A composite nitrogen source suitable for efficient proliferation of Lactobacillus crispatus and its application

Abstract

The invention discloses a composite nitrogen source suitable for efficient proliferation of Lactobacillus crispatus and its application, belonging to the technical field of microorganisms. The invention prepares a composite nitrogen source by using yeast extract FM502, bone peptone and whey hydrolyzate in a specific ratio, and adds Lactobacillus crispatus CCFM1298 to a culture medium with the composite nitrogen source as the sole nitrogen source. The proliferation effect of Lactobacillus crispatus CCFM1298 is significantly better than that of a culture medium with three single nitrogen sources as the sole nitrogen source. When the nitrogen source addition amount is increased from 1 g / L to 25 g / L of MRS addition amount, the OD of the optimal nitrogen source combination is 5.6 times that of the MRS nitrogen source, and the number of viable bacteria of the optimal nitrogen source combination is 12.7 times that of the MRS nitrogen source, which is conducive to the industrialization of Lactobacillus crispatus CCFM1298, thereby improving industrial benefits.

Description

Technical Field

[0001] The invention relates to a composite nitrogen source suitable for efficient proliferation of Lactobacillus crispatus and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Lactobacillus is an important flora of the female reproductive tract. It accounts for more than 95% of the vaginal secretions of healthy women of childbearing age, and Lactobacillus crispatus is the most abundant lactobacillus in the vagina of healthy women. It is a marker of the healthy female vaginal microenvironment. Lactobacillus crispatus is closely related to the health of the female reproductive tract. Its reduced abundance can easily cause a variety of female reproductive tract diseases such as bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). Therefore, Lactobacillus crispatus plays an important role in maintaining the balance of the female vaginal microecology. At present, antibiotics are commonly used drugs for the treatment of female vaginal diseases, but long-term use of antibiotics may disrupt the patient's vaginal flora, inhibit the growth of beneficial bacteria such as lactic acid bacteria, and make the normal vaginal flora in an unbalanced state; at the same time, the abuse of antibiotics will also increase the drug resistance of pathogens, leading to problems such as ineffective treatment. Therefore, it is urgent to find a safer treatment method. As one of the most promising probiotics for treating vaginal diseases such as BV and VVC, Lactobacillus crispatus can inhibit the proliferation of pathogens without destroying the balance of its own beneficial bacteria, thereby maintaining the health of the reproductive tract. Due to its significant probiotic effects, the market demand for Lactobacillus crispatus continues to increase. However, the main fermentation method of Lactobacillus crispatus is currently based on traditional static culture, which has the problem of low bacterial density in the fermentation liquid, and the number of viable bacteria is generally less than 10 8 ~10 9 CFU / ml, which makes Lactobacillus crispatus unfavorable for industrialization and large-scale production. At present, there are relatively few studies on the optimization of Lactobacillus crispatus culture, so it is of great significance to increase the number of viable Lactobacillus crispatus through optimization.

[0003] Nitrogen source is a key factor restricting the high-density culture of lactic acid bacteria. Microbial cell growth metabolism and product synthesis all require nitrogen source. Different types of nitrogen sources will affect the growth status and metabolic activity of Lactobacillus crispatus. By adjusting the supply of nitrogen source, the fermentation process of Lactobacillus crispatus can be optimized and its product yield can be increased. At present, the screening of nitrogen sources at home and abroad is based on batch culture mode. High-quality nitrogen sources are screened by single factor test and orthogonal experiment. However, the proliferation efficiency of nitrogen source per unit mass cannot be accurately analyzed by traditional methods to determine the most suitable nitrogen source for the growth of Lactobacillus crispatus. On the one hand, the nitrogen source addition amount of batch culture mode is often excessive. When Lactobacillus crispatus grows to the maximum amount, it may be due to the decrease of pH or lack of carbon source, and the available effective nitrogen components in the nitrogen source are not fully utilized; on the other hand, due to the low utilization of nitrogen source in the culture medium, the environmental osmotic pressure of the culture medium is increased, which leads to a low fermentation density of Lactobacillus crispatus. Therefore, the most suitable nitrogen source for the growth of Lactobacillus crispatus can be determined by screening nitrogen source by the proliferation efficiency of nitrogen source per unit mass to achieve the best culture effect. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a composite nitrogen source suitable for the efficient proliferation of Lactobacillus crispatus and its application, aiming to solve the problem that the proliferation of Lactobacillus crispatus is affected by the low utilization rate of the nitrogen source during the cultivation process of Lactobacillus crispatus.

[0005] The first technical solution provided by the present invention is a composite nitrogen source for Lactobacillus crispatus, wherein the composite nitrogen source consists of yeast extract powder FM502, bone peptone and whey protein hydrolyzate.

[0006] In certain embodiments, the mass ratio of the yeast extract FM502, bone peptone and whey protein hydrolysate is 3:(1-2):(1-2).

[0007] In certain embodiments, the nitrogen source screening method comprises: replacing the composite nitrogen source in the MRS medium with different types of single nitrogen sources to prepare different nitrogen source screening mediums;

[0008] The seed liquid of Lactobacillus crispatus activated to the third generation was inoculated into different single nitrogen source culture media respectively. After culturing to the stable period, the proliferation amount of different single nitrogen sources was determined by measuring the OD600nm value.

[0009] The second technical solution provided by the present invention is a culture medium for promoting the proliferation of Lactobacillus crispatus, wherein the optimal nitrogen source screened out by the first technical solution is compounded into a composite nitrogen source in different proportions and then replaced with the nitrogen source in the culture medium.

[0010] In certain embodiments, the culture medium contains a complex nitrogen source, glucose, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate monohydrate, Tween 80, and cysteine ​​hydrochloride.

[0011] In certain embodiments, the components in the culture medium are 1 g / L complex nitrogen source, 6 g / L glucose, 7 g / L dipotassium hydrogen phosphate, 7 g / L disodium hydrogen phosphate, 0.25 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, 1 g / L Tween 80, and 1 g / L cysteine ​​hydrochloride.

[0012] In certain embodiments, the culture medium contains a complex nitrogen source, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate heptahydrate, manganese sulfate monohydrate, Tween-80, and cysteine ​​hydrochloride.

[0013] In certain embodiments, the composition of the culture medium is 25 g / L complex nitrogen source, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.25 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, 1 g / L Tween 80, and 1 g / L cysteine ​​hydrochloride.

[0014] In certain embodiments, the Lactobacillus crispatus is Lactobacillus crispatus CCFM1298, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO:62275.

[0015] The third technical solution provided by the present invention is a culture method for promoting the proliferation of Lactobacillus crispatus. The method comprises culturing Lactobacillus crispatus in a culture system containing the composite nitrogen source described in the first technical solution.

[0016] In certain embodiments, the culture system contains 1 g / L of complex nitrogen source, 6 g / L of glucose, 7 g / L of dipotassium hydrogen phosphate, 7 g / L of disodium hydrogen phosphate, 0.25 g / L of magnesium sulfate heptahydrate, 0.05 g / L of manganese sulfate monohydrate, 1 g / L of Tween 80, and 1 g / L of cysteine ​​hydrochloride.

[0017] In certain embodiments, the culture system contains 25 g / L of complex nitrogen source, 20 g / L of glucose, 2 g / L of dipotassium hydrogen phosphate, 2 g / L of diammonium hydrogen citrate, 5 g / L of sodium acetate, 0.25 g / L of magnesium sulfate heptahydrate, 0.05 g / L of manganese sulfate monohydrate, 1 g / L of Tween 80, and 1 g / L of cysteine ​​hydrochloride.

[0018] In certain embodiments, the seed solution of Lactobacillus crispatus is inoculated into the culture system at an inoculation rate of 3% and cultured, and the culture is stopped after the culture reaches a stable period.

[0019] In certain embodiments, the method for preparing the Lactobacillus crispatus seed liquid is: streak culture of Lactobacillus crispatus on MRS solid culture medium, pick a single colony of Lactobacillus crispatus and culture it in MRS liquid culture medium for 15 to 18 hours, inoculate the bacterial liquid in the liquid culture medium into the MRS liquid culture medium at an inoculation rate of 3%, and continue culturing for 15 to 18 hours to obtain the seed liquid of Lactobacillus crispatus.

[0020] The fourth technical solution provided by the present invention is the use of the composite nitrogen source described in the first technical solution or the culture medium described in the second technical solution in culturing Lactobacillus crispatus in the fields of food and medicine.

[0021] The technical effects of the present invention are as follows:

[0022] The invention provides a nitrogen source screening method for promoting efficient proliferation of Lactobacillus crispatus CCFM1298. After Lactobacillus crispatus CCFM1298 is cultured by using the culture medium, the pH value of the fermentation liquid is not less than 4, and the glucose content is not less than 2g / L after the fermentation is completed, indicating that the growth of the strain stops because the nitrogen source in the culture medium is completely used up. At this time, the nitrogen source consumption is 1g / L. By measuring the OD600nm value of the bacterial liquid, the proliferation effect of the strain on different nitrogen sources per unit mass can be accurately obtained, and then the optimal single nitrogen source can be screened out.

[0023] The invention prepares a composite nitrogen source by preparing yeast extract FM502, bone peptone and whey protein hydrolysate in a specific ratio, and adds Lactobacillus crispatus CCFM1298 to a culture medium with the composite nitrogen source as the sole nitrogen source. The proliferation effect of Lactobacillus crispatus CCFM1298 is significantly better than that of culture mediums with three single nitrogen sources as the sole nitrogen source, and the proliferation effect of the composite nitrogen source is significantly better than that of MRS. When the nitrogen source addition amount is increased from 1 g / L to 25 g / L of MRS, the OD of the optimal nitrogen source combination is 5.6 times that of MRS, and the viable count of the optimal nitrogen source combination is 12.7 times that of MRS. The composite nitrogen source is conducive to the industrialization of Lactobacillus crispatus CCFM1298, thereby improving industrial benefits. DETAILED DESCRIPTION

[0024] 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.

[0025] Test method:

[0026] Glucose determination: S-10 biosensor analyzer was used to determine the glucose concentration in the fermentation broth. According to the instructions of the manual, the specific operations were as follows: the sampler accurately aspirated 25 μL of standard solution for calibration, the fermentation broth was centrifuged, and after centrifugation, it was diluted according to the residual glucose concentration. When the residual sugar content was higher than 0.8, it was necessary to dilute it. The sampler accurately aspirated 25 μL of supernatant and inserted it into the reaction cell after the instrument issued a "beep" prompt sound when the sample was ready. The glucose concentration displayed by the instrument multiplied by the dilution factor was the remaining amount of glucose in the fermentation broth.

[0027] Maximum biomass determination: The detection wavelength of the UV spectrophotometer was set to 600 nm, and the OD600nm value of the fermentation broth was measured. When the absorbance value exceeded 0.8, the fermentation broth was diluted a certain multiple so that the absorbance value after dilution was within the range of 0.2 to 0.8. The OD600nm value of the fermentation broth was the measured absorbance value multiplied by the dilution multiple.

[0028] pH determination: pH was measured using a precision pH meter.

[0029] Live bacteria count: After oscillating and mixing the fermentation liquid, pipette 0.5 ml into 4.5 ml PBS, dilute it tenfold in sequence, select the appropriate dilution gradient, pipette 1 ml of the dilution liquid into a plate, add the modified MRS-L solid culture medium, shake well and wait for solidification before placing in a 37°C incubator for 48-72 hours.

[0030] The raw materials used in the embodiment:

[0031] The Lactobacillus crispatus CCFM1298 involved in the following examples is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 62275, which has been disclosed in the patent document CN116694498A. The casein peptone, bone peptone, brewer's yeast extract powder, and soy peptone in the following examples were purchased from Shanghai Yuanpeptide Biotechnology Co., Ltd.; peptone Y001A and yeast extract powder Y017B were purchased from Beijing Hongrun Baoshun Technology Co., Ltd.; tryptone LP0042B and whey protein hydrolysate were purchased from OXOID, UK; yeast peptone FD103, fish peptone FP351, peptone FP326, yeast extract powder FM502, yeast extract powder FM902, and beef extract powder were purchased from Angel Yeast Co., Ltd.; casein was purchased from Bofei Meike; tryptone was purchased from Beyotime Biotechnology Co., Ltd.; pancreatic casein peptone 70169 was purchased from Sigma-Aldrich, USA, and yeast peptone and yeast powder were purchased from Lesaffre Group, France.

[0032] PBS (g / L): NaCl 8.5g / L, KH2PO4 4.3g / L, Na2HPO4 5g / L, cysteine ​​hydrochloride 0.3g / L, distilled water 1000g / L.

[0033] Improved MRS-L liquid culture medium (g / L): glucose 20 g / L, peptone 10 g / L, yeast extract 8 g / L, beef extract powder 4 g / L, anhydrous sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4 2 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·H2O 0.05 g / L, Tween-80 1 g / L, cysteine ​​hydrochloride 1 g / L, and distilled water 1000 g / L.

[0034] Improved MRS-L solid culture medium (g / L): glucose 20 g / L, peptone 10 g / L, yeast extract 8 g / L, beef extract powder 4 g / L, anhydrous sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, K2HPO4 2 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·H2O 0.05 g / L, Tween-80 1 g / L, cysteine ​​hydrochloride 1 g / L, distilled water 1000 g / L, agar 20 g / L.

[0035] Nitrogen source preferred culture medium (g / L): nitrogen source 1g / L, glucose 6g / L, K2HPO47 g / L, Na2HPO47 g / L, MgSO4·7H2O 0.25g / L, MnSO4·H2O 0.05g / L, Tween-80 1g / L, cysteine ​​hydrochloride 1g / L, distilled water 1000g / L.

[0036] Example 1: Screening of single nitrogen source for Lactobacillus crispatus CCFM1298

[0037] Different single nitrogen source culture media were prepared: nitrogen source 1g / L (single nitrogen source: casein peptone, bone peptone, brewer's yeast extract powder, soy peptone, peptone Y001A, yeast extract powder Y017B, tryptone LP0042B, whey protein hydrolysate, yeast peptone FD103, fish peptone FP351, peptone FP326, yeast extract powder FM502, yeast extract powder FM902, beef extract powder, casein, tryptone, pancreatic digested casein peptone 70169, yeast peptone and yeast powder), glucose 6g / L, K2HPO4 7 g / L, Na2HPO4 7 g / L, MgSO4·7H2O 0.25g / L, MnSO4·H2O 0.05g / L, Tween 80 1g / L, cysteine ​​hydrochloride 1g / L, distilled water 1000g / L. After preparation, adjust the pH to 6.5 and sterilize at 121℃ for 15min.

[0038] Activation culture of bacteria: The activated Lactobacillus crispatus CCFM1298 seed solution was inoculated into different nitrogen source culture media at an inoculation rate of 3%, and cultured in a 37° C. incubator for 15 h.

[0039] Fermentation density detection: Determine the OD600nm value and pH value of the fermentation broth with different nitrogen sources, and judge the strain's utilization effect of different nitrogen sources based on the OD600nm value. At the same time, take a small amount of fermentation broth and centrifuge it to determine the glucose content in the supernatant.

[0040] Table 1 The utilization ability of Lactobacillus crispatus CCFM1298 on different nitrogen sources

[0041]

[0042] The test results are shown in Table 1. It can be seen from the results that when Lactobacillus crispatus CCFM1298 was cultured in 1g / L different nitrogen source mediums, the content of glucose was sufficient, and the pH value was higher than 4.0 when Lactobacillus crispatus CCFM1298 stopped growing, indicating that the strain stopped growing because the nitrogen source was fully utilized, not due to the influence of carbon source utilization and low pH. The final concentration of the fermentation liquid at the end of the culture was the proliferation amount of the strain using the unit mass nitrogen source, and the proliferation amount of the strain using different nitrogen sources was obtained accordingly. Among them, yeast extract FM502, bone peptone and whey protein hydrolyzate were the single nitrogen sources with the best proliferation effect of Lactobacillus crispatus CCFM1298 using the unit mass nitrogen source.

[0043] Example 2: A composite nitrogen source that promotes efficient proliferation of Lactobacillus crispatus CCFM1298

[0044] Medium preparation: nitrogen source yeast extract powder FM502, bone peptone, whey protein hydrolyzate, the mass ratio of which is 3:1:1, the total amount of nitrogen source added is 1g / L, glucose 6g / L, K2HPO47 g / L, Na2HPO47 g / L, MgSO4·7H2O0.25g / L, MnSO4·H2O 0.05g / L, Tween 80 1g / L, cysteine ​​hydrochloride 1g / L, distilled water 1000g / L. After preparation, adjust the pH to 6.5 and sterilize at 121℃ for 15min.

[0045] Activation culture of bacteria: inoculate the activated Lactobacillus crispatus CCFM1298 seed liquid into the culture medium at an inoculation rate of 3%, and culture it in a 37° C. incubator for 15 hours.

[0046] Fermentation density detection: Determine the OD600nm value and pH value of the fermentation broth with different nitrogen sources, and judge the strain's utilization effect of the nitrogen source combination based on the OD600nm value. At the same time, take a small amount of fermentation broth and centrifuge it to determine the glucose content in the supernatant.

[0047] Table 2 The utilization ability of Lactobacillus crispatus CCFM1298 on compound nitrogen sources

[0048]

[0049] The test results are shown in Table 2. It can be seen from the results that when the optimal nitrogen source combination ratio is set to 3:1:1, the OD value of Lactobacillus crispatus CCFM1298 is 1.102, which is 1.61 times the OD value of the MRS control, and the fermentation density is improved.

[0050] Example 3: A composite nitrogen source for promoting efficient proliferation of Lactobacillus crispatus CCFM1298

[0051] Medium preparation: Compound nitrogen source yeast extract powder FM502, bone peptone, whey protein hydrolyzate, the mass ratio is 3:2:1, the total amount of nitrogen source added is 1g / L, glucose 6g / L, K2HPO47 g / L, Na2HPO47 g / L, MgSO4·7H2O0.25g / L, MnSO4·H2O 0.05g / L, Tween 80 1g / L, cysteine ​​hydrochloride 1g / L, distilled water 1000g / L. After preparation, adjust the pH to 6.5 and sterilize at 121℃ for 15min.

[0052] Activation culture of bacteria: inoculate the activated Lactobacillus crispatus CCFM1298 seed liquid into the culture medium at an inoculation rate of 3%, and culture it in a 37° C. incubator for 15 hours.

[0053] Fermentation density detection: Determine the OD600nm value and pH value of the fermentation broth with different nitrogen sources, and judge the strain's utilization effect of the nitrogen source combination based on the OD600nm value. At the same time, take a small amount of fermentation broth and centrifuge it to determine the glucose content in the supernatant.

[0054] Table 3 Utilization ability of Lactobacillus crispatus CCFM1298 on compound nitrogen sources

[0055]

[0056] The test results are shown in Table 3. It can be seen from the results that when the nitrogen source combination ratio is set to 3:2:1, the OD value of Lactobacillus crispatus CCFM1298 is significantly improved, reaching 2.935, indicating that this ratio of composite nitrogen source can provide sufficient nutrients and growth-promoting factors for Lactobacillus crispatus, and the fermentation density of Lactobacillus crispatus CCFM1298 is significantly improved.

[0057] Example 4: A composite nitrogen source for promoting efficient proliferation of Lactobacillus crispatus CCFM1298

[0058] Medium preparation: nitrogen source yeast extract powder FM502, bone peptone, whey protein hydrolyzate, the mass ratio of which is 3:1:2, the total amount of nitrogen source added is 1g / L, glucose 6g / L, K2HPO47 g / L, Na2HPO47 g / L, MgSO4·7H2O0.25g / L, MnSO4·H2O 0.05g / L, Tween 80 1g / L, cysteine ​​hydrochloride 1g / L, distilled water 1000g / L. After preparation, adjust the pH to 6.5 and sterilize at 121℃ for 15min.

[0059] Activation culture of bacteria: inoculate the activated Lactobacillus crispatus CCFM1298 seed liquid into the culture medium at an inoculation rate of 3%, and culture it in a 37° C. incubator for 15 hours.

[0060] Fermentation density detection: Determine the OD600nm value and pH value of the fermentation broth with different nitrogen sources, and judge the effect of the strain on the nitrogen source combination according to the OD600nm value. At the same time, take a small amount of fermentation broth and centrifuge it to determine the glucose content in the supernatant.

[0061] Table 4 The utilization ability of Lactobacillus crispatus CCFM1298 on compound nitrogen sources

[0062]

[0063] The test results are shown in Table 4. It can be seen from the results that when the optimal nitrogen source combination ratio is set to 3:1:2, the OD value of Lactobacillus crispatus CCFM1298 is 1.142, which is 1.66 times the OD value of the MRS control, and the fermentation density is improved.

[0064] Example 5: A composite nitrogen source for promoting efficient proliferation of Lactobacillus crispatus CCFM1298

[0065] Medium preparation: nitrogen source yeast extract powder FM502, bone peptone, whey protein hydrolyzate, the mass ratio of which is 3:2:2, the total amount of nitrogen source added is 1g / L, glucose 6g / L, K2HPO47 g / L, Na2HPO47 g / L, MgSO4·7H2O0.25g / L, MnSO4·H2O 0.05g / L, Tween 80 1g / L, cysteine ​​hydrochloride 1g / L, distilled water 1000g / L. After preparation, adjust the pH to 6.5 and sterilize at 121℃ for 15min.

[0066] Activation culture of bacteria: inoculate the activated Lactobacillus crispatus CCFM1298 seed liquid into the culture medium at an inoculation rate of 3%, and culture it in a 37° C. incubator for 15 hours.

[0067] Fermentation density detection: Determine the OD600nm value and pH value of the fermentation broth with different nitrogen sources, and judge the effect of the strain on the nitrogen source combination according to the OD600nm value. At the same time, take a small amount of fermentation broth and centrifuge it to determine the glucose content in the supernatant.

[0068] Table 5 The utilization ability of Lactobacillus crispatus CCFM1298 on compound nitrogen sources

[0069]

[0070] The test results are shown in Table 5. It can be seen from the results that when the optimal nitrogen source combination ratio is set to 3:2:2, the OD value of Lactobacillus crispatus CCFM1298 is 1.088, which is 1.59 times the OD value of the MRS control, and the fermentation density is improved.

[0071] Example 6 A composite nitrogen source for promoting efficient proliferation of Lactobacillus crispatus CCFM1298

[0072] Preparation of culture medium: The culture medium was prepared according to the formula of MRS culture medium. The nitrogen source was yeast extract powder FM502, bone peptone, and whey protein hydrolyzate in a mass ratio of 3:2:1. The total amount of nitrogen source added was 25g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, MgSO4·7H2O 0.25g / L, MnSO4·H2O 0.05g / L, Tween 801g / L, cysteine ​​hydrochloride 1g / L, and distilled water 1000g / L. After preparation, the pH was adjusted to 6.5 and sterilized at 121℃ for 15min.

[0073] Activation culture of bacteria: inoculate the activated Lactobacillus crispatus CCFM1298 seed liquid into the culture medium at an inoculation rate of 3%, and culture it in a 37° C. incubator for 15 hours.

[0074] Fermentation density detection: Determine the OD600nm value and pH value of the optimal compound nitrogen source fermentation liquid with the same total amount of nitrogen source added as the MRS medium. At the same time, take a small amount of fermentation liquid and centrifuge it to determine the glucose content in the supernatant.

[0075] Live bacteria count: Pipette 1 ml of fermentation liquid into 9 ml PBS for gradient dilution, select appropriate dilution gradient for counting, culture at 37°C for 48 to 72 hours, and count after cultivation.

[0076] Table 6 Utilization ability of Lactobacillus crispatus CCFM1298 on the optimal composite nitrogen source

[0077]

[0078] The test results are shown in Table 6. It can be seen from the results that according to Examples 2 to 5, the composite nitrogen source with the best nitrogen source proliferation effect per unit mass of Lactobacillus crispatus was obtained. When the total amount of the optimal composite nitrogen source was added according to the total amount of nitrogen source added in MRS, that is, when the composite nitrogen source (FM502: bone peptone: whey protein hydrolyzate = 3:2:2) was added according to the MRS addition amount of 25g / L, the OD value of Lactobacillus crispatus CCFM1298 was 9.43, and the number of viable bacteria was 6.1×10 9 , the fermentation density was significantly improved.

[0079] Comparative Example 1

[0080] MRS medium was prepared, with a total nitrogen source addition of 25 g / L. The remaining ingredients were the same as those of MRS, that is, consistent with the formulation of Example 6, with only the nitrogen source replaced. The OD, pH value, glucose content and viable count of the fermentation broth were measured.

[0081] Table 7 Utilization ability of Lactobacillus crispatus CCFM1298 on MRS composite nitrogen source

[0082]

[0083] From the results in Table 7, it can be seen that the OD of Lactobacillus crispatus CCFM1298 after culture in MRS medium was 2.695, and the number of viable bacteria was 4.8×10 8 CFU / ml, while the OD of the optimal nitrogen source combination in Example 6 is 5.6 times that of the MRS nitrogen source, and the number of viable bacteria of the optimal nitrogen source combination is 12.7 times that of the MRS nitrogen source. It can be seen that the utilization efficiency and proliferation effect of the optimal composite nitrogen source screened by this method are better than those of the MRS culture medium.

[0084] Comparative Example 2

[0085] Preparation of culture medium: The composite nitrogen source is the four nitrogen sources with higher OD screened in Example 1: yeast extract FM502, bone peptone, whey protein hydrolyzate, and pancreatic casein peptone 70169, which are prepared in a mass ratio of 3:2:1:1, and the total amount of nitrogen source added is 1g / L, glucose 6g / L, K2HPO47 g / L, Na2HPO47 g / L, MgSO4·7H2O 0.25g / L, MnSO4·H2O0.05g / L, Tween 80 1g / L, cysteine ​​hydrochloride 1g / L, and distilled water 1000g / L. After preparation, adjust the pH to 6.5 and sterilize at 121°C for 15min.

[0086] Activation culture of bacteria: inoculate the activated Lactobacillus crispatus CCFM1298 seed liquid into the culture medium at an inoculation rate of 3%, and culture it in a 37° C. incubator for 15 hours.

[0087] The OD600nm value and pH value of the fermentation broth with different nitrogen sources were measured, and the effect of the strain on the nitrogen source combination was judged according to the OD600nm value. At the same time, a small amount of fermentation broth was centrifuged and the glucose content in the supernatant was measured.

[0088] Table 8 Utilization ability of Lactobacillus crispatus CCFM1298 on four composite nitrogen sources

[0089]

[0090] The test results are shown in Table 8. It can be seen from the results that when the four different optimal nitrogen sources screened in Example 1 are compounded at a composite mass ratio of 3:2:1:1 of yeast extract powder FM502: bone peptone: whey protein hydrolysate: pancreatic digested casein peptone 70169, the OD value of Lactobacillus crispatus CCFM1298 is 1.023, indicating that increasing the number of nitrogen sources does not enhance the proliferation effect of Lactobacillus crispatus CCFM1298, and the proliferation effect of Lactobacillus crispatus CCFM1298 is the strongest when the mass ratio of the three nitrogen sources, yeast extract powder FM502: bone peptone: whey protein hydrolysate, is 3:2:1.

[0091] Comparative Example 3

[0092] Preparation of culture medium: according to the composite nitrogen source of Example 3: yeast extract FM502, bone peptone, whey protein hydrolyzate, mass ratio of 3:2:1, total nitrogen source added is 1g / L, glucose 6g / L, K2HPO47 g / L, Na2HPO47 g / L, MgSO4·7H2O 0.25g / L, MnSO4·H2O 0.05g / L, Tween 80 1g / L, cysteine ​​hydrochloride 1g / L, distilled water 1000g / L. After preparation, adjust pH to 6.5, sterilize at 121°C for 15min.

[0093] Activation culture of strains: Inoculate the activated Lactobacillus crispatus CCFM1339 seed liquid into the culture medium at a 3% inoculation rate and culture it in a 37°C incubator for 15 hours. The preservation number of Lactobacillus crispatus CCFM1339 is GDMCCNO.63795, which has been disclosed in the patent document with publication number CN117701462A.

[0094] The OD600nm value and pH value of the fermentation broth with different nitrogen sources were measured, and the effect of the strain on the nitrogen source combination was judged according to the OD600nm value. At the same time, a small amount of fermentation broth was centrifuged and the glucose content in the supernatant was measured.

[0095] Table 9 Utilization ability of Lactobacillus crispatus CCFM1339 on three kinds of composite nitrogen sources

[0096]

[0097] The test results are shown in Table 9. It can be seen from the results that when the composite nitrogen source of Example 2-5 is used to culture the same species of Lactobacillus crispatus CCFM1339, the nitrogen source with the highest OD is yeast extract FM502: bone peptone: whey protein hydrolysate = 3:2:2, and the OD value only reaches 0.923. The OD of the composite nitrogen source whose nitrogen source is yeast extract FM502: bone peptone: whey protein hydrolysate = 3:2:1 is 0.798, indicating that this composite nitrogen source is not the optimal composite nitrogen source for Lactobacillus crispatus CCFM1339. In summary, the composite nitrogen source whose nitrogen source is yeast extract FM502: bone peptone: whey protein hydrolysate = 3:2:1 is the optimal composite nitrogen source for Lactobacillus crispatus CCFM1298, which significantly improves its proliferation effect. This composite nitrogen source is not the universal optimal composite nitrogen source for all Lactobacillus crispatus.

[0098] 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 composite nitrogen source for Lactobacillus crispatus CCFM1298, characterized in that: The composite nitrogen source consists of yeast extract FM502, bone peptone and whey protein hydrolysate, wherein the mass ratio of the yeast extract FM502, bone peptone and whey protein hydrolysate is 3:(1-2):(1-2); the preservation number of the crispatus Lactobacillus CCFM1298 is GDMCCNO:62275.

2. A culture medium for promoting the proliferation of Lactobacillus crispatus CCFM1298, characterized in that: The nitrogen source in the culture medium is replaced by the composite nitrogen source according to claim 1.

3. The culture medium according to claim 2, characterized in that The culture medium contains a complex nitrogen source, glucose, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate heptahydrate, manganese sulfate monohydrate, Tween 80 and cysteine ​​hydrochloride.

4. The culture medium according to claim 3, characterized in that The ingredients in the culture medium are 1 g / L complex nitrogen source, 6 g / L glucose, 7 g / L dipotassium hydrogen phosphate, 7 g / L disodium hydrogen phosphate, 0.25 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, 1 g / L Tween 80, and 1 g / L cysteine ​​hydrochloride.

5. The culture medium according to claim 2, characterized in that The culture medium contains a composite nitrogen source, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate heptahydrate, manganese sulfate monohydrate, Tween-80, and cysteine ​​hydrochloride.

6. The culture medium according to claim 5, characterized in that The ingredients in the culture medium are 25 g / L complex nitrogen source, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.25 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate monohydrate, 1 g / L Tween-80, and 1 g / L cysteine ​​hydrochloride.

7. A method for promoting the proliferation of Lactobacillus crispatus CCFM1298, characterized in that: The method comprises culturing Lactobacillus crispatus CCFM1298 in a culture system containing the composite nitrogen source according to claim 1.

8. The method according to claim 7, characterized in that The culture system contains 1 g / L of composite nitrogen source, 6 g / L of glucose, 7 g / L of dipotassium hydrogen phosphate, 7 g / L of disodium hydrogen phosphate, 0.25 g / L of magnesium sulfate heptahydrate, 0.05 g / L of manganese sulfate monohydrate, 1 g / L of Tween 80, and 1 g / L of cysteine ​​hydrochloride. Alternatively, the culture system contains 25 g / L of complex nitrogen source, 20 g / L of glucose, 2 g / L of dipotassium hydrogen phosphate, 2 g / L of diammonium hydrogen citrate, 5 g / L of sodium acetate, 0.25 g / L of magnesium sulfate heptahydrate, 0.05 g / L of manganese sulfate monohydrate, 1 g / L of Tween-80, and 1 g / L of cysteine ​​hydrochloride.

9. The method according to claim 7, characterized in that: The seed solution of Lactobacillus crispatus CCFM1298 was inoculated into the culture system at an inoculation rate of 3% and cultured. After the culture reached the stable period, the culture was stopped.

10. Use of the composite nitrogen source according to claim 1 or the culture medium according to claim 2 in culturing Lactobacillus crispatus in the fields of food and medicine.

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