CN114854647A - Lactobacillus fermentum and culture and application thereof - Google Patents
Lactobacillus fermentum and culture and application thereof Download PDFInfo
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- CN114854647A CN114854647A CN202210683693.3A CN202210683693A CN114854647A CN 114854647 A CN114854647 A CN 114854647A CN 202210683693 A CN202210683693 A CN 202210683693A CN 114854647 A CN114854647 A CN 114854647A
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- lactobacillus fermentum
- fermentation
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- lactobacillus
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Abstract
The invention discloses Lactobacillus fermentum (SS-31). The results of the study show that Lactobacillus fermentum SS-31 of the present invention is capable of reducing the secretion of NO in cells, and inflammatory factors IL-1 beta, IL-6, and IL-10. Therefore, the lactobacillus fermentum SS-31 has stronger anti-inflammatory activity. In addition, the inventor also develops a high-density fermentation medium specially used for the lactobacillus fermentum SS-31 and a culture method thereof, the medium has simple components, does not need to add beef powder and peptone, and has simple and convenient preparation, quick operation and good application prospect; the culture method improves the utilization efficiency of fermentation equipment, and can realize high-density enrichment culture, thereby obviously reducing the fermentation cost. In conclusion, the invention realizes the high-density fermentation culture of the lactobacillus fermentum SS-31, so as to be applied to further development of medicines, health products, foods or feeds and the like in the future.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to lactobacillus fermentum and culture and application thereof.
Background
Inflammatory bowel disease is a chronic recurrent disease, which is slow in onset, has an unknown etiology, is easy to relapse, and is mostly difficult to completely cure. The means for treating inflammatory bowel diseases mainly include drug treatment and surgical treatment. Among them, although drug therapy can effectively alleviate symptoms, long-term drug therapy can cause pathogenic bacteria to have drug resistance to antibiotics, and surgical treatment is expensive. Therefore, the search for a new safe and reliable treatment method is a problem to be solved urgently. With the development of the research, the probiotic preparation is considered as a novel adjuvant therapy. The probiotics can play a role in regulating intestinal flora, has remarkable effects of preventing and treating the inflammatory bowel diseases, does not generate drug resistance, adverse reactions and other advantages, and is expected to become one of important means for preventing or assisting in treating the inflammatory bowel diseases.
The lactobacillus can regulate intestinal flora balance, induce nonspecific activation of host immune system, resist oxidation, reduce blood sugar, resist enteritis, and regulate immunity. Many studies for developing lactic acid bacteria strains having probiotic functions and being safe to humans have been reported in recent years, and these strains have been applied to pharmaceuticals or functional foods. For example:
chinese patent 'application of lactobacillus plantarum CQPC02 in preparation of food or medicine for preventing liver oxidative damage' (patent number 201811639687.8 published as 2019, 04 and 16)
Chinese patent "lactic acid bacteria, natural immunostimulant derived from the same, agent for preventing/treating infectious diseases, and food and drink" (patent No. 201980004577.0 published Japanese 2020, 06, 23)
Chinese patent 'a plant lactobacillus plantarum capable of relieving hyperuricemia and application thereof' (patent No. 202011515393.1 published Japanese 2021, 05 and 14)
Chinese patent "novel Lactobacillus plantarum and Lactobacillus composition and its use for treating or preventing heavy metal-related diseases" (patent No. 201911052816.8 publication No. 2020, 05, 26)
However, there is no report on the prevention or treatment of inflammatory bowel disease by lactic acid bacteria, and lactic acid bacteria can produce certain probiotic effect when the amount in the body reaches a certain level. The current low fermentation level of the lactic acid bacteria limits the expansion application of the lactic acid bacteria, so that the probiotic bacteria can be added into functional food and feed taking the lactic acid bacteria as main components by utilizing the high-density culture technology of the bacterial strains to reach the corresponding quantity. The core of realizing high-density culture of the strain is the optimization of a fermentation medium and fermentation conditions. The growth and propagation of lactic acid bacteria require nutrient substances such as carbon sources, nitrogen sources, inorganic salts, nutritional factors and the like, and the lactic acid bacteria need to continuously exchange substance energy with the outside in the growth process and are influenced by the outside environment (temperature, pH and the like). The high-density culture technology is a method for making the density of thalli cultured in a liquid culture exceed that of a common culture method by changing culture conditions or adding other reagents through certain culture technologies and equipment. Compared with the common culture, the high-density culture can not only improve the thallus density more quickly, shorten the production period, but also reduce the production cost of equipment. Therefore, the high-density fermentation enrichment culture of the lactic acid bacteria is a key technology for the current development process of the probiotic product with anti-inflammatory activity.
Disclosure of Invention
The invention aims to solve the technical problem of providing a lactobacillus fermentum and culture and application thereof, in particular to a lactobacillus strain with anti-inflammatory activity and fermentation culture, and application thereof in developing functional health-care food, feed and feed leavening agent.
In order to solve the technical problems, the invention adopts the following technical scheme:
a Lactobacillus fermentum strain is Lactobacillus fermentum (SS-31) with the preservation number of CGMCC NO:24925。
the lactobacillus fermentum 16S rDNA gene has a base sequence of a sequence table SEQ.ID.NO. 1.
In the high-density fermentation medium for the lactobacillus fermentum, the carbon source in the medium is one or more of glucose, maltose, sucrose and lactose, the nitrogen source is one or more of peptone, yeast extract powder, beef peptone and tryptone, and the inorganic salt is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium citrate and sodium acetate.
In the culture medium, the carbon source is maltose, the nitrogen source is yeast extract powder, and the inorganic salt is dipotassium hydrogen phosphate.
The high-density fermentation medium of the lactobacillus fermentum is an MRS liquid medium, and each 1L of the MRS liquid medium comprises the following components in percentage by weight: 15.00g of maltose, 20.00g of yeast extract powder, 9.00g of dipotassium phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate, 801.00 g of Tween and 1000mL of water, adjusting the pH value to 6.5, and carrying out autoclaving at 115 ℃ for 20 min.
The high-density fermentation culture method of the lactobacillus fermentum comprises the steps of inoculating lactobacillus fermentum seed liquid into the high-density fermentation culture medium according to claim 5, wherein the inoculation amount is 1-5% (v/v), adjusting the initial fermentation pH value to 5.8-7.8, controlling the fermentation broth pH value to 6.8 +/-0.02 by using 15-25% ammonia water solution after fermentation, and culturing for 12-24h at 32-42 ℃.
The lactobacillus fermentum is applied to medicines, health products, foods or feeds.
The medicine is anti-inflammatory.
Lactobacillus beverage containing the Lactobacillus fermentum with viable count of 10 8 CFU/mL or more.
The biological feed leaven contains fermentation liquor of the lactobacillus fermentum, and a carbon source in the fermentation culture liquor is a compound of maltose and sucrose and contains cellulase.
In previous studies, the inventors isolated Lactobacillus fermentum SS-31 from sour bamboo shoot in Guangxi Liuzhou. A cell inflammation model is established by adopting RAW264.7 macrophages induced by lipopolysaccharide, the cell activity is measured by adopting a CCK-8 method, and the anti-inflammatory activity is evaluated by measuring the release amounts of NO, IL-1 beta, IL-6 and IL-10 by an ELISA kit. The results show that Lactobacillus fermentum SS-31 of the present invention is able to reduce the secretion of NO in cells, as well as the inflammatory factors IL-1 β, IL-6, and IL-10. Therefore, the lactobacillus fermentum SS-31 has stronger anti-inflammatory activity. In addition, the inventor also develops a high-density fermentation medium specially used for the lactobacillus fermentum SS-31 and a culture method thereof, the medium has simple components, does not need to add beef powder and peptone, and has simple and convenient preparation, quick operation and good application prospect; the culture method improves the utilization efficiency of fermentation equipment, and can realize high-density enrichment culture, thereby obviously reducing the fermentation cost.
In conclusion, the functional research of the lactobacillus of the bamboo shoot sour Liuzhou source is enriched, the probiotic effect is exerted, and meanwhile, the strain resource of the Guangxi special fermented food is promoted; meanwhile, the invention realizes the high-density fermentation culture of the lactobacillus fermentum SS-31, so as to be applied to further developing medicines, health products, foods or feeds and the like in the future.
Drawings
FIG. 1 is an agarose gel electrophoresis of the 16S rDNA PCR amplification product of Lactobacillus fermentum SS-31, showing: 1Marker, 2 Lactobacillus fermentum SS-31.
FIG. 2 is a graph showing the effect of different concentrations of Lactobacillus fermentum SS-31 and LPS treatment on the viability of RAW.264.7 cells.
FIG. 3 is a graph showing the results of the content of NO produced by Lactobacillus fermentum SS-31 in LPS-stimulated RAW264.7 cells.
FIG. 4 is a graph showing the results of the IL-1. beta. content of Lactobacillus fermentum SS-31 produced by RAW264.7 cells stimulated by LPS.
FIG. 5 is a graph showing the results of IL-6 content of Lactobacillus fermentum SS-31 produced by RAW264.7 cells stimulated by LPS.
FIG. 6 is a graph showing the results of IL-10 content of Lactobacillus fermentum SS-31 produced by RAW264.7 cells stimulated by LPS.
P < 0.05 in FIGS. 2 to 6 indicates the difference from the normal group
Description of preservation information
Lactobacillus fermentum (SS-31 with the preservation number of CGMCC NO:24925the preservation date: 2022 year _05_ month _19_ day, the preservation address is: west road No.1, north zhou zhao yang district, beijing, institute of microbiology, china academy of sciences, zip code 100101, depository: china general microbiological culture Collection center.
Preservation conditions are as follows: mixing the enriched culture solution with sterilized glycerol to control the final concentration to 20-30%, packaging into glycerol tubes, and storing in-80 deg.C refrigerator or liquid nitrogen tank.
Detailed Description
Example 1 evaluation and identification of anti-inflammatory Activity of Lactobacillus fermentum SS-31
1. Sequencing identification of Lactobacillus fermentum SS-31
(1) Activation of a glycerol tube: lactobacillus fermentum SS-31 preserved in glycerol tubes was streaked on MRS solid medium plates and cultured at 37 ℃ for 48 h. Each 1L of MRS solid culture medium consists of the following components in percentage by weight: 1000mL of MRS liquid medium and 15g of agar. Each 1L of MRS liquid culture medium comprises the following components by weight: 20.00g of carbon source, 20.00g of tryptone, 9.00g of dipotassium phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate, 801.00 g of Tween and 1000mL of water, adjusting the pH value to 6.5, and carrying out autoclaving at 115 ℃ for 20 min.
(2) Liquid activation: and selecting a single colony, inoculating the single colony in a test tube containing 10mL of MRS liquid culture medium for static culture, performing primary activation at 37 ℃ for 24h, inoculating the strain suspension liquid in an inoculum size of 2% (v/v) in the MRS liquid culture medium containing 100mL, and performing secondary activation at 37 ℃ for static culture for 24h to obtain a seed solution.
(3) And (3) identification: inoculating the seed solution into a proliferation culture medium seed in an inoculation amount of 2% (v/v), adjusting the initial pH to 6.8, placing the seed solution in a 37 ℃ incubator for culturing for 24h, streaking the seed solution to an MRS solid culture medium for culturing for 48h, and selecting a single colony for PCR. 16S rRNA gene amplification was performed using a reverse primer 1492R (5'-AGAGTTTGATTTGATCCTGGCTAG-3', SEQ.ID.NO.2) and a forward primer 27F (5'-GGTTACCTTGTTACGACTT-3', SEQ.ID.NO.3), and PCR amplification was performed in a 25. mu.L reaction system by pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 1min, annealing at 64 ℃ for 1min, extension at 72 ℃ for 2min, 35 cycles, and storage at 4 ℃. After the amplification, 4. mu.L of the PCR product was subjected to 1% agarose gel electrophoresis and observed, and the result is shown in FIG. 1. Sequencing, carrying out homologous comparison analysis on the sequence obtained by sequencing and a GenBank database of NCBI (national center for Biotechnology information) to show that the strain SS-31 is lactobacillus fermentum, the 16s rDNA sequence is shown as SEQ. ID.NO.1 and 2. the determination of the anti-inflammatory activity of the lactobacillus
(1) And (3) culturing the cells: RAW264.7 macrophages were cultured in DMEM containing 10% fetal bovine serum. Cells in log phase at 2.5X 10 5 one/mL of the cells were seeded in 24-well plates at 500. mu.L/well and 5% CO at 37 ℃ 2 Was cultured overnight in a humidified incubator of (1) for subsequent experiments.
(2) Determination of cell viability: the concentration of the pancreatin-digested RAW264.7 cell suspension was adjusted to 1.0 × 10 by a blood cell count method 4 Inoculating to 96-well plate at 37 deg.C and 5% CO 2 Culturing in an incubator to a fusion state. After 24h stimulation with 1. mu.g/mL LPS, 10. mu.L of activated different concentrations (1X 10) 5 、1×10 6 、1×10 7 、1×10 8 、1×10 9 CFU/mL) for 3 h. 10 μ L of CCK-8 solution was added to each well, and after incubation at 37 ℃ for 1h, absorbance values were measured at 450nm to evaluate the effect of different concentrations of lactic acid bacteria on RAW264.7 cells. The cell viability calculation formula is as follows:
in the formula: the As experimental group contains cell culture medium, CCK-8 and lactobacillus; the Ac model group contains a cell culture medium, CCK-8 and lactobacillus-free bacteria; ab control group did not contain cell and lactic acid bacteria medium, CCK-8.
(4) Determination of NO: subjecting the pancreatin-digested RAW264.7 cell suspension to hemocytometerThe concentration is adjusted to 2.5 × 10 by a numerical method 5 cell/mL, evenly inoculated in a 24-well plate, at 37 ℃ and 5% CO 2 Culturing in an incubator to a fusion state. Adding 1 μ g/mL LPS for 24h, and adding 50 μ L of activated concentration of 1 × 10 8 Culturing the bacterial solution in the concentration of CFU/mL for 3h, and collecting the supernatant. According to the operation of the NO detection kit specification, the formula for calculating the mass concentration of NO is as follows:
note: in the formula: c represents a standard (sodium nitrite standard solution, concentration 20. mu. mol/L); n is the dilution factor (n-4).
As can be seen from FIG. 2, the cell growth promoting effect was gradually increased as the concentration of lactic acid bacteria was increased. When the cell concentration is 10 in comparison with the normal group 9 The survival rate of SS-31 to cells is 111.61% at CFU/mL, and the difference is significant (P < 0.05); when the concentration of the bacteria is 10 8 The cell survival rate of SS-31 at CFU/mL is 97.05% but not significant (P is more than 0.05); the cell concentration was 10 in comparison with that in the normal group 5 、10 6 、10 7 The cell survival rate is obviously reduced (P < 0.05) at CFU/mL, and the cell survival is inhibited. In conclusion, the cell concentration was 10 8 CFU/mL was used as the optimal working concentration for the subsequent experiments.
As can be seen from FIG. 3, the levels of NO secreted by RAW264.7 after treatment with Lactobacillus fermentum SS-31 and LPS. LPS induces RAW264.7 cells to secrete a large amount of NO (P is less than 0.05), lactobacillus SS-31 can remarkably inhibit NO secretion (P is less than 0.05), and the inhibition rate is 84.31%.
(5) Measurement of inflammatory factors IL-1. beta., IL-6, and IL-10: adjusting the cell suspension of RAW264.7 after trypsinization to 2.5X 10 5 cell/mL, evenly inoculated in a 24-well plate, 37 ℃ and 5% CO 2 Culturing in an incubator to a fusion state. LPS was added at a concentration of 1. mu.g/mL for 24h, followed by 50. mu.L of activated concentration of 1X 10 8 Culturing the bacterial solution in the concentration of CFU/mL for 3h, and collecting the supernatant. The content of inflammatory factors IL-1 beta, IL-6 and IL-10 in cell supernatant is determined according to the operating procedures of the ELISA kit instructionAmount of the compound (A).
As can be seen from FIG. 4, LPS induces RAW264.7 cells to secrete a large amount of inflammatory factor IL-1 beta (P < 0.05), and lactobacillus SS-31 can remarkably inhibit IL-1 beta secretion (P < 0.05), with an inhibition rate of 49.21%.
As can be seen from FIG. 5, LPS induces RAW264.7 cells to secrete a large amount of inflammatory factor IL-6(P < 0.05), and lactobacillus SS-31 can remarkably inhibit IL-6 secretion (P < 0.05), with an inhibition rate of 14.12%.
As can be seen from FIG. 6, LPS induces RAW264.7 cells to reduce the secretion of proinflammatory factor IL-10 (P < 0.05), and lactobacillus SS-31 can remarkably promote the secretion of IL-10 (P < 0.05), with the promotion rate of 86.80%.
The research results show that the lactobacillus fermentum has obvious anti-inflammatory effect in vitro and can be applied to anti-inflammatory drugs and functional fermented foods.
Example 2 optimization of fermentation Medium composition
(1) Influence of the type of carbon Source in the fermentation Medium
The culture was carried out in a fermentation medium according to the method of example 1, containing the following composition: 20.00g/L of carbon source, 20.00g of tryptone, 9.00g of dipotassium phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate and 801.00 g of Tween, adjusting the pH value to 6.5, and carrying out autoclaving at 115 ℃ for 20 min. Wherein the carbon source is glucose, maltose, sucrose, lactose, respectively. The number of viable bacteria in the obtained fermentation liquid is shown in table 1, and the difference of the capital letters in table 1 indicates the significance of the survival rate difference between different strains (P < 0.05).
TABLE 1 Effect of different carbon sources on the growth of Lactobacillus fermentum SS-31
(2) Influence of the type of Nitrogen Source in the fermentation Medium
The culture was carried out in a fermentation medium according to the method of example 1, containing the following composition: 20.00g/L of maltose, 20.00g of nitrogen source, 9.00g of dipotassium phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate and 801.00 g of Tween, adjusting the pH value to 6.5, and carrying out autoclaving at 115 ℃ for 20 min. Wherein the nitrogen source is peptone, yeast extract powder, beef peptone and tryptone respectively. The number of viable bacteria in the obtained fermentation liquid is shown in table 2, and the difference of the lower case letters in table 2 indicates the significance of the survival rate difference between different strains (P < 0.05).
TABLE 2 Effect of different nitrogen sources on the growth of Lactobacillus fermentum SS-31
(3) Influence of fermentation Medium inorganic salt species
The culture was carried out in a fermentation medium according to the method of example 1, containing the following composition: 20.00g/L of maltose, 20.00g of yeast extract powder, 9.00g of inorganic salt, 0.50g of manganese sulfate, 1.00g of magnesium sulfate and 801.00 g of Tween, adjusting the pH value to 6.5, and carrying out autoclaving at 115 ℃ for 20 min. Wherein the inorganic salt is dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium citrate, and sodium acetate. The number of viable bacteria in the obtained fermentation broth is shown in Table 3, and the difference of the capital letters in the Table 3 indicates the significance of the survival rate difference between different strains (P < 0.05).
TABLE 3 Effect of different inorganic salts on the growth of Lactobacillus fermentum SS-31
(4) Influence of addition amounts of maltose, yeast extract powder and dipotassium hydrogen phosphate in fermentation medium
The cultures were carried out in the fermentation medium of the respective formulations of Table 4, according to the method of example 1. The number of viable bacteria in the obtained fermentation broth is shown in Table 5.
TABLE 4 culture media with different carbon source, nitrogen source and inorganic salt content ratios
| Species of | Maltose | Yeast extract powder | Dipotassium hydrogen phosphate | Manganese sulfate |
| Tween | 80 |
|
|
5 | 20 | 9 | 0.5 | 1 | 1 | |
| Formulation 2 | 10 | 20 | 9 | 0.5 | 1 | 1 | |
| Formulation 3 | 15 | 20 | 9 | 0.5 | 1 | 1 | |
| Formulation 4 | 20 | 20 | 9 | 0.5 | 1 | 1 | |
|
|
25 | 20 | 9 | 0.5 | 1 | 1 | |
|
|
15 | 5 | 9 | 0.5 | 1 | 1 | |
| Formulation 7 | 15 | 10 | 9 | 0.5 | 1 | 1 | |
| Formulation 8 | 15 | 15 | 9 | 0.5 | 1 | 1 | |
| Formulation 9 | 15 | 20 | 9 | 0.5 | 1 | 1 | |
|
|
15 | 25 | 9 | 0.5 | 1 | 1 | |
| Formulation 11 | 15 | 20 | 3 | 0.5 | 1 | 1 | |
| Formulation 12 | 15 | 20 | 5 | 0.5 | 1 | 1 | |
| Formulation 13 | 15 | 20 | 7 | 0.5 | 1 | 1 | |
| Formulation 14 | 15 | 20 | 9 | 0.5 | 1 | 1 | |
|
|
15 | 20 | 11 | 0.5 | 1 | 1 |
TABLE 5 Effect of different formulations on the growth of Lactobacillus fermentum SS-31
As can be seen from Table 5, the carbon source is the main component constituting the medium, and the concentration of the carbon source plays a key role in the growth of lactic acid bacteria. A carbon source suitable for fermentation of the lactobacillus fermentum is selected to be maltose with the content of 15g/L through research. When the sugar concentration is too high, the strain is proliferated and grown in a large amount at the early stage, and a large amount of acid is generated by fermentation, so that the culture medium environment is acidic, and the growth and metabolism of the strain are inhibited. The nitrogen source provides essential elements for the growth and metabolism of the thalli and is rich in amino acid, inorganic salt and vitamins. The nitrogen source suitable for the lactobacillus fermentum is determined by research to be yeast extract powder with the content of 20 g/L. When the concentration of the nitrogen source at the early stage is too low, the nutrient substances in the culture medium are insufficient, and the thalli grow slowly; when the concentration of the nitrogen source is too high, the thalli grow too fast, so that the thalli are aged and autolyzed. Inorganic salts are also important factors in the growth and metabolism of the bacterial cells, and can constitute cellular materials and regulate osmotic pressure. The inorganic salt suitable for L.fermentum was found by investigation to be dipotassium hydrogen phosphate, at a level of 9 g/L. The lactic acid bacteria decompose saccharides during the growth and metabolism process to produce a large amount of lactic acid, and the lactic acid is accumulated in a large amount to reduce the pH value in the culture medium along with the culture, so that the growth of the lactic acid bacteria is inhibited. The presence of inorganic salts can adjust the pH of the culture medium and neutralize peracid, thereby promoting the growth of the bacterial cells. And inorganic ions in the inorganic salt are absorbed by lactobacillus with cations and anions to carry out a series of biosynthetic metabolism, enzyme activity activation and the like, so that trace elements are supplemented for the growth of the lactobacillus.
Example 3 Effect of culture conditions for Lactobacillus fermentum SS-31
The culture was carried out in a fermentation medium according to the method of example 1, containing the following composition: 15.00g/L of maltose, 20.00g of yeast extract powder, 9.00g of dipotassium phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate and 801.00 g of Tween, adjusting the pH value to 6.5, and carrying out autoclaving at 115 ℃ for 20 min. The lactobacillus fermentum SS-31 after the third generation activation is added into a proliferation culture medium by 3 percent (v/v) inoculation amount, the initial pH is adjusted to 6.8, the pH of the culture medium is maintained to be 6.8 +/-0.02 by feeding neutralizing agent ammonia water, the culture medium is placed in a constant temperature box at 37 ℃ for 24 hours, and the viable count is measured. The number of viable bacteria in the obtained fermentation broth is shown in Table 6, and the difference of the lower case letters in the Table 6 indicates the significance of the survival rate difference between different strains (P < 0.05).
TABLE 6 viable count of Lactobacillus fermentum SS-31 after 24h incubation with ammonia
| Name of neutralizer | Without addition of a neutralizing agent | Aqueous ammonia |
| Viable count (. times.10) 10 CFU/mL) | 0.80 b | 1.19 a |
The results of the above examples canKnowing: the carbon source for fermenting the lactobacillus fermentum SS-31 is maltose, the nitrogen source is yeast extract powder, the inorganic salt is dipotassium hydrogen phosphate, high-density culture of the lactobacillus fermentum SS-31 is facilitated, the lactobacillus fermentum SS-31 is cultured by the culture medium, and the viable count is higher than that of other culture medium types. The high-density fermentation medium provided by the invention is used under the fermentation conditions that: the fermentation temperature is 37 ℃, the inoculum size is 3 percent, the initial pH value is pH 6.8, the pH value of the fermentation liquor is kept to be 6.8 +/-0.02 by adding ammonia water in the period, and the viable bacteria concentration in the fermentation liquor of the lactobacillus fermentum SS-31 can reach 1.19 multiplied by 10 after the fermentation liquor is cultured for 24 hours at 37 DEG C 10 CFU/mL, approximately 23 times higher than MRS medium, is more suitable for culturing Lactobacillus fermentum SS-31 than other media types.
Example 4 use of Lactobacillus fermentum SS-31
(1) Method for preparing fermented milk containing the lactobacillus
Heating fresh milk at 100 deg.C for 15 min or 140 deg.C for 3-5s, cooling to 35-37 deg.C, inoculating 3-5% of lactobacillus to make its starter concentration reach 10% 8 Fermenting at 35-37 deg.C above CFU/mL to pH 4.2-4.5 to obtain lactobacillus milk beverage containing the lactobacillus.
(2) Preparing lactobacillus fermentation powder:
collecting lactobacillus fermentation liquor, centrifuging at 4000r/min for 20 minutes at 4 ℃, discarding supernatant, collecting thallus precipitate, eluting the precipitate with a freeze-drying protective agent, wherein the protective agent comprises: precipitate-8: the formula of the protective agent is as follows: 10% of skim milk, 3% of trehalose, 1% of sodium L-glutamate and 1% of tween 80, collecting a mixture of a protective agent and bacterial sludge, concentrating in vacuum, and spray drying to obtain the lactobacillus ferments SS-31 leavening agent powder.
(3) The lactobacillus-containing biological feed starter
10mL of fermentation broth of Lactobacillus fermentum SS-31 was added with 1g of biological feed. The fermentation culture solution comprises the following components: 15.00g/L of maltose, 20.00g/L of cane sugar, 20.00g/L of yeast extract powder, 9.00g/L of dipotassium phosphate, 0.50g/L of manganese sulfate, 1.00g/L of magnesium sulfate, 8015.00 g/L of Tween and 20.00g/L of cellulase, culturing for 18-24h, vacuum concentrating and spray drying to obtain the biological feed leaven containing lactobacillus fermentum SS-31.
Sequence listing
<110> Guangxi university
<120> Lactobacillus fermentum and culture and application thereof
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<170> SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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cttgcacctg attgattttg gtcgccaacg agtggcggac gggtgagtaa cacgtacgta 120
acctgcccag aagcggggga caacatttgg aaacagatgc taataccgca taacaacgtt 180
gttcgcatga acaacgctta aaagatggct tctcgctatc acttctggat ggacctgcgg 240
tgcattagct tgttggtggg gtaacggcct accaaggcga tgatgcatag ccgagttgag 300
agactgatcg gccacaatgg gactgagaca cggcccatac tcctacggga ggcagcagta 360
gggaatcttc cacaatgggc gcaagcctga tggagcaaca ccgcgtgagt gaagaagggt 420
ttcggctcgt aaagctctgt tgttaaagaa gaacacgtat gagagtaact gttcatacgt 480
tgacggtatt taaccagaaa gtcacggcta actacgtgcc agcagccgcg gtaatacgta 540
ggtggcaagc gttatccgga tttattgggc gtaaagagag tgcaggcggt tttctaagtc 600
tgatgtgaaa gccttcggct taaccggaga agtgcatcgg aaactggata acttgagtgc 660
agaagagggt agtggaactc catgtgtagc ggtggaatgc gtagatatat ggaagaacac 720
cagtggcgaa ggcggctacc tggtctgcaa ctgacgctga gactcgaaag catgggtagc 780
gaacaggatt agataccctg gtagtccatg ccgtaacgat gagtgctagg tgttggaggg 840
tttccgccct tcagtgccgg agctaacgca ttaagcactc cgcctggggg agtacgaccg 900
caaggttgaa actcaaggaa ttgacggggg ccccgcacaa gcggtggagc atgtggttta 960
attcgaagct acgcgaagaa ccttaccagg tcttgacatc ttgcgccaat cctagagata 1020
gggcgttcct tcggaacgca atgacagggt ggtgccatgg tcc 1063
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agagtttgat ttgatcctgg ctag 24
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggttaccttg ttacgactt 19
Claims (10)
1. A Lactobacillus fermentum is characterized by being Lactobacillus fermentum SS-31 with the preservation number of CGMCC NO:24925。
2. lactobacillus fermentum according to claim 1, characterized in that the 16S rDNA gene has the base sequence of seq id No.1 of the sequence listing.
3. A high-density fermentation medium of Lactobacillus fermentum according to claim 1, wherein the carbon source is one or more of glucose, maltose, sucrose, and lactose, the nitrogen source is one or more of peptone, yeast extract, beef peptone, and tryptone, and the inorganic salt is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium citrate, and sodium acetate.
4. A high-density fermentation medium of Lactobacillus fermentum according to claim 3, characterized in that the carbon source in the medium is maltose, the nitrogen source is yeast extract powder, and the inorganic salt is dipotassium hydrogen phosphate.
5. A high-density fermentation medium of Lactobacillus fermentum according to claim 4, characterized in that it is MRS liquid medium, comprising the following components in each 1L of MRS liquid medium: 15.00g of maltose, 20.00g of yeast extract powder, 9.00g of dipotassium phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate, 801.00 g of Tween and 1000mL of water, adjusting the pH value to 6.5, and carrying out autoclaving at 115 ℃ for 20 min.
6. The method for high-density fermentation culture of lactobacillus fermentum according to claim 1, wherein lactobacillus fermentum seed solution is inoculated into the high-density fermentation medium according to claim 5, the inoculum size is 1% -5%, the initial fermentation pH is adjusted to 5.8-7.8, after fermentation, the pH of the fermentation broth is controlled to 6.8 ± 0.02 by using 15% -25% ammonia water solution, and the fermentation broth is cultured for 12-24h at 32-42 ℃.
7. Use of the lactobacillus fermentum of claim 1 in a medicament, nutraceutical, food or feed.
8. Use according to claim 7, characterized in that the drug is an anti-inflammatory drug.
9. A lactic acid bacterium drink characterized by containing the Lactobacillus fermentum of claim 1 and having a viable count of up to 10 8 CFU/mL or more.
10. A biological feed starter culture comprising a fermentation broth of the Lactobacillus fermentum of claim 1, wherein the carbon source in the fermentation broth is a complex of maltose and sucrose, and the fermentation broth comprises cellulase.
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| CN117802010A (en) * | 2024-02-28 | 2024-04-02 | 广州集妍化妆品科技有限公司 | Lactobacillus fermentum fermentation filtrate, preparation method and application thereof |
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