CN114854647B - Lactobacillus fermentum and culture and application thereof - Google Patents
Lactobacillus fermentum and culture and application thereof Download PDFInfo
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- CN114854647B CN114854647B CN202210683693.3A CN202210683693A CN114854647B CN 114854647 B CN114854647 B CN 114854647B CN 202210683693 A CN202210683693 A CN 202210683693A CN 114854647 B CN114854647 B CN 114854647B
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- lactobacillus fermentum
- fermentation
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- lactobacillus
- lactic acid
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Classifications
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Abstract
Lactobacillus fermentum (Lactobacillus fermentum) SS-31 is disclosed. The research result shows that the lactobacillus fermentum SS-31 of the invention can reduce the secretion of NO in cells and inflammatory factors IL-1 beta, IL-6 and IL-10. Thus, lactobacillus fermentum SS-31 has strong anti-inflammatory activity. In addition, the inventor also develops a high-density fermentation medium special for fermenting lactobacillus SS-31 and a culture method thereof, and the medium has simple components, simple preparation, quick operation and good application prospect without adding beef powder and peptone; 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 high-density fermentation culture of lactobacillus fermentum SS-31, so that the lactobacillus fermentum is applied to further development of medicines, health products, foods or feeds and the like in 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 mostly difficult to cure completely due to slow onset, unclear etiology, and easy recurrent attacks. The means for treating inflammatory bowel disease mainly include drug treatment and surgical treatment. Among them, although the drug treatment can effectively relieve symptoms, the long-term drug treatment can lead to resistance of pathogenic bacteria to antibiotics, and the operation treatment is expensive. Therefore, the search for new safe and reliable treatments is a major issue. With the intensive development of research, probiotic formulations are considered as a novel adjuvant therapy. The probiotics can play a role in regulating intestinal flora, have remarkable prevention and treatment effects on inflammatory bowel diseases, do not generate drug resistance, adverse reaction and the like, and are expected to become one of important means for preventing or assisting in treating inflammatory bowel diseases.
Lactic acid bacteria can regulate intestinal flora balance, induce nonspecific activation of host immune system, and has antioxidant, blood sugar reducing, enteritis resisting, and immunity regulating effects. In recent years, many studies have been reported to develop lactic acid strains which have a probiotic function and are safe for humans, and there have been applications of these strains to drugs or functional foods. For example:
application of Chinese patent lactobacillus plantarum CQPC02 in preparing food or medicine for preventing liver oxidative damage (patent number 201811639687.8 publication date 2019, 04 month 16)
Chinese patent "lactic acid bacteria, natural immune activator derived from the same, agent for preventing and treating infectious diseases and food and beverage" (patent No. 201980004577.0 publication day 2020, month and 23)
Chinese patent "Lactobacillus plantarum capable of relieving hyperuricemia and application thereof" (patent number 202011515393.1 publication day 2021, 05, 14 days)
Chinese patent 'novel lactobacillus plantarum, lactobacillus composition and use thereof for treating or preventing heavy metal related diseases' (patent number 201911052816.8 publication day 2020, month 26)
However, there is no report on the prevention or treatment of inflammatory bowel disease by lactic acid bacteria, and the amount of lactic acid bacteria in the body reaches a certain level to produce a certain probiotic effect. However, the current low fermentation level of lactic acid bacteria limits the expansion application of the lactic acid bacteria, so that the high-density culture technology of the strain is utilized to realize the purpose of adding probiotics into functional foods and feeds taking the lactic acid bacteria as main components to reach the corresponding quantity. The core for realizing the high-density culture of the strain is the optimization of a fermentation medium and fermentation conditions. The growth and propagation of lactobacillus require nutrient substances such as carbon source, nitrogen source, inorganic salt, nutrient factors and the like, and the growth process needs to continuously exchange the energy of the substances with the outside and is influenced by the outside environment (temperature, pH and the like). The high-density culture technique is a method of changing culture conditions by a certain culture technique and equipment or adding other reagents so that the density of the liquid cultured thalli exceeds that of the common culture. Compared with common culture, the high-density culture can not only improve the thallus density more rapidly and shorten the production period, but also reduce the production cost of equipment. Thus, high-density fermentation enrichment culture of lactic acid bacteria is currently a key technology in developing probiotic products with anti-inflammatory activity.
Disclosure of Invention
The invention aims to solve the technical problem of providing lactobacillus fermentum, and culture and application thereof, in particular to lactobacillus with anti-inflammatory activity, fermentation culture and application thereof in developing functional health-care food, feed and feed starter.
In order to solve the technical problems, the invention adopts the following technical scheme:
the lactobacillus fermentum is lactobacillus fermentum (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.
The carbon source in the high-density fermentation culture medium of the lactobacillus fermentum 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 respectively.
The carbon source in the culture medium 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 MRS liquid medium, and each 1L of MRS liquid medium consists of the following components in percentage by weight: 15.00g of maltose, 20.00g of yeast extract powder, 9.00g of dipotassium hydrogen phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate, 1.00g of Tween 80, 1000mL of water, adjusting the pH value to 6.5 and sterilizing at 115 ℃ for 20min under high pressure.
The high-density fermentation culture method of the lactobacillus fermentum comprises the steps of inoculating lactobacillus fermentum seed solution into the high-density fermentation culture medium of claim 5, adjusting the initial fermentation pH value to 5.8-7.8, controlling the pH value of the fermentation liquor to 6.8+/-0.02 by adopting 15-25% ammonia water solution after fermentation, and culturing for 12-24 hours at 32-42 ℃.
The lactobacillus fermentum is applied to medicines, health products, foods or feeds.
The medicine is anti-inflammatory medicine.
Lactic acid bacteria beverage containing the above Lactobacillus fermentum with viable count of 10 8 CFU/mL or more.
A biological feed starter comprises the fermentation liquid of the lactobacillus fermentum, wherein the carbon source in the fermentation culture liquid is a maltose and sucrose complex and contains cellulase.
In the previous study, the inventors isolated lactobacillus fermentum (Lactobacillus fermentum) SS-31 of the present invention from sour bamboo shoots in Guangxi willow. The anti-inflammatory activity is evaluated by establishing a cell inflammation model by inducing RAW264.7 macrophages by lipopolysaccharide, measuring cell viability by a CCK-8 method, and measuring the release amounts of NO, IL-1 beta, IL-6 and IL-10 by an ELISA kit. The results show that the lactobacillus fermentum SS-31 of the invention can reduce the secretion of NO in cells and inflammatory factors IL-1 beta, IL-6 and IL-10. Thus, lactobacillus fermentum SS-31 has strong anti-inflammatory activity. In addition, the inventor also develops a high-density fermentation medium special for fermenting lactobacillus SS-31 and a culture method thereof, and the medium has simple components, simple preparation, quick operation and good application prospect without adding beef powder and peptone; 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 enriches the functional research of the lactic acid bacteria of the sour bamboo shoot source in willow, plays the effect of probiotics and is beneficial to the popularization of the strain resources of the Guangxi special fermented food; meanwhile, the invention realizes high-density fermentation culture of the lactobacillus fermentum SS-31, so that the lactobacillus fermentum is applied to further development of medicines, health-care products, foods or feeds and the like in future.
Drawings
FIG. 1 is an agarose gel electrophoresis chart of a 16S rDNA PCR amplification product of Lactobacillus fermentum SS-31, in which: 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 RAW.264.7 cell viability.
FIG. 3 is a graph showing the results of the stimulation of RAW264.7 cells by LPS by Lactobacillus fermentum SS-31.
FIG. 4 is a graph showing the results of the production of IL-1β by Lactobacillus fermentum SS-31 on LPS-stimulated RAW264.7 cells.
FIG. 5 is a graph showing the results of the production of IL-6 by Lactobacillus fermentum SS-31 on LPS-stimulated RAW264.7 cells.
FIG. 6 is a graph showing the results of the production of IL-10 by Lactobacillus fermentum SS-31 on LPS-stimulated RAW264.7 cells.
P < 0.05 in FIGS. 2 to 6 represents the difference compared to the normal group
Description of preservation information
Lactobacillus fermentum (Lactobacillus fermentum) SS-31 with the preservation number of CGMCC NO:24925preservation date: 2022 year_05_month_19_day, the deposit address is: beijing, chaoyang area, north Chen Xi Lu 1, 3, china academy of sciences microbiological institute, postal code 100101, preservation unit: china general microbiological culture Collection center.
Preservation conditions: and (3) fully mixing the enriched and cultured bacterial liquid with the killed glycerol to control the final concentration to be between 20 and 30 percent, subpackaging the bacterial liquid into glycerol pipes, and finally placing the glycerol pipes in a refrigerator or a liquid nitrogen tank at the temperature of minus 80 ℃ for preservation.
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) Glycerol tube activation: the Lactobacillus fermentum SS-31 deposited in the glycerol tubes was streaked on MRS solid medium plates and incubated at 37℃for 48h. Every 1L of MRS solid culture medium consists of the following components in percentage by weight: MRS liquid culture medium 1000mL, agar 15g. Every 1L of MRS liquid culture medium consists of the following components in percentage by weight: 20.00g of carbon source, 20.00g of tryptone, 9.00g of dipotassium hydrogen phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate, 1.00g of Tween 80, 1000mL of water, adjusting the pH value to 6.5 and sterilizing at 115 ℃ for 20min under high pressure.
(2) Liquid activation: single colony is selected and inoculated in a test tube containing 10mL of MRS liquid culture medium for static culture, the culture is carried out for 24 hours at 37 ℃ for primary activation, bacterial suspension is inoculated in 100mL of MRS liquid culture medium with an inoculum size of 2% (v/v), and the static culture is carried out for 24 hours at 37 ℃ for secondary activation, so as to obtain seed liquid.
(3) And (3) identification: the seed solution was inoculated into a proliferation medium seed at an inoculum size of 2% (v/v), the initial pH was adjusted to 6.8, the culture was carried out in an incubator at 37℃for 24 hours, streaked to MRS solid medium for 48 hours, and single colony was picked up for PCR. The 16S rRNA gene amplification is carried out by adopting a reverse primer 1492R (5'-AGAGTTTGATTTGATCCTGGCTAG-3', SEQ.ID.NO.2) and a forward primer 27F (5'-GGTTACCTTGTTACGACTT-3', SEQ.ID.NO.3), a 25 mu L reaction system is used for PCR amplification, 94 ℃ pre-denaturation is adopted for 5min, 94 ℃ denaturation is adopted for 1min, 64 ℃ annealing is adopted for 1min, 72 ℃ extension is carried out for 2min,35 cycles are carried out, and the PCR amplification is preserved at 4 ℃. After the amplification, 4. Mu.L of the PCR product was subjected to 1% agarose gel electrophoresis, and the result was shown in FIG. 1. Sequencing, carrying out homologous comparison analysis on the sequence obtained by sequencing and a GenBank database of NCBI, and determining that the strain SS-31 is lactobacillus fermentum and the 16s rDNA sequence is shown as SEQ ID No.1 and the anti-inflammatory activity of the lactobacillus is 2
(1) Culturing cells: RAW264.7 macrophages were cultured in DMEM containing 10% fetal bovine serum. Cells in log phase were grown at 2.5X10 5 Each mL was inoculated into 24-well plates at 500. Mu.L per well and 5% CO at 37 ℃ 2 Overnight culture in humidified incubator for subsequent useAnd (5) experiment.
(2) Measurement of cell viability: the concentration of the suspension of RAW264.7 cells after pancreatin digestion was adjusted to 1.0X10 by the cytometry method 4 Uniformly inoculating into 96-well plate at 37deg.C with 5% CO 2 Culturing in an incubator until the mixture is fused. After 24h of 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) was cultured for 3 hours. After adding 10. Mu.L of CCK-8 solution to each well and incubating for 1h at 37℃absorbance was measured at 450nm and the effect of different concentrations of lactic acid bacteria on RAW264.7 cells was assessed. The cell viability was calculated as follows:
wherein: as experimental group contains cell culture medium, CCK-8 and lactobacillus; the Ac model group contains cell culture medium, CCK-8 and lactic acid bacteria; ab control group was free of cell and lactic acid bacteria medium, CCK-8.
(4) Determination of NO: the concentration of the suspension of RAW264.7 cells after pancreatin digestion was adjusted to 2.5X10 by the cytometry method 5 cell/mL, uniformly inoculating in 24-well plate, 37 ℃ and 5% CO 2 Culturing in an incubator until the mixture is fused. 1. Mu.g/mL LPS was added to stimulate for 24h, followed by 50. Mu.L of activated concentration 1X 10 8 The CFU/mL bacterial solution was cultured for 3 hours, and the supernatant was collected. According to the operation of the instruction book of the NO detection kit, the mass concentration formula of the calculated NO is as follows:
note that: wherein: 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 enhanced as the concentration of lactic acid bacteria was increased. When the cell concentration of the cell was 10 as compared with the normal group 9 At CFU/mL, SS-31 pairs were fineCell viability was 111.61%, with significant differences (P < 0.05); when the concentration of the bacterial cells is 10 8 At CFU/mL, the cell viability of SS-31 was 97.05% but not significant (P > 0.05); cell concentration 10 compared to the normal group 5 、10 6 、10 7 Cell viability was significantly reduced (P < 0.05) at CFU/mL and cell viability was inhibited. To sum up, the concentration of the selected bacterial cells is 10 8 CFU/mL was used as the optimal concentration for subsequent experiments.
As can be seen from FIG. 3, the level of NO secretion 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 < 0.05), and lactobacillus SS-31 can remarkably inhibit NO secretion (P < 0.05), and the inhibition rate is 84.31%.
(5) Measurement of inflammatory factors IL-1 beta, IL-6 and IL-10: the suspension of the RAW264.7 cells after pancreatin digestion was adjusted to 2.5X10 5 cell/mL, uniformly inoculating in 24-well plate, 37 ℃ and 5% CO 2 Culturing in an incubator until the mixture is fused. LPS at a concentration of 1. Mu.g/mL was added for 24h and then 50. Mu.L of activated 1X 10 was added 8 The CFU/mL bacterial solution was cultured for 3 hours, and the supernatant was collected. The content of inflammatory factors IL-1 beta, IL-6 and IL-10 in the cell supernatant was determined according to the procedure of ELISA kit instructions.
From FIG. 4, it can be seen that LPS induces RAW264.7 cells to secrete a large amount of inflammatory factors IL-1β (P < 0.05), and lactic acid bacteria SS-31 can significantly inhibit IL-1β 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 factors IL-6 (P < 0.05), and lactic acid bacteria SS-31 can remarkably inhibit IL-6 secretion (P < 0.05), and the inhibition rate is 14.12%.
As can be seen from FIG. 6, LPS induced RAW264.7 cells reduced the secretion of the pro-inflammatory factor IL-10 (P < 0.05), and lactic acid bacteria SS-31 was able to significantly promote IL-10 secretion (P < 0.05), with a rate of 86.80%.
The research results show that the lactobacillus fermentum has obvious effect on in vitro anti-inflammatory and can be applied to anti-inflammatory drugs and functional fermented foods.
Example 2 optimization of fermentation Medium Components
(1) Influence of the carbon source species of the fermentation Medium
According to the method in example 1, the culture is carried out in a fermentation medium having the following composition: 20.00g/L of carbon source, 20.00g of tryptone, 9.00g of dipotassium hydrogen phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate and 1.00g of Tween 80, adjusting the pH value to 6.5, and sterilizing at 115 ℃ for 20min. Wherein the carbon sources are glucose, maltose, sucrose and lactose respectively. The number of viable bacteria in the obtained fermentation broth is shown in Table 1, and the differences in the lower-case letters in Table 1 indicate the significance of the difference in survival rate between different strains (P < 0.05).
TABLE 1 influence on the growth of Lactobacillus fermentum SS-31 under different carbon sources
(2) Influence of the nitrogen source species of the fermentation Medium
According to the method in example 1, the culture is carried out in a fermentation medium having the following composition: maltose 20.00g/L, nitrogen source 20.00g, dipotassium hydrogen phosphate 9.00g, manganese sulfate 0.50g, magnesium sulfate 1.00g, tween 80 1.00g, regulating pH value to 6.5, and sterilizing at 115 ℃ for 20min. Wherein the nitrogen source is peptone, yeast extract powder, beef peptone, and tryptone respectively. The number of viable bacteria in the obtained fermentation broth is shown in Table 2, and the differences in the lower-case letters in Table 2 indicate the significance of the difference in survival rate between different strains (P < 0.05).
TABLE 2 Effect of different Nitrogen sources on Lactobacillus fermentum SS-31 growth
(3) Influence of inorganic salt species of fermentation Medium
According to the method in example 1, the culture is carried out in a fermentation medium having the following composition: maltose 20.00g/L, yeast extract 20.00g, inorganic salt 9.00g, manganese sulfate 0.50g, magnesium sulfate 1.00g, tween 80 1.00g, regulating pH to 6.5, and sterilizing at 115 ℃ for 20min under high pressure. Wherein the inorganic salts are dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium citrate and sodium acetate respectively. The number of viable bacteria in the obtained fermentation broth is shown in Table 3, and the differences in the lower-case letters in Table 3 indicate the significance of the difference in survival rate between different strains (P < 0.05).
TABLE 3 influence of different inorganic salts on the growth of Lactobacillus fermentum SS-31
(4) Influence of the addition of maltose, yeast extract and dipotassium phosphate in the fermentation Medium
The culture was carried out in the fermentation medium of each formulation having the composition shown in Table 4 according to the method in 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 type | Maltose | Yeast extract powder | Dipotassium hydrogen phosphate | Manganese sulfate | Magnesium sulfate | Tween 80 |
| Formulation 1 | 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 |
| Formulation 5 | 25 | 20 | 9 | 0.5 | 1 | 1 |
| Formulation 6 | 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 |
| Formulation 10 | 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 |
| Formulation 15 | 15 | 20 | 11 | 0.5 | 1 | 1 |
TABLE 5 Effect of different formulations on Lactobacillus fermentum SS-31 growth
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. The carbon source fermented by the proper lactobacillus fermentum is selected to be maltose, and the content is 15g/L. When the sugar concentration is too high, the strain grows in a large amount in the early stage, and a large amount of acid is produced by fermentation, so that the environment of the culture medium is slightly acidic, and the growth metabolism of the strain is 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 fermenting lactobacillus is yeast extract powder with the content of 20g/L. When the concentration of the nitrogen source in the early stage is too low, the nutrient substances in the culture medium are insufficient, so that the growth of thalli is slow; when the nitrogen source concentration is too high, the thallus grows too fast to cause the thallus to age and autolyze. Inorganic salts are also important factors in the growth and metabolism of cells, and can constitute cellular materials and regulate osmotic pressure. The inorganic salt suitable for lactobacillus fermentum is dipotassium hydrogen phosphate with the content of 9g/L. Lactic acid bacteria decompose saccharides to produce a large amount of lactic acid in the growth metabolism process, and the pH in the culture medium is reduced due to the accumulation of a large amount of lactic acid during the time culture, so that the growth of the lactic acid bacteria is inhibited. The pH of the culture medium can be adjusted by the existence of inorganic salt, and peracid is neutralized, so that the growth of thalli is promoted. And inorganic ions in the inorganic salt are absorbed by lactobacillus of cations and anions to carry out a series of biosynthesis metabolism, enzyme activity activation and the like, so that microelements are supplemented for the growth of the inorganic salt.
EXAMPLE 3 Effect of culture conditions of Lactobacillus fermentum SS-31
According to the method in example 1, the culture is carried out in a fermentation medium having the following composition: 15.00g/L maltose, 20.00g yeast extract powder, 9.00g dipotassium hydrogen phosphate, 0.50g manganese sulfate, 1.00g magnesium sulfate and 1.00g Tween 80, adjusting the pH value to 6.5, and sterilizing at 115 ℃ for 20min. Inoculating activated lactobacillus fermentum SS-31 in 3% (v/v) amount into proliferation culture medium, regulating initial pH to 6.8, maintaining pH of culture medium with ammonia water as neutralizing agent to 6.8+ -0.02, culturing in a 37 deg.C incubator for 24 hr, and measuring viable count. The number of viable bacteria in the obtained fermentation broth is shown in Table 6, and the differences in the lower-case letters in Table 6 indicate the significance of the difference in survival rate between the different strains (P < 0.05).
TABLE 6 viable count of Lactobacillus fermentum SS-31 after 24h of culture with fed-batch ammonia
| Neutralizer designation | No-flow-addition neutralizer | Ammonia water |
| Viable count (. Times.10) 10 CFU/mL) | 0.80 b | 1.19 a |
From the results of the above examples, it can be seen that: the carbon source of fermentation of the lactobacillus fermentum SS-31 is maltose, the nitrogen source is yeast extract powder, the inorganic salt is dipotassium hydrogen phosphate, which is favorable for high-density culture of the lactobacillus fermentum SS-31, and the number of viable bacteria is higher than that of other culture medium types when the lactobacillus fermentum SS-31 is cultured by the culture medium. The high-density fermentation medium of the invention is used under the fermentation conditions that: the fermentation temperature is 37 ℃, the inoculation amount is 3%, the initial pH value is 6.8, the pH value of the fermentation liquor is kept to be 6.8+/-0.02 by ammonia water added during the period, and after 24 hours of culture at 37 ℃, the viable bacteria concentration in the fermentation liquor of the lactobacillus fermentum SS-31 can reach 1.19x10 10 CFU/mL was approximately 23-fold higher than MRS medium, which was more suitable for culturing Lactobacillus fermentum SS-31 than other medium types.
EXAMPLE 4 use of Lactobacillus fermentum SS-31
(1) Method for preparing fermented milk containing the lactobacillus
Fresh cow milk is processed at 100deg.CHeating for 15 min or 140 deg.C for 3-5s, cooling to 35-37 deg.C, inoculating lactobacillus with an inoculum size of 3% -5% to make the concentration of fermenting agent reach 10 8 Fermenting at 35-37deg.C to pH of 4.2-4.5 with CFU/mL or above to obtain lactobacillus milk beverage containing the lactobacillus.
(2) Preparation of lactobacillus fermentation powder:
collecting lactobacillus fermentation liquor, centrifuging at 4000r/min for 20min at 4deg.C, discarding supernatant, collecting thallus precipitate, eluting precipitate with freeze-drying protecting agent, and protecting agent: precipitation = 8:1, the formula of the protective agent is as follows: 10% of skim milk, 3% of trehalose, 1% of L-sodium glutamate and 1% of Tween 80, collecting a mixture of a protective agent and bacterial sludge, concentrating in vacuum, and spray drying to obtain lactobacillus fermentum SS-31 starter powder.
(3) Biological feed starter containing the lactobacillus
10mL of fermented lactobacillus SS-31 fermentation broth is taken and added with 1g of biological feed. The fermentation culture solution comprises the following components: 15.00g/L maltose, 20.00g/L sucrose, 20.00g/L yeast extract powder, 9.00g/L dipotassium hydrogen phosphate, 0.50g/L manganese sulfate, 1.00g/L magnesium sulfate, 15.00g/L tween 80 and 20.00g/L cellulase, culturing for 18-24 hours, concentrating in vacuum, and spray drying to obtain the biological feed starter containing lactobacillus fermentum SS-31.
Sequence listing
<110> university of Guangxi
<120> Lactobacillus fermentum and culture and use thereof
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<170> SIPOSequenceListing 1.0
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<211> 1063
<212> DNA
<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 (3)
1. A strain of lactobacillus fermentum is characterized in that the strain is lactobacillus fermentum (Lactobacillus fermentum) SS-31 with the preservation number of CGMCC NO: 24925.
2. The high-density fermentation culture method of the lactobacillus fermentum according to claim 1, which is characterized in that lactobacillus fermentum seed liquid is inoculated into a high-density fermentation culture medium, the inoculum size is 1% -5%, the initial fermentation pH value is adjusted to be 5.8-7.8, 15% -25% ammonia water solution is adopted to control the pH value of the fermentation liquid to be 6.8+/-0.02 after fermentation, and the fermentation liquid is cultured for 12-24 hours at the temperature of 32-42 ℃; the high-density fermentation medium is composed of the following content components in per 1L: 15.00g of maltose, 20.00g of yeast extract powder, 9.00g of dipotassium hydrogen phosphate, 0.50g of manganese sulfate, 1.00g of magnesium sulfate, 1.00g of Tween 80, 1000mL of water, adjusting the pH value to 6.5 and sterilizing at 115 ℃ for 20min under high pressure.
3. A lactic acid bacterium beverage comprising the fermented lactic acid bacterium according to claim 1, wherein the viable count of the fermented lactic acid bacterium is up to 10 8 CFU/mL or more.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967454A (en) * | 2009-07-27 | 2011-02-09 | 上海斯哒米纳生物科技有限公司 | High-intensity culture method for lactobacilli |
| CN109481476A (en) * | 2018-12-29 | 2019-03-19 | 重庆第二师范学院 | Application of the lactobacillus fermenti CQPC04 in the food or drug that preparation improves ulcerative colitis |
| CN112501046A (en) * | 2020-09-11 | 2021-03-16 | 杭州娃哈哈科技有限公司 | Lactobacillus fermentum with weight-losing function and application thereof |
| CN113234612A (en) * | 2021-02-05 | 2021-08-10 | 重庆第二师范学院 | Lactobacillus fermentum ZS40 having preventive effect on colitis |
| CN114191490A (en) * | 2022-01-28 | 2022-03-18 | 善恩康生物科技（苏州）有限公司 | Lactobacillus fermentum and application of lactobacillus fermentum product in oxidation resistance and tumor resistance |
| KR20220050257A (en) * | 2020-10-15 | 2022-04-25 | 충북대학교 산학협력단 | Novel starter of Lactobacillus fermentum EFEL6800 with probiotic activity |
| KR20220075622A (en) * | 2020-11-30 | 2022-06-08 | 나눔제약 주식회사 | Lactobacillus fermentum OKBL-L.FE 1 strain having anti-inflammatory activity and antimicrobial activity against pathogenic microorganism and uses thereof |
-
2022
- 2022-06-16 CN CN202210683693.3A patent/CN114854647B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967454A (en) * | 2009-07-27 | 2011-02-09 | 上海斯哒米纳生物科技有限公司 | High-intensity culture method for lactobacilli |
| CN109481476A (en) * | 2018-12-29 | 2019-03-19 | 重庆第二师范学院 | Application of the lactobacillus fermenti CQPC04 in the food or drug that preparation improves ulcerative colitis |
| CN112501046A (en) * | 2020-09-11 | 2021-03-16 | 杭州娃哈哈科技有限公司 | Lactobacillus fermentum with weight-losing function and application thereof |
| KR20220050257A (en) * | 2020-10-15 | 2022-04-25 | 충북대학교 산학협력단 | Novel starter of Lactobacillus fermentum EFEL6800 with probiotic activity |
| KR20220075622A (en) * | 2020-11-30 | 2022-06-08 | 나눔제약 주식회사 | Lactobacillus fermentum OKBL-L.FE 1 strain having anti-inflammatory activity and antimicrobial activity against pathogenic microorganism and uses thereof |
| CN113234612A (en) * | 2021-02-05 | 2021-08-10 | 重庆第二师范学院 | Lactobacillus fermentum ZS40 having preventive effect on colitis |
| CN114191490A (en) * | 2022-01-28 | 2022-03-18 | 善恩康生物科技（苏州）有限公司 | Lactobacillus fermentum and application of lactobacillus fermentum product in oxidation resistance and tumor resistance |
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