
Aiming at common mould causing food spoilage, aspergillus flavus and penicillium are selected as bacteriostatic indicator strains.
Respectively transferring Aspergillus flavus, Penicillium and Cladosporium species preserved in 20% glycerol to PDA solid culture medium, and performing activated culture at 30 deg.C. Then transferring the activated mould spores to PDA slant, culturing at 30 deg.C for 7d, washing the spores from the culture medium with ME medium containing 0.1% Tween 80 (malt extract 20g/L, sucrose 20g/L, peptone 1g/L, natural pH), and counting by using blood cell plate counting method to obtain the product with spore content of 1 × 105Aspergillus flavus and Penicillium spores.
2.2 preparation of Lactobacillus plantarum fermented extract
(1) Preparing a seed solution: and (3) selecting the activated lactobacillus plantarum single colony, inoculating the lactobacillus plantarum single colony into an MRS liquid culture medium, and carrying out anaerobic culture at 25-30 ℃ for 12-20 hours respectively. Sampling, and performing microscopic examination to remove foreign bacteria for later use.
(2) Liquid anaerobic fermentation: inoculating the prepared seed liquid into a liquid culture medium which contains 10g/L yeast powder, 20g/L peptone, 20g/L glucose and 20g/L calcium carbonate and has initial pH of 7.0-7.2 at the temperature of 30 ℃ in a 10% inoculation amount for fermentation culture. When the biomass did not increase any more after 48h, the fermentation was stopped. Centrifuging at room temperature at 4000rpm for 20min to obtain supernatant as lactobacillus plantarum fermented extract.
2.3 preparation of fermented extract of Propionibacterium freudenreichii
(1) Preparing a seed solution: and (3) selecting the activated single bacterial colony of the propionibacterium freudenreichii, inoculating the single bacterial colony into an SLB liquid culture medium, and carrying out anaerobic culture at the temperature of 30 ℃ for 45 hours respectively. Sampling, and performing microscopic examination to remove foreign bacteria for later use.
(2) Liquid anaerobic fermentation: inoculating the prepared seed liquid into a liquid culture medium which contains 10g/L yeast powder, 20g/L peptone, 20g/L glucose and 20g/L calcium carbonate and has the initial pH of 7.0-7.2 for fermentation culture at the temperature of 30 ℃ in 10 percent of the total inoculation amount. Fermenting for 96h until the biomass is not increased any more, and finishing the fermentation. Centrifuging at room temperature at 4000rpm for 20min to obtain supernatant as fermented extract of Propionibacterium freudenreichii.
2.4 Observation of the bacteriostatic Effect
Respectively taking 50mL of distilled water and 50mL of lactobacillus plantarum and propionibacterium freudenreichii which are fermented independently and preparing mixed fermentation liquor by the mixed fermentation, adding a proper amount of potato glucose agar to melt, adjusting the pH value of a culture medium to 6, sterilizing by high-pressure steam, and pouring into a flat plate. After the plate solidified, 10uL of 1 × 10 solution was added dropwise5Culturing the Penicillium and Aspergillus flavus spore suspension at 30 ℃. The growth of the mold was observed.
As can be seen from FIG. 1, the blue mold on the B plate containing the mixed fermentation extract was almost completely inhibited, while the colony of Aspergillus flavus on the B plate was smaller than that of the A plate of Lactobacillus plantarum fermented alone and that of the C plate of Propionibacterium freudenreichii fermented liquid, and the color of the colony of Aspergillus flavus on the B plate was lighter than that of the A plate and that of the C plate, indicating that Aspergillus flavus was also significantly inhibited. Therefore, the mixed fermentation liquid has higher bacteriostatic activity than single fermentation extracts of lactobacillus plantarum and propionibacterium freudenreichii.
Example 3 Effect of the inoculation ratio of Lactobacillus plantarum and Propionibacterium on the bacteriostatic Activity of the fermentation broth
(1) Preparing a seed solution:
and (3) selecting the activated lactobacillus plantarum colony, inoculating the lactobacillus plantarum colony into an MRS liquid culture medium, carrying out static culture at the temperature of 30-37 ℃, carrying out culture for 12-20 h, and sampling to carry out microscopic examination when the OD of the culture medium reaches 2-4 so as to obtain a sterile lactobacillus plantarum strain for later use. The MRS culture medium contains 3-7 g of peptone, 5-9 g of beef extract, 5-9 g of yeast powder, 10-15 g of glucose, 1-2 g of sodium acetate, 1-2 g of diamine citrate, 800.2-0.5 g of tween, 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 per liter.
And (3) selecting the activated single bacterial colony of the propionibacterium freudenreichii, inoculating the single bacterial colony into an SLB liquid culture medium, and statically culturing at the temperature of 25-32 ℃ for 30-50 h. When the OD of the culture medium reaches 1-2, sampling and performing microscopic examination to remove foreign bacteria for later use. Wherein each liter of the SLB culture 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.
(2) Liquid anaerobic fermentation: inoculating the prepared seed liquid with 12 percent of total inoculation amount, wherein the volume ratio of lactobacillus plantarum to propionibacterium is (1: 4), (2: 3), (1: 1), (3: 2) and (4: 1); inoculating the seed liquid into a new liquid culture medium for fermentation culture, wherein the liquid culture medium contains 10g/L yeast powder, 20g/L peptone, 20g/L glucose and 20g/L calcium carbonate, and the initial pH is 7.0-7.2. The fermentation temperature was 30 ℃.
After fermenting for 24h, glucose is supplemented in batches until the final concentration is 1%, and the sugar supplementing interval is 10h each time. And when the biomass in the fermentation liquor does not increase any more after the fermentation is carried out for 80 hours, ending the fermentation. And centrifuging the fermentation liquor at 4000rpm for 20min, and obtaining the supernatant fluid which is the mixed fermentation liquor.
The results of comparing the bacteriostatic activity of the mixed fermentation broth with different inoculation ratios according to the method of example 2.4 are shown in fig. 2, and it can be seen that different inoculation ratios have certain influence on the bacteriostatic activity of the mixed fermentation broth. With the increase of the inoculation ratio of the lactobacillus plantarum, the antibacterial activity of the fermentation liquor to the penicillium and the aspergillus flavus is increased and then decreased. When the inoculation ratio of the lactobacillus plantarum to the propionibacterium is 3:2, the antibacterial activity of the fermentation liquor is strongest.
Example 4 Effect of sugar supplementation regimen on the bacteriostatic Activity of metabolites
(1) Preparing a seed solution:
and (3) selecting the activated lactobacillus plantarum colony, inoculating the lactobacillus plantarum colony into an MRS liquid culture medium, carrying out static culture at the temperature of 30-37 ℃, carrying out culture for 12-20 h, and sampling to carry out microscopic examination when the OD of the culture medium reaches 2-4 so as to obtain a sterile lactobacillus plantarum strain for later use. The MRS culture medium contains 3-7 g of peptone, 5-9 g of beef extract, 5-9 g of yeast powder, 10-15 g of glucose, 1-2 g of sodium acetate, 1-2 g of diamine citrate, 800.2-0.5 g of tween, 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 per liter.
And (3) selecting the activated single bacterial colony of the propionibacterium freudenreichii, inoculating the single bacterial colony into an SLB liquid culture medium, and statically culturing at the temperature of 25-32 ℃ for 30-50 h. When the OD of the culture medium reaches 1-2, sampling and performing microscopic examination to remove foreign bacteria for later use. Wherein each liter of the SLB culture 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.
(2) Liquid anaerobic fermentation: inoculating the prepared seed liquid by 12 percent of total inoculation amount, wherein the volume ratio of the lactobacillus plantarum to the propionibacterium is 1: 3; inoculating the seed liquid into a new liquid culture medium for fermentation culture, wherein the liquid culture medium contains 10g/L yeast powder, 20g/L peptone, 20g/L glucose and 20g/L calcium carbonate, and the initial pH is 7.0-7.2. The fermentation temperature was 30 ℃. The fermentation process is carried out by the following four sugar supplement modes.
A. After fermenting for 24h, glucose is supplemented in three batches until the final concentration is 1%, and the sugar supplementing interval is 6 h.
B. After fermenting for 24 hours, glucose is supplemented in three batches until the final concentration is 1 percent, and the sugar supplementing interval is 10 hours each time.
C. After fermenting for 24h, glucose is supplemented in three batches until the final concentration is 1%, and the sugar supplementing interval is 20h each time.
D. After fermenting for 24h, glucose is supplemented in two batches until the final concentration is 1.5%, and the sugar supplementing interval is 10h each time.
E. After fermenting for 24h, glucose is supplemented in two batches until the final concentration is 1.5%, and the sugar supplementing interval is 20h each time.
The results of comparing the mixed fermentation liquors of different sugar-supplementing methods according to the method of example 2.4 are shown in fig. 3, the mixed fermentation liquors obtained by several different sugar-supplementing methods have little difference in inhibition effect on penicillium, but have obvious advantage on the inhibition effect on aspergillus niger group B, so after 24h of fermentation, glucose is supplemented to the final concentration of 1% in three batches, and the optimal sugar-supplementing scheme is that the sugar-supplementing interval is 10h every time.
On the basis that two products with synergistic antibacterial effects are found, the mixed fermentation inoculation ratio and the sugar supplement mode are optimized, and the antibacterial activity of the fermentation liquor is further improved, so that a good foundation is laid for industrial development of the natural anticorrosive product.
Claims (10)
1. The mixed fermentation liquid with improved antibacterial activity is characterized in that the mixed fermentation liquid is obtained by mixing and fermenting lactobacillus plantarum and propionibacterium freudenreichii subspecies, wherein the preservation number of the lactobacillus plantarum is CGMCC NO.18027, and the strain number of the propionibacterium freudenreichii subspecies is ATCC 9614.
2. The mixed fermentation broth with improved bacteriostatic activity according to claim 1, wherein the mixed fermentation process of lactobacillus plantarum and propionibacterium freudenreichii subspecies schie comprises the following steps:
(1) respectively preparing liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schleriensler;
(2) liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schilder were mixed according to the ratio of 1: inoculating the mixture into a liquid fermentation culture medium in a volume ratio of 1-10, and performing mixed fermentation;
(3) adding glucose into the fermentation liquor in a grading manner, wherein the final concentration of the glucose reaches 0.5-1.5%;
(4) when the bacterial mass in the fermentation liquor stops increasing, the fermentation is finished;
(5) centrifuging the fermentation liquor, and taking supernatant to obtain the mixed fermentation liquor.
3. The mixed fermentation liquor with improved bacteriostatic activity according to claim 2, wherein after 24 hours of mixed fermentation, 25-30% of glucose solution is added into the fermentation liquor in portions until the final concentration of glucose in the fermentation liquor reaches 0.5-1.5%, wherein the time interval for supplementing the glucose solution is 5-12 hours.
4. The mixed fermentation liquid with improved bacteriostatic activity according to claim 2, wherein in step (2), the total inoculation amount of the liquid seeds of lactobacillus plantarum and the liquid seeds of propionibacterium freudenreichii subspecies schildersha is 5-20%; the mixed fermentation condition is anaerobic culture at the temperature of 30 ℃.
5. The mixed fermentation broth with improved bacteriostatic activity according to claim 2, wherein in step (2), the liquid fermentation medium comprises 5-10 g/L yeast powder, 15-25 g/L peptone, 15-30 g/L glucose, 10-25 g/L calcium carbonate, and initial pH is 7.0-7.2.
6. A method of preparing the mixed fermentation broth with improved bacteriostatic activity according to claim 1, wherein the method comprises the steps of:
(1) respectively preparing liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schleri, wherein the preservation number of the lactobacillus plantarum is CGMCC NO.18027, and the strain number of the propionibacterium freudenreichii subspecies schleri is ATCC 9614;
(2) liquid seeds of lactobacillus plantarum and liquid seeds of propionibacterium freudenreichii subspecies schilder were mixed according to the ratio of 1: inoculating the mixture into a liquid fermentation culture medium in a volume ratio of 1-10 for mixed fermentation;
(3) adding glucose into the fermentation liquor in a grading manner, wherein the final concentration of the glucose reaches 0.5-1.5%;
(4) when the bacterial mass in the fermentation liquor stops increasing, the fermentation is finished;
(5) and centrifuging the fermentation liquor to obtain supernatant, namely the mixed fermentation liquor.
7. The method for preparing the mixed fermentation liquor with the improved antibacterial activity according to claim 6, wherein after 24 hours of mixed fermentation, 25% -30% of glucose solution is added into the fermentation liquor in a divided manner until the final concentration of glucose in the fermentation liquor reaches 0.5-1.5%, wherein the time interval for supplementing the glucose solution is 5-12 hours.
8. The method for preparing a mixed fermentation broth with improved bacteriostatic activity according to claim 6, wherein the total inoculation amount of the liquid seeds of Lactobacillus plantarum and the liquid seeds of Propionibacterium freudenreichii subspecies Sheli is 5-20%; the mixed fermentation condition is anaerobic culture at the temperature of 30 ℃.
9. The method for preparing a mixed fermentation broth with improved antibacterial activity according to claim 6, wherein in the step (2), the liquid fermentation medium comprises 5-10 g/L of yeast powder, 15-25 g/L of peptone, 15-30 g/L of glucose, 10-25 g/L of calcium carbonate, and the initial pH is 7.0-7.2.
10. The use of the mixed fermentation broth with improved bacteriostatic activity according to claim 1 for inhibiting penicillium, aspergillus, and cladosporium.
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