High-density culture method, fermentation process and application of plant lactobacillus CCFM8661

Abstract

The invention discloses a high-density culture method, a fermentation process and application of plant lactobacillus CCFM8661, and belongs to the technical field of microorganisms. According to the invention, the culture medium is optimally designed and mainly comprises cane sugar, peptone, beef extract, yeast powder, Tween 80, magnesium sulfate heptahydrate, manganese sulfate monohydrate and Na2HPO4-citric acid buffer salt, and the components are optimized. According to the method, the growth rate and the viable count of the plant lactobacillus CCFM8661 are increased, the plant lactobacillus CCFM8661 enters a stable phase after being cultured for 21 hours, and the viable count of thalli of the obtained plant lactobacillus CCFM8661 reaches 1.5 * 10 < 12 > CFU / g. Compared with the original MRS culture medium, the OD600nm value is increased by 2.8 times. The plant lactobacillus CCFM8661 is not only suitable for food processing, but also can be applied to the fields of health care products and medicines, so that the culture medium disclosed by the invention has a wide market application prospect.

Description

Technical Field

[0001] The invention relates to a high-density culture method, a fermentation process and application of lactobacillus plantarum CCFM8661, and belongs to the technical field of microorganisms. Background Art

[0002] As people's health awareness continues to improve, the application of probiotics is becoming more and more extensive, especially in the fields of functional foods and health products. Probiotics refer to living microorganisms that can have beneficial effects on the human body when taken in appropriate amounts. They mainly promote health by improving the balance of the host's intestinal microbial community, including but not limited to enhancing immunity, regulating the intestines, promoting nutrient absorption, and eliminating harmful factors. Probiotics mainly include Lactobacillus, Bifidobacterium, etc. Lactic acid bacteria are a type of bacteria that can produce lactic acid during metabolism. They are widely present in dairy products and the human intestine and play an important role in human health. Lactobacillus plantarum is a subspecies of lactic acid bacteria. It is widely distributed in the natural environment, including plants, soil, and fermented foods. Lactobacillus plantarum is widely used in the food industry, especially in the production of fermented foods, because of its good tolerance and probiotic properties.

[0003] High-density fermentation refers to a fermentation process that obtains the highest possible cell concentration in a unit volume of culture medium. This technology is very important in the industrial production of probiotics and lactic acid bacteria because it can significantly improve production and economic benefits, while also ensuring the consistency of product quality. Lactobacillus plantarum CCFM8661, also known as Lactobacillus plantarum CCFM8661, is a high-efficiency probiotic strain discovered in recent years. It is widely used in the food industry because of its good stability, acid resistance and bile resistance. Although Lactobacillus plantarum CCFM8661 has many advantages, it still faces some challenges in industrial production, mainly in terms of the difficult control of the culture conditions of the strain, the long fermentation cycle, the low cell density and the high production cost. For example, the fermentation cycle is long: traditional fermentation processes often require a long fermentation cycle, which not only increases production costs, but also may cause the cell to be contaminated during the fermentation process, affecting the safety of the product. For example, the cell density is not high: in the high-density fermentation process, the cell density is an important indicator for measuring fermentation efficiency. In the prior art, due to the limitations of culture conditions and fermentation processes, it is often difficult to achieve an ideal cell density, thereby limiting the increase in production. Such as production cost is higher: because culture condition is complicated, fermentation cycle is long and cell density is not high, cause the production cost of plant lactobacillus CCFM8661 to be higher, this limits its widespread use in fields such as functional foods, health products and medicines to a certain extent.For the above-mentioned defect existing in the prior art, the present invention aims to develop a kind of efficient culture method and fermentation process that are applicable to plant lactobacillus CCFM8661 growth.By optimizing culture condition, shortening fermentation cycle, improving cell density and reducing production cost, give full play to the potential of plant lactobacillus CCFM8661, promote its widespread use in fields such as functional foods, health products and medicines. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a high-density culture method, fermentation process and application of Lactobacillus plantarum CCFM8661. The purpose of the present invention is to increase the number of viable bacteria and shorten the fermentation cycle, thereby improving production efficiency.

[0005] The first technical solution provided by the present invention is a method for culturing plant lactobacillus, wherein the plant lactobacillus is cultured in a culture system with maltose or sucrose as a carbon source and yeast powder as a nitrogen source, and the culture system also contains Na2HPO4-citrate buffer salt, magnesium sulfate, manganese sulfate and Tween 80.

[0006] In certain embodiments, in the culture system, the concentration of sucrose is 10-35 g / L, and the concentration of yeast powder is 5-20 g / L.

[0007] In certain embodiments, in the culture system, the concentration of Na2HPO4-citrate buffer is 0.05-0.20 mol / L, the concentration of magnesium sulfate is 0-1.2 g / L, the concentration of manganese sulfate is 0.05-0.4 g / L, and the concentration of Tween 80 is 0.5-4 mL / L.

[0008] In certain embodiments, the culture system contains sucrose, peptone, beef extract, yeast powder, Tween, magnesium sulfate heptahydrate, manganese sulfate monohydrate, and Na2HPO4-citrate buffer.

[0009] In certain embodiments, the culture system contains 25 g / L sucrose, 10 g / L peptone, 5 g / L beef extract, 10 g / L yeast powder, 1 mL / L Tween 80, 0.2 g / L magnesium sulfate heptahydrate, 0.1 g / L manganese sulfate monohydrate, and 0.1 mol / L Na2HPO4-citrate buffer.

[0010] In certain embodiments, a 2% bacterial solution of Lactobacillus plantarum is added to the culture system and cultured at 37° C. for not less than 20 hours.

[0011] In certain embodiments, the plant lactobacillus includes but is not limited to Lactobacillus plantarum CCFM8661, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of CGMCC No.5494 and a deposit date of November 29, 2011, and is recorded in the patent document with a publication number of CN111869735A.

[0012] The second technical solution provided by the present invention is a culture medium, which consists of sucrose, peptone, beef extract, yeast powder, Tween, magnesium sulfate heptahydrate, manganese sulfate monohydrate, and Na2HPO4-citrate buffer.

[0013] In certain embodiments, the working concentrations of the components in the culture medium are sucrose 10-35 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 15-20 g / L, Tween 80 0.5-4 mL / L, magnesium sulfate heptahydrate 0-1.2 g / L, manganese sulfate monohydrate 0.05-0.4 g / L, and Na2HPO4-citrate buffer 0.05-0.20 mol / L.

[0014] In certain embodiments, the working concentrations of the components in the culture medium are sucrose 25 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 10 g / L, Tween 80 1 mL / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.1 g / L, and Na2HPO4-citrate buffer 0.1 mol / L.

[0015] The third technical solution provided by the present invention is the use of the culture medium described in the second technical solution in culturing plant lactobacillus in the fields of food and medicine.

[0016] In certain embodiments, the Lactobacillus plantarum includes but is not limited to Lactobacillus plantarum CCFM8661, which is described in the patent document with publication number CN111869735A.

[0017] In certain embodiments, the food includes, but is not limited to, health food, regular food, and food for special dietary uses.

[0018] The technical effects of the present invention are as follows:

[0019] By optimizing the fermentation conditions, the growth rate and viable count of Lactobacillus plantarum CCFM8661 were significantly improved. After 21 hours of cultivation, the culture entered the stable period and the viable count of Lactobacillus plantarum CCFM8661 reached 1.5*10 12 CFU / g. Compared with the original MRS medium, OD 600nm The value was increased by 2.8 times. Since Lactobacillus plantarum CCFM8661 is not only suitable for food processing, but also can be used in the fields of health care products and medicine, the culture medium of the present invention has a wide market application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of different carbon sources on the growth of Lactobacillus plantarum CCFM8661.

[0021] Figure 2 The effect of different sucrose addition amounts on the growth of Lactobacillus plantarum CCFM8661.

[0022] Figure 3 The effect of different yeast powder addition amounts on the growth of Lactobacillus plantarum CCFM8661.

[0023] Figure 4 Effects of different buffer salt systems and concentrations on the growth of Lactobacillus plantarum CCFM8661.

[0024] Figure 5 The effect of different magnesium sulfate addition amounts on the growth of Lactobacillus plantarum CCFM8661.

[0025] Figure 6 This is the effect of different manganese sulfate addition amounts on the growth of Lactobacillus plantarum CCFM8661.

[0026] Figure 7 The effect of different Tween 80 addition amounts on the growth of Lactobacillus plantarum CCFM8661.

[0027] Figure 8This is the growth curve of Lactobacillus plantarum CCFM8661 in the optimized culture medium.

[0028] Fig. 9 These are the growth curves of Lactobacillus plantarum CCFM8661 in the optimized medium and MRS medium, respectively. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0030] The Lactobacillus plantarum CCFM8661 involved in the following examples is Lactobacillus plantarum CCFM8661, with a deposit number of CGMCC No.5494, which has been disclosed in the patent application document with publication number CN111869735A.

[0031] The content of each component in the plant lactobacillus CCFM8661 MRS liquid culture medium described below is: glucose 20g / L, peptone 10g / L, beef extract 5g / L, yeast powder 4g / L, Tween 80 1mL / L, dipotassium hydrogen phosphate heptahydrate 2g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate monohydrate 0.05g / L, diammonium hydrogen citrate 2g / L, sodium acetate trihydrate 5g / L, pH value is 6.2. 115℃ high pressure steam sterilization for 20min. 15g agar powder is added to the solid culture medium.

[0032] Example 1: Optimization of high-density fermentation medium (determination of carbon source type)

[0033] The strains stored in -80℃ refrigerator glycerol were streaked on MRS solid culture medium, and single colonies were picked into MRS liquid culture medium, and cultured in a constant temperature shaker at 37℃ and 50r / min for 12h as seed liquid. The glucose in MRS liquid culture medium was replaced with 20g / L maltose, sucrose, α-lactose, and starch in turn. Inoculate into a 250mL conical flask containing 50mL of different carbon source culture medium at an inoculum volume of 2% (V / V), culture in a constant temperature incubator at 37℃ for 24h, and then measure the cell density of the bacterial liquid. Set up the following culture medium:

[0034] Culture medium 1: maltose 20 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0035] Culture medium 2: sucrose 20 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0036] Culture medium 3: α-lactose 20 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0037] Culture medium 4: starch 20 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0038] Effects of carbon source types on the growth of Lactobacillus plantarum CCFM8661 Figure 1 .

[0039] Depend on Figure 1 It can be seen that when 20g / L maltose is used as the carbon source, OD 600nm The highest value is 2.4, followed by sucrose and the lowest is starch, indicating that Lactobacillus plantarum CCFM8661 has the best utilization efficiency of maltose. Although Lactobacillus plantarum CCFM8661 has the best utilization efficiency of maltose, it is expensive. According to the principle of cost saving, sucrose will be selected as the optimal carbon source for the culture medium in the future.

[0040] After screening out the carbon source, the effect of the amount of carbon source added (10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L) on the bacterial density was investigated. The following culture medium was set:

[0041] Culture medium 4: sucrose 10 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0042] Culture medium 5: sucrose 15 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0043] Culture medium 6: sucrose 20 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0044] Culture medium 7: sucrose 25 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0045] Culture medium 8: sucrose 30 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0046] Culture medium 9: sucrose 35 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0047] Effects of carbon source addition on the growth of Lactobacillus plantarum CCFM8661 Figure 2 .

[0048] Depend on Figure 2 It can be seen that when the amount of sucrose added was 25 g / L, the OD 600nm The maximum value reached 2.0. If the amount of sucrose added continued to increase, OD 600nm The value decreased instead. This may be due to the high concentration of carbon source, which inhibited the growth and metabolism of Lactobacillus plantarum CCFM8661. Therefore, the optimal addition amount of sucrose was determined to be 25g / L.

[0049] Example 2: Optimization of high-density fermentation medium (determination of nitrogen source concentration)

[0050] Lactobacillus plantarum CCFM8661 This example investigates the effect of the amount of yeast powder added on Lactobacillus plantarum CCFM8661. Yeast powder was added at 0, 5 g / L, 10 g / L, 15 g / L, and 20 g / L, and inoculated into a 250 mL conical flask containing a yeast powder culture medium of different concentrations at an inoculum amount of 2% (V / V), and cultured in a 37°C constant temperature incubator for 24 h, and then the cell density of the bacterial solution was determined. The following culture medium was set:

[0051] Culture medium 10: sucrose 25 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 0 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0052] Culture medium 11: sucrose 25 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 5 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0053] Culture medium 12: sucrose 25 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 10 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0054] Culture medium 13: sucrose 25 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 15 g / L, Tween 80 1 g / L, dipotassium hydrogen phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0055] Culture medium 14: sucrose 25 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 20 g / L, Tween 80 1 g / L, dipotassium hydrogen phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L.

[0056] Effects of nitrogen source addition on the growth of Lactobacillus plantarum CCFM8661 Figure 3 .

[0057] Depend on Figure 3 It can be seen that with the increase of yeast powder addition, the OD of Lactobacillus plantarum CCFM8661 600nm When the yeast powder addition amount was 15 g / L, the OD value of Lactobacillus plantarum CCFM8661 600nm The highest value was 2.4. When the yeast powder addition amount was 10 g / L, the OD 600nm The value is 2.3 because when the yeast powder addition amount is 10g / L and 15g / L, OD 600nmThe values ​​are not much different. Considering economic factors, the optimal addition amount of yeast powder is determined to be 10g / L.

[0058] Example 3: Determination of buffer salt type and concentration

[0059] On the basis of the optimization of carbon and nitrogen sources, the buffer salts (sodium acetate trihydrate, dipotassium hydrogen phosphate heptahydrate, diammonium hydrogen citrate) in the MRS liquid culture medium were replaced by NaH2PO4-Na2HPO4, KH2PO4-Na2HPO4, and Na2HPO4-citric acid buffer salt systems in turn. The concentrations of the three buffer salt systems were selected as 0, 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, and 0.20 mol / L, respectively. The inoculation amount was 2% (V / V) and inoculated into a 250 mL conical flask containing a buffer salt culture medium of different concentrations, and the culture was statically cultured in a 37°C constant temperature incubator for 24 hours, and then the cell density of the bacterial solution was measured to screen out the optimal buffer salt.

[0060] Effects of different buffer salt systems and concentrations on the growth of Lactobacillus plantarum CCFM8661 Figure 4 .

[0061] Depend on Figure 4 It can be seen that in the Na2HPO4-citric acid buffer system, the bacterial density of Lactobacillus plantarum CCFM8661 was significantly higher than that of the other two buffer salts. When its concentration was 0.1 mol / L, the bacterial density reached the highest, and OD 600nm The value is 3.3. Citric acid, as an organic acid, has significant oxidizing ability. In the fermentation process, organic acids are effectively utilized, which will cause the pH value of the fermentation environment to rise. In particular, when organic acid salts undergo oxidation reactions, alkaline substances will be generated, further promoting the increase in pH. This change effectively balances the effect of lactic acid production on the pH value of the system during the fermentation process, creating a more favorable environment for the growth of bacteria. Therefore, Na2HPO4-citric acid buffer salt will be selected as the optimal buffer salt system in the future.

[0062] Example 4: Determination of trace element addition amount

[0063] Determination of trace element addition: Based on the optimal carbon, nitrogen source, and buffer salt, the effects of magnesium sulfate addition (0, 0.2g / L, 0.4g / L, 0.8g / L, 1.2g / L), manganese sulfate addition (0.05g / L, 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L), and Tween 80 addition (0.5mL / L, 1.0mL / L, 2.0mL / L, 3.0mL / L, 4.0mL / L) on bacterial density were investigated in turn. The effects of different magnesium sulfate additions on the growth of Lactobacillus plantarum CCFM8661 are shown in Figure 5; Effects of different manganese sulfate additions on the growth of Lactobacillus plantarum CCFM8661 Figure 6 Effects of different Tween 80 additions on the growth of Lactobacillus plantarum CCFM8661 Figure 7 .

[0064] according to Figure 5 It can be seen that when the addition amount of magnesium sulfate is 0.2g / L, the cell density of Lactobacillus plantarum CCFM8661 reaches the peak value, and OD 600nm The value is 3.4. Therefore, the optimum addition amount of magnesium sulfate is determined to be 0.2 g / L. Figure 6 It can be seen that when the addition amount of manganese sulfate was 0.1 g / L, the cell density of Lactobacillus plantarum CCFM8661 reached its peak value, and OD 600nm The value was 3.6. When the amount of manganese sulfate added exceeded this level, the bacterial density decreased slightly. This phenomenon may be attributed to the excessively high concentration of manganese ions in the culture medium, which inhibited the growth of Lactobacillus plantarum CCFM8661. Therefore, we determined that the optimal amount of manganese sulfate added was 0.1 g / L. Figure 7 It can be seen that when the addition amount of Tween 80 was 1 mL / L, the bacterial density reached the maximum value, and OD 600nm The value is 3.64. When the amount of Tween 80 added exceeds this value, the bacterial density begins to decrease. Based on this observation, we determined that the optimal amount of Tween 80 added is 1mL / L.

[0065] In summary, by analyzing the data in each chart, we concluded that the optimal addition amounts of magnesium sulfate, manganese sulfate and Tween 80 for the growth of Lactobacillus plantarum CCFM8661 are 0.2 g / L, 0.1 g / L and 1 mL / L, respectively.

[0066] Example 5 Fermentation production of Lactobacillus plantarum CCFM8661

[0067] Preparation of seed solution: Inoculate the selected single colony of Lactobacillus plantarum CCFM8661 into MRS liquid culture medium, and culture it in a constant temperature shaker at 37°C and 50 r / min for 12 hours as seed solution.

[0068] The contents of each component in the MRS liquid culture medium are: glucose 20 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 4 g / L, Tween 80 1 g / L, dipotassium phosphate heptahydrate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate trihydrate 5 g / L, and the pH value is 6.2.

[0069] Fermenter inoculation: Inoculate the seed solution into the fermenter containing the optimized fermentation medium and continue to culture at 37°C. The fermenter is a device used for large-scale fermentation, which can provide a suitable environment and conditions to achieve large-scale growth of strains.

[0070] The content of each component in the optimized culture medium is: sucrose 25g / L, peptone 10g / L, beef extract 5g / L, yeast powder 10g / L, Tween 80 1mL / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate monohydrate 0.1g / L, Na2HPO4-citrate buffer 0.1mol / L

[0071] Bacteria density determination: During the fermentation process, samples were taken every 3 hours to test OD 600nm The growth curve of Lactobacillus plantarum CCFM8661 in the optimized culture medium was plotted. Figure 8 .

[0072] Depend on Figure 8 It can be seen that Lactobacillus plantarum CCFM8661 enters the stable phase after 21 hours of culture in the optimized medium. 600nm The value is 4.91.

[0073] End of fermentation: The fermentation liquid was separated, purified and dried to obtain the bacteria of Lactobacillus plantarum CCFM8661. The number of viable bacteria was measured and reached 1.5*10 12 CFU / g.

[0074] Example 6: Preparation of capsules using Lactobacillus plantarum CCFM8661

[0075] The fermentation process of the present invention is used to ferment Lactobacillus plantarum CCFM8661. This step ensures the activity and quantity of the strain, and provides a basis for the subsequent capsule product. Prebiotics, magnesium stearate and microcrystalline cellulose are added, and the mixed powder is loaded into the plant capsule shell to obtain the capsule product. The operation points are as follows:

[0076] Mixing: Mix the fermented Lactobacillus plantarum CCFM8661 with prebiotics, magnesium stearate and microcrystalline cellulose. Prebiotics, as food for probiotics, help the growth and activity of strains in the intestines; magnesium stearate, as a lubricant, helps the uniform dispersion of the mixture and the smooth progress of the capsule filling process; microcrystalline cellulose, as a filler, can increase the volume of the capsule contents, and also help improve the stability and disintegration of the capsule. Mix the fermented Lactobacillus plantarum CCFM8661 with prebiotics, magnesium stearate, microcrystalline cellulose and other excipients in a predetermined ratio. During the mixing process, ensure that the mixture is uniform and free of lumps, and that the components are evenly distributed.

[0077] Capsule filling: Professional capsule filling equipment is used to fill the mixed powder into the plant capsule shell. The plant capsule shell is made of natural plant materials and has the characteristics of being biodegradable, non-toxic, and easy to swallow. The filled capsules are quality inspected, including appearance inspection (to ensure that the capsule shape is complete and not damaged), sealing inspection (to ensure that the capsule shell is tightly closed and there is no powder leakage), weight inspection (to ensure that the amount of powder in each capsule is accurate and uniform), etc., to ensure the qualified rate and stability of the product.

[0078] Packaging and storage: Qualified capsule products should be packaged with packaging materials that have good sealing and moisture resistance to protect the capsule products from the influence of the external environment. The packaged capsule products should be stored in a cool, dry and ventilated place, avoiding direct sunlight and high temperature and high humidity environment to ensure the long-term stability and effectiveness of the product.

[0079] Through the above steps, we can obtain a capsule product containing active plant lactobacillus CCFM8661, which has a high probiotic content and significant probiotic effect; the auxiliary materials are reasonably proportioned, which improves the stability and bioavailability of the product; the use of plant capsule shells increases the safety and convenience of the product; the preparation method is simple and feasible, and is easy to industrialize.

[0080] Comparative Example 1

[0081] The optimized culture medium in the fermenter in Example 5 was replaced with MRS culture medium, and other conditions or parameters were consistent with those in Example 5.

[0082] Bacteria density determination: During the fermentation process, samples were taken every 3 hours to test OD 600nm The growth curves of Lactobacillus plantarum CCFM8661 in the optimized medium and MRS medium were plotted. Fig. 9 .

[0083] Depend on Fig. 9 It can be seen that Lactobacillus plantarum CCFM8661 entered the stable phase after 21 hours of culture in both the optimized medium and the MRS medium. At this stage, the number of cells basically reached the maximum value and remained stable. In the optimized medium, when the culture time was 21 hours, the OD 600nm The value was 4.91; in MRS medium, the OD was measured when the culture time was 21h. 600nm The value was 1.78. Compared with the original MRS medium, in the optimized medium, OD 600nm The value increased by 2.8 times. This shows that in the optimized culture medium, the density of the bacteria is higher and the growth is more vigorous. Fermentation in MRS medium ends: the fermentation liquid is separated, purified and dried to obtain the bacteria of Lactobacillus plantarum CCFM8661. The number of viable bacteria is measured, reaching 6*10 11CFU / g. In the optimized culture medium, bacterial activity increased by 2.5 times. This result further confirmed the effectiveness of the optimized culture medium in increasing the activity and quantity of Lactobacillus plantarum CCFM8661.

[0084] This shows that in the optimized culture medium, the cell concentration of Lactobacillus plantarum CCFM8661 is high and the growth condition is good. The optimized culture medium provides nutrients and growth environment that are more suitable for the growth of Lactobacillus plantarum CCFM8661, thereby promoting its growth and reproduction.

[0085] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for culturing Lactobacillus plantarum, characterized in that: The method comprises the following steps: culturing plant lactobacillus in a culture system with 10 to 35 g / L maltose or sucrose as a carbon source and 5 to 20 g / L yeast powder as a nitrogen source; the culture system further comprises 0.05 to 0.20 mol / L Na2HPO4-citrate buffer salt, 0 to 1.2 g / L magnesium sulfate, 0.05 to 0.4 g / L manganese sulfate and 0.5 to 4.0 mL / L Tween 80.

2. The method according to claim 1, characterized in that The culture system contains sucrose, peptone, beef extract, yeast powder, Tween, magnesium sulfate heptahydrate, manganese sulfate monohydrate, and Na2HPO4-citric acid buffer salt.

3. The method according to claim 1 or 2, characterized in that: The culture system contains 25 g / L sucrose, 10 g / L peptone, 5 g / L beef extract, 10 g / L yeast powder, 1 mL / L Tween 80, 0.2 g / L magnesium sulfate heptahydrate, 0.1 g / L manganese sulfate monohydrate, and 0.1 mol / L Na2HPO4-citrate buffer.

4. The method according to any one of claims 1 to 3, characterized in that: Add 2% of Lactobacillus plantarum bacterial liquid into the culture system and culture at 37° C. for not less than 20 hours.

5. The method according to any one of claims 1 to 3, characterized in that: The plant lactobacillus includes but is not limited to Lactobacillus plantarum CCFM8661, and the deposit number of the plant lactobacillus CCFM8661 is CGMCC No.5494.

6. A culture medium, characterized in that The culture medium consists of sucrose, peptone, beef extract, yeast powder, Tween, magnesium sulfate heptahydrate, manganese sulfate monohydrate and Na2HPO4-citrate buffer salt.

7. The culture medium according to claim 6, characterized in that The working concentrations of the components in the culture medium are sucrose 10-35 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 15-20 g / L, Tween 80 0.5-4.0 mL / L, magnesium sulfate heptahydrate 0-1.2 g / L, manganese sulfate monohydrate 0.05-0.4 g / L, and Na2HPO4-citrate buffer 0.05-0.20 mol / L.

8. The culture medium according to claim 6 or 7, characterized in that The working concentrations of the components in the culture medium are sucrose 25 g / L, peptone 10 g / L, beef extract 5 g / L, yeast powder 10 g / L, Tween 80 1 mL / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.1 g / L, and Na2HPO4-citrate buffer 0.1 mol / L.

9. Use of the culture medium according to any one of claims 6 to 8 in culturing Lactobacillus plantarum in the fields of food and medicine.

10. The use according to claim 9, characterized in that: The plant lactobacillus includes but is not limited to Lactobacillus plantarum CCFM8661, and the deposit number of the plant lactobacillus CCFM8661 is CGMCC No.5494.

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