Method for improving fermentation level of lactobacillus acidophilus based on culture medium improvement and pH regulation

Abstract

The invention discloses a method for improving the fermentation level of lactobacillus acidophilus based on culture medium improvement and pH regulation and control, and belongs to the technical field of microbial fermentation. A seed solution of lactobacillus acidophilus is inoculated into an improved culture medium without buffer salt, fermentation is performed at the temperature of 37 DEG C under the constant pH range of 4.5-4.6, in the fermentation process, 2mol / L sodium hydroxide is fed into the solution to keep the pH constant, the fermentation level of lactobacillus acidophilus can be remarkably improved, the viable count of lactobacillus acidophilus CCFM137 fermentation liquor can reach 3.37 * 10 < 9 > CFU / mL, and the pH value of the lactobacillus acidophilus CCFM137 fermentation liquor can reach 3.7 * 10 < 9 > CFU / mL. Meanwhile, the raw material utilization rate and the freeze-drying survival rate are improved, and certain guiding significance is achieved for 100L or larger-scale lactobacillus acidophilus fermentation. The lactobacillus acidophilus culture medium has the advantages of simple formula, high nutrient substance utilization rate, high fermentation efficiency, short fermentation time and the like, and is beneficial to industrial production of lactobacillus acidophilus.

Description

Technical Field

[0001] The invention relates to a method for improving the fermentation level of Lactobacillus acidophilus based on culture medium improvement and pH control, and belongs to the technical field of microbial fermentation. Background Art

[0002] Lactobacillus acidophilus is an important probiotic that colonizes the human oral cavity and gastrointestinal tract. It has multiple probiotic functions, such as regulating intestinal flora, alleviating lactose intolerance, inhibiting pathogens, preventing obesity, preventing cancer, and enhancing immunity. It is now widely used in food, medicine, and industrial fields. Due to the urgent demand for such probiotics from consumers, high-density fermentation technology is particularly important for the efficient production of live bacterial preparations of this bacterium. However, due to the differences in Lactobacillus acidophilus strains and its high requirements for nutritional conditions and fermentation environment, its industrial production is greatly restricted.

[0003] Optimizing the composition of the culture medium is a common strategy to improve the fermentation effect, mainly including the optimization of carbon source and nitrogen source. The carbon sources suitable for the growth of Lactobacillus acidophilus are mainly glucose, sucrose and lactose, but the main function of the carbon source is to provide energy for bacterial metabolism. Therefore, the optimization of the carbon source is mainly to adjust its addition amount. There are many types of nitrogen sources, and different nitrogen sources have different effects on the growth of Lactobacillus acidophilus. Martin Senz et al. (Control of cellmorphology of probiotic Lactobacillus acidophilus for enhanced cell stability during industrial processing, 2015) used different nitrogen sources to culture Lactobacillus acidophilus NCFM, and the final optimized viable count was 1.02×10 9 CFU / mL; Wu Yexu (Effects of yeast extract on the morphology and freeze-dried survival rate of Lactobacillus acidophilus, 2023) used a variety of yeast extract powders containing different growth factors to culture Lactobacillus acidophilus, and finally showed that a suitable nitrogen source had a significant promoting effect on the cell division of Lactobacillus acidophilus, and the final viable count was 2.53×10 9 CFU / mL.

[0004] In addition to the composition of the culture medium, the pH environment during high-density fermentation also plays a decisive role in the fermentation level of Lactobacillus acidophilus. Cui Shumao (Factors and Laws Affecting Growth Inhibition and Freeze-dried Survival of Lactobacillus acidophilus, 2016) conducted a batch-fed fermentation of Lactobacillus acidophilus CCFM137 at a constant pH of 7, and the final number of viable bacteria was only 3.1×10 8CFU / mL; Chinese patent application CN108559717A discloses a high-density fermentation medium for Lactobacillus acidophilus and a method for preparing its powder. The results show that the optimal growth pH of Lactobacillus acidophilus is 5.7-6.0, which is also the commonly used pH range for lactic acid bacteria fermentation; Zhao Yanxia (Research on High-density Culture Process of Lactobacillus acidophilus IMAU30067, 2018) showed that the optimal constant pH of Lactobacillus acidophilus is 5.0, and Wang Tianyun (Research on Growth and Survival of Lactobacillus acidophilus under Different pH Conditions, 2000) used soybean juice as the culture medium and adjusted the initial pH value to 4.5 for the cultivation of Lactobacillus acidophilus, but as the pH continued to decrease, the final number of viable bacteria was only 4.47×10 8 CFU / mL. Summary of the invention

[0005] [Technical issues]

[0006] The technical problem to be solved by the present invention is to improve the high-density fermentation level of Lactobacillus acidophilus CCFM137.

[0007] [Technical solution]

[0008] One of the purposes of the present invention is to provide a modified culture medium suitable for high-density fermentation of Lactobacillus acidophilus CCFM137, which is obtained by improving the type of nitrogen source and the concentration of carbon and nitrogen sources from MRS culture medium, can improve the fermentation effect and nutrient utilization rate of Lactobacillus acidophilus, and is conducive to large-volume fermentation.

[0009] The improved culture medium comprises: (1) an improved culture medium A for fermentation of Lactobacillus acidophilus CCFM137 seed liquid, (2) an improved culture medium B without buffer salt for constant pH fermentation of Lactobacillus acidophilus CCFM137 in a fermentation tank;

[0010] in,

[0011] (1) Improved medium A for fermentation of Lactobacillus acidophilus CCFM137 seed liquid, comprising the following ingredients by weight: 3.8% glucose, 3.0% yeast extract FM503, 0.5% anhydrous sodium acetate, 0.5% K 2 HPO 4 0.2%, ammonium citrate 0.2%, MgSO 4 7H 2 O 0.01%, MnSO 4 ·H 2 O 0.005%, L-cysteine ​​hydrochloride 0.1%, Tween-80 0.1%, the balance is water;

[0012] (2) A modified medium B without buffer salt for constant pH fermentation of Lactobacillus acidophilus CCFM137 in a fermenter, wherein the raw materials include the following components by weight percentage: 3.4-3.8% glucose, 3.0% yeast extract FM503, MgSO 4 7H 2 O0.01%, MnSO 4 ·H 2 O 0.005%, L-cysteine ​​hydrochloride 0.05%, Tween-80 0.12%, and the balance water.

[0013] A second object of the present invention is to provide a Lactobacillus acidophilus CCFM137 fermentation tank fermentation method, which mainly comprises the following steps:

[0014] The seed liquid of Lactobacillus acidophilus CCFM137 is inoculated into the improved culture medium B at 37°C, anaerobic, fermentation time 10h, stirring speed 15r / min, and pH is maintained constant at 4.5-4.6 by adding 2mol / L sodium hydroxide solution during the fermentation process, providing a more suitable pH environment for the growth of Lactobacillus acidophilus, thereby achieving high-density fermentation of Lactobacillus acidophilus. Using 2mol / L sodium hydroxide solution can avoid uneven alkali supplementation caused by excessively high sodium hydroxide solution concentration, that is, excessively high pH in a short time in some areas, and can also avoid the need for a larger fermentation tank volume due to too low concentration.

[0015] [Beneficial Effects]

[0016] The present invention combines improved culture medium, clarified the optimal growth pH of the strain and controlled the pH environment during the fermentation process to be constant within a corresponding range, which can promote the rapid growth and reproduction of bacteria, thereby improving the fermentation level and efficiency, shortening the production cycle, reducing equipment investment and lowering production costs, and has important practical value.

[0017] The improved culture medium of the present invention simplifies the three nitrogen sources in MRS into yeast extract powder FM503, and adjusts the addition amount of carbon and nitrogen sources. It has the advantages of simple ingredients, simple preparation, and high utilization rate of carbon and nitrogen sources. Under the fermentation conditions of 20mL system containing buffer salt, the number of viable bacteria in the fermentation broth of the improved culture medium reaches 1.87×10 9 CFU / mL, which is 9.03 times higher than that of conventional MRS culture medium, the reducing sugar utilization rate is 52.2%, and the amino acid nitrogen utilization rate is 23.08%, which has certain guiding significance for the cultivation of Lactobacillus acidophilus in small systems.

[0018] The present invention uses a modified culture medium to culture Lactobacillus acidophilus CCFM137 in a 100L large system, and combines the fermentation conditions with a constant pH range of 4.5 to 4.6 to solve the problems of excessively long cell morphology, low viable count, and low freeze-drying survival rate caused by a constant pH of 5.7. The final viable count of the fermentation liquid in this method reaches 3.37×10 9 CFU / mL, which is 16.28 times higher than that of conventional MRS culture medium, the utilization rate of reducing sugar is as high as 88.38%, the utilization rate of amino acid nitrogen is 33.90%, and the freeze-dried survival rate is 77.83%, which is significantly higher than that at pH 5.7. This culture method realizes the pilot-scale production of Lactobacillus acidophilus, improves the utilization rate of raw materials in the production process, and the fermentation time is only 10 hours, which has certain guiding significance for the fermentation of Lactobacillus acidophilus of the same or larger scale. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Growth curve (A) and cell morphology (B) of Lactobacillus acidophilus CCFM137 in 20 mL of MRS medium and optimized medium.

[0020] Figure 2 The process flow of 100L fermentation.

[0021] Figure 3 Growth curves (A) and cell morphology (B) of Lactobacillus acidophilus CCFM137 at different constant pH values ​​under 100 L system and optimized culture medium conditions. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific implementation methods.

[0023] 1. The culture medium involved in the following comparative examples and embodiments is as follows:

[0024] (1) MRS medium: 2% glucose, 1% soy peptone, 1% beef extract, 0.5% yeast extract, 0.5% anhydrous sodium acetate, K 2 HPO 4 0.2%, diammonium hydrogen citrate 0.2%, MgSO 4 7H 2 O 0.01%, MnSO 4 ·H 2 O 0.005%, L-cysteine ​​hydrochloride 0.1%, Tween-80 0.1%, all of the above reagents were of analytical grade.

[0025] (2) Solid culture medium: MRS medium, 1.0% agar powder, used for strain streaking and counting.

[0026] (3) Improved medium A: glucose 3.4-3.8%, yeast extract FM503 3.0%, anhydrous sodium acetate 0.5%, diammonium hydrogen citrate 0.2%, K 2 HPO 4 0.2%, MgSO 4 7H 2 O 0.01%, MnSO 4 ·H 2 O 0.005%, Tween 80 0.1%, L-cysteine ​​hydrochloride 0.1%, all raw materials are food grade, and the rest is water. They are sterilized at 121°C for 15 min in an autoclave and used after cooling for the preparation of seed solution.

[0027] (4) Improved medium B without buffer salt: glucose 3.4-3.8%, yeast extract FM503 3.0%, MgSO 4 7H 2 O0.01%, MnSO 4 ·H 2 O 0.005%, L-cysteine ​​hydrochloride 0.05%, Tween-80 0.12%, all raw materials are food grade, and the rest is water. After sterilization and cooling, it is used for fermentation in a 100L fermenter.

[0028] Yeast extract powder FM503, FM502, FM985, FM803, FM860 are all produced by Angel Yeast Co., Ltd. and sold by Huaian Tianyulong Trading Co., Ltd. (17768293416). Analytical grade raw materials were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0029] 2. The detection methods involved in the following comparative examples and embodiments are as follows:

[0030] (1) Identification of Lactobacillus acidophilus CCFM137: 16S rDNA method was used to perform PCR amplification reaction with 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers. The obtained sequence results were compared with the NCBI database, and the homology result was 98.30%, which was confirmed to be Lactobacillus acidophilus.

[0031] (2) Determination of viable bacterial count: Counting by pouring plate method after gradient dilution.

[0032] (3) Determination of amino acid nitrogen in fermentation broth: Formaldehyde titration method was used. For details, see the reference: Cui Shumao. Factors and rules affecting growth inhibition and freeze-dried survival of lactic acid bacteria [D]. Jiangnan University, 2017.

[0033] Amino acid nitrogen utilization rate (%) = (initial amino acid nitrogen - remaining amino acid nitrogen) / initial amino acid nitrogen × 100%.

[0034] (4) Determination of reducing sugar in fermentation broth: 3,5-dinitrosalicylic acid (DNS) method. For details, see the reference: Wang Mingrui, Hou Yanxi, Zhang Yadan. Determination of polysaccharide content in fermentation broth by 3,5-dinitrosalicylic acid method [J]. Modern Food, 2020, 04(2): 16-222.

[0035] Reducing sugar utilization rate (%) = (initial reducing sugar concentration - residual reducing sugar concentration) / initial reducing sugar concentration × 100%.

[0036] (5) Determination of freeze-dried survival rate: The bacterial sludge collected by centrifugation was mixed with a protective agent to form an emulsion, and the emulsion was freeze-dried to form a freeze-dried powder. The emulsion (CFU / mL) and freeze-dried powder (CFU / g) were counted by the plate pouring method. The freeze-dried survival rate (%) = (live bacteria count of freeze-dried powder (CFU / g) × freeze-dried powder mass (g)) / (live bacteria count of emulsion (CFU / mL) × emulsion volume (mL)) × 100%.

[0037] 3. The culture methods involved in the following comparative examples and embodiments are as follows:

[0038] (1) Activation of strains:

[0039] Streak Lactobacillus acidophilus frozen at -80°C on a solid culture medium plate and culture it anaerobically at 37°C for 48 hours; pick a single colony and inoculate it in MRS culture medium and culture it anaerobically at 37°C for 24 hours, repeat the inoculation of 5% (v / v) into MRS culture medium for more than 3 times to obtain an activated fermentation liquid.

[0040] (2) Preparation of seed solution:

[0041] The activated fermentation liquid of Lactobacillus acidophilus in step (1) is inoculated into improved culture medium A. After 3 to 4 subcultures, it is inoculated into 5 to 7 L of improved culture medium A at an inoculum amount of 2% (v / v). After anaerobic culture at 37° C. for 9 hours, a seed liquid is obtained for fermentation in a 100 L system.

[0042] (3) Inoculation and fermentation:

[0043] 20mL system (Comparative Examples 1 to 19): The activated fermentation broth of step (1) was inoculated at a rate of 5% (v / v) into MRS and culture media containing different types and amounts of nitrogen sources, i.e., Comparative Examples 1 to 17. The fermentation temperature was 37°C, the anaerobic fermentation time was 12h, and the number of viable cells was determined after the fermentation was completed.

[0044] 100L system (Comparative Examples 20-25, Examples 1-3): The seed liquid of step (2) was inoculated into a 100L system (tank capacity 120L, liquid volume 100L) at an inoculum amount of 5-7% (v / v), the fermentation temperature was 37°C, the pH was constant, the nitrogen pressure was maintained, the rotation speed was 15r / min, the fermentation time was 10h, and samples were taken during the fermentation period for the determination of viable bacteria count, reducing sugar content and amino acid nitrogen.

[0045] (4) Bacteria collection and freeze-drying:

[0046] After the fermentation is completed, the cells are collected using a centrifuge, mixed with protective agents (maltodextrin, inulin, oligofructose, skim milk powder), and then freeze-dried for storage.

[0047] 4. The following comparative examples and examples used: Lactobacillus acidophilus CCFM1200, deposited in Guangdong Microbial Culture Collection Center, with a deposit number of GDMCC No: 62128; Lactobacillus acidophilus CCFM137, deposited in Yangzhou Jiangda Shisheng Yisheng Food Co., Ltd., sales link: https: / / item.jd.com / 10021424590527.html, product model: JUP#A7, is an active probiotic powder containing Lactobacillus acidophilus CCFM137.

[0048] Comparative Example 1

[0049] The conventional MRS culture system was used to culture Lactobacillus acidophilus CCFM137 in a 20 mL system. The specific components are shown in Table 1. In addition to the components in the table, the following components were included: 0.5% anhydrous sodium acetate, K 2 HPO 4 0.2%, diammonium hydrogen citrate 0.2%, MgSO4·7H2O 0.01%, MnSO4·H2O 0.005%, Tween-80 0.1%, L-cysteine ​​hydrochloride 0.1%.

[0050] (1) Activation of strains:

[0051] The Lactobacillus acidophilus CCFM137 frozen at -80°C was streaked onto a solid culture medium plate and anaerobically cultured at 37°C for 48 hours; a single colony was picked and inoculated into an MRS culture medium and anaerobically cultured at 37°C for 24 hours, and the inoculation into the MRS culture medium was repeated more than 3 times at an inoculation amount of 5% (v / v) to obtain an activated fermentation liquid.

[0052] (2) Inoculation and fermentation:

[0053] The activated fermentation liquid of step (1) was inoculated into MRS medium at a rate of 5% (v / v), the fermentation temperature was 37° C., the anaerobic fermentation time was 12 h, and the number of viable cells was determined after the fermentation was completed.

[0054] Comparative Examples 2 to 15

[0055] On the basis of Comparative Example 1, the type of nitrogen source was adjusted to form various comparative examples, as shown in Table 1.

[0056] Comparative Example 16

[0057] On the basis of Comparative Example 7, the addition amount of nitrogen source yeast extract powder FM503 was adjusted to 3.0%.

[0058] Comparative Example 17

[0059] On the basis of Comparative Example 16, the added amount of glucose was adjusted to 3.8%.

[0060] Comparative Example 18

[0061] On the basis of comparative example 1, Lactobacillus acidophilus CCFM1200 was used for the experiment.

[0062] (1) Activation of strains:

[0063] The Lactobacillus acidophilus CCFM1200 frozen at -80°C was streaked onto a solid culture medium plate and anaerobically cultured at 37°C for 48 hours; a single colony was picked and inoculated into an MRS culture medium and anaerobically cultured at 37°C for 24 hours, and the inoculation into the MRS culture medium was repeated more than 3 times at an inoculation amount of 5% (v / v) to obtain an activated fermentation liquid.

[0064] (2) Inoculation and fermentation:

[0065] The activated fermentation liquid of step (1) was inoculated into MRS medium at an inoculation rate of 5% (v / v), the fermentation temperature was 37° C., the anaerobic fermentation time was 12 h, and the number of viable cells was determined after the fermentation was completed.

[0066] Comparative Example 19

[0067] On the basis of Comparative Example 17, Lactobacillus acidophilus CCFM1200 was used for the experiment.

[0068] Table 1 20mL system comparative example raw materials and addition amount

[0069]

[0070] Table 2 Number of viable cells in 20 mL comparative fermentation broth

[0071]

[0072]

[0073] Comparative Example 20

[0074] Comparative Example 20: Lactobacillus acidophilus CCFM137 fermentation was carried out in a 100 L fermentation tank system with a constant pH of 5.7, as shown in Table 3.

[0075] (1) Activation of strains:

[0076] The Lactobacillus acidophilus CCFM137 frozen at -80°C was streaked onto a solid culture medium plate and anaerobically cultured at 37°C for 48 hours; a single colony was picked and inoculated into an MRS culture medium and anaerobically cultured at 37°C for 24 hours, and the inoculation into the MRS culture medium was repeated more than 3 times at an inoculation amount of 5% (v / v) to obtain an activated fermentation liquid.

[0077] (2) Preparation of seed solution:

[0078] The activated fermentation liquid of Lactobacillus acidophilus CCFM137 in step (1) is inoculated into improved culture medium A. After 3 to 4 subcultures, it is inoculated into 5 to 7 L of improved culture medium A at an inoculum amount of 2% (v / v). After anaerobic culture at 37° C. for 9 hours, a seed liquid is obtained for fermentation in a 100 L system.

[0079] (3) Inoculation and fermentation:

[0080] The seed liquid of step (2) is inoculated into a 100L system at an inoculum amount of 5-7% (v / v) (5-7L), the fermentation temperature is 37°C, the pH is constant at 5.7, the nitrogen pressure is maintained, the rotation speed is 15r / min, the fermentation time is 10h, and samples are taken during the fermentation to determine the viable bacteria count, reducing sugar content and amino acid nitrogen.

[0081] Comparative Example 21

[0082] Comparative Example 21 differs from Comparative Example 20 in that the constant pH of Comparative Example 21 is 4.7.

[0083] Comparative Example 22

[0084] Comparative Example 22 differs from Comparative Example 21 in that the constant pH of Comparative Example 22 is 4.4.

[0085] Comparative Example 23

[0086] Comparative Example 23 differs from Comparative Example 22 in that the constant pH of Comparative Example 23 is 4.3.

[0087] Comparative Example 24

[0088] The difference between Comparative Example 24 and Comparative Example 20 is that Comparative Example 24 uses Lactobacillus acidophilus CCFM1200 and the constant pH is 5.7.

[0089] Comparative Example 25

[0090] Comparative Example 25 differs from Comparative Example 24 in that the constant pH of Comparative Example 25 is 4.5.

[0091] Example 1

[0092] The difference between Example 1 and Comparative Example 23 is that the constant pH of Example 1 is 4.6, and the strain is Lactobacillus acidophilus CCFM137.

[0093] Example 2

[0094] The difference between Example 2 and Example 1 is that the constant pH of Example 2 is 4.5.

[0095] Example 3

[0096] The difference between Example 3 and Example 2 is that the amount of glucose added in Example 3 is 3.4%.

[0097] Table 3 100L system comparative examples and examples raw materials and addition amounts, fermentation time

[0098]

[0099] Table 4 Live bacterial count, reducing sugar utilization rate and amino acid nitrogen utilization rate of fermentation broth in 100L system comparative example

[0100]

[0101] Table 5 Live bacteria count, reducing sugar utilization rate and amino acid nitrogen utilization rate of fermentation broth in 100L system example

[0102]

[0103] From Comparative Example 1, it can be seen that the number of viable bacteria in the fermentation broth was 2.07×10 8 CFU / mL; Comparative Examples 2 to 15 used different nitrogen sources. The results showed that Comparative Example 7 had the best fermentation effect, in which the nitrogen source used was yeast extract powder FM503, and the number of viable bacteria in the fermentation liquid was 9.90×10 8 CFU / mL; Comparative Example 16 increased the amount of yeast extract FM503 added to 3% compared with Comparative Example 7, and the number of viable bacteria in the fermentation liquid was 1.49×10 9 CFU / mL; Comparative Example 17 increased the amount of glucose added to 3.8% compared to Comparative Example 16, and the number of viable bacteria after culture was stabilized at 1.87×10 9 CFU / mL increased by 9.03 times compared with MRS medium, and the cells were significantly shortened.

[0104] Comparative Example 18 is the same as Comparative Example 1. Lactobacillus acidophilus CCFM1200 is cultured using MRS medium. The number of viable bacteria after culture is stable at 6.30×10 8 CFU / mL; The culture of comparative example 19 is the same as that of comparative example 17, but Lactobacillus acidophilus CCFM1200 is used, and the number of viable bacteria after culture is stable at 1.75×10 9 CFU / mL only increased by 2.78 times compared with MRS culture medium.

[0105] The 100L fermentation tank system is a constant pH fermentation. By adding 2 mol / L sodium hydroxide solution to remove acid inhibition, the bacteria can continue to proliferate, and a better fermentation effect can be obtained. In this system, the constant pH value of comparative example 20 is 5.7, and the final viable count of Lactobacillus acidophilus CCFM137 in the fermentation liquid is 7.60×10 8 CFU / mL, the utilization rate of reducing sugar was 67.20%, the utilization rate of amino acid was 24.45%, and the number of viable bacteria in freeze-dried powder was 1.91×10 10 CFU / g, the freeze-dried survival rate is only 45.75%; Figure 3 It can be seen that under this pH condition, the addition of NaOH solution will make the cells of Lactobacillus acidophilus CCFM137 appear chain-like, and the number of viable bacteria is significantly lower than that of Comparative Example 17, which indicates that a constant pH of 5.7 does not have a good fermentation effect; Figure 1 The growth curve of Lactobacillus acidophilus CCFM137 in the buffered salt-modified culture medium shows that it has the fastest growth rate in 4 to 10 hours, and the corresponding pH range is 4.1 to 5.3, indicating that Lactobacillus acidophilus can obtain better fermentation effect within this pH range.

[0106] In Comparative Example 21, the constant pH value was reduced to 4.7, and the pH was still maintained with 2 mol / L NaOH solution. The final number of viable bacteria in the fermentation broth was 2.53×10 9 CFU / mL, the utilization rate of reducing sugar was 90.53%, the utilization rate of amino acid was 28.18%, and the number of viable bacteria in lyophilized powder was 4.95×10 10 CFU / g, the freeze-dried survival rate was 46.97%. Compared with comparative example 20, the cell division was more complete, the cells were significantly shortened, and the number of viable bacteria increased significantly.

[0107] The constant pH value of comparative example 22 was 4.4, and the number of viable bacteria in the final fermentation liquid was 2.46×10 9 CFU / mL, the utilization rate of reducing sugar was 82.04%, the utilization rate of amino acid was 34.23%, and the number of viable bacteria in lyophilized powder was 8.47×10 10 CFU / g, and the freeze-dried survival rate was 84.07%.

[0108] The constant pH value of Comparative Example 23 was 4.3, and the final number of viable bacteria in the fermentation liquid was 2.57×10 9 CFU / mL, the utilization rate of reducing sugar was 82.89%, the utilization rate of amino acid was 29.90%, and the number of viable bacteria in lyophilized powder was 6.05×10 10 CFU / g, and the freeze-dried survival rate was 61.64%.

[0109] Comparative Example 24 was fermented with Lactobacillus acidophilus CCFM1200 on the basis of Comparative Example 20, with a constant pH value of 5.7 and a final viable count of 9.0×10 8 CFU / mL, the utilization rate of reducing sugar was 55.58%, the utilization rate of amino acid was 21.11%, and the number of viable bacteria in lyophilized powder was 2.27×10 10 CFU / g, and the freeze-dried survival rate was 58.87%.

[0110] The difference between Comparative Example 25 and Comparative Example 24 is that the constant pH value is 4.5, Lactobacillus acidophilus CCFM1200 is still used, and the final viable bacterial count of the fermentation liquid is 3.23×10 9 CFU / mL, the utilization rate of reducing sugar was 91.76%, the utilization rate of amino acid was 31.67%, and the number of viable bacteria in lyophilized powder was 1.73×10 11 CFU / g, and the freeze-dried survival rate was 80.18%, which shows that Lactobacillus acidophilus CCFM1200 can also obtain better results under this pH condition than at pH 5.7, but compared with pH 5.7, the number of viable bacteria only increased by 3.59 times, and compared with MRS medium, it only increased by 5.13 times. This shows that this method does not improve the fermentation effect of CCFM1200 as much as CCFM137. This method is a high-density fermentation technology that is more suitable for the latter, but it also has a certain improvement effect on the fermentation of other Lactobacillus acidophilus strains.

[0111] The constant pH value of Example 1 was 4.6, and the final number of viable bacteria in the fermentation broth was 2.75×10 9 CFU / mL, the utilization rate of reducing sugar was 88.38%, the utilization rate of amino acid was 28.73%, and the number of viable bacteria in lyophilized powder was 5.45×10 10 CFU / g, the freeze-drying survival rate was 50.68%, and the fermentation effect was significantly improved compared with comparative example 21.

[0112] The constant pH value of Example 2 was 4.5, and the final number of viable bacteria in the fermentation broth was 3.37×10 9 CFU / mL, the utilization rate of reducing sugar was 83.93%, the utilization rate of amino acid was 33.90%, and the number of viable bacteria in lyophilized powder was 1.32×10 11CFU / g, and the freeze-dried survival rate was 77.83%. The fermentation effect was significantly improved compared with Example 1, and the freeze-dried survival rate was also significantly improved.

[0113] The constant pH value of Example 3 was 4.5, the amount of glucose added was 3.4%, and the final number of viable bacteria in the fermentation broth was 3.16×10 9 CFU / mL, the utilization rate of reducing sugar was 86.62%, the utilization rate of amino acid was 30.72%, and the number of viable bacteria in lyophilized powder was 1.05×10 11 CFU / g, the freeze-drying survival rate was 75.84%, and the number of viable fermentation bacteria was not significantly different from that in Example 2.

[0114] Based on comparative examples 20 to 23 and examples 1 to 3, Lactobacillus acidophilus CCFM137 obtained a better fermentation effect in examples 1 to 3, and its highest viable count was 16.28 times higher than that of the MRS culture medium in comparative example 1, and 4.43 times higher than that of comparative example 18. Comparative example 25 also shows that the fermentation effect of Lactobacillus acidophilus CCFM1200 can also be improved under this condition. The above results show that using an optimized culture medium in a fermenter and keeping the pH constant in the range of 4.5 to 4.6 can achieve the best fermentation effect of Lactobacillus acidophilus CCFM137.

[0115] This experiment found that when the constant pH was 5.7, Lactobacillus acidophilus CCFM137 would have problems such as excessive cell growth, insufficient division, and low viable count. However, unexpectedly, when the constant pH was in the range of 4.5 to 4.6, it was clearly observed that the cell division was more complete and the viable count increased significantly, indicating that the optimal growth pH range of Lactobacillus acidophilus CCFM137 is 4.5 to 4.6, which also provides a new fermentation idea for strains that have poor fermentation effects at pH 5.7.

[0116] 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. An improved culture medium for high-density fermentation of Lactobacillus acidophilus, characterized in that: include: (1) a modified medium A used as a Lactobacillus acidophilus seed culture medium, (2) a modified medium B without buffer salts used for constant pH fermentation of Lactobacillus acidophilus in a fermenter; in, (1) Improved medium A, comprising the following ingredients by weight: 3.8% glucose, 3.0% yeast extract FM50, 0.5% anhydrous sodium acetate, 0.2% K2HPO4, 0.2% ammonium citrate, 0.01% MgSO4·7H2O, 0.005% MnSO4·H2O, 0.1% L-cysteine ​​hydrochloride, 0.1% Tween-80, and the balance water; (2) Improved culture medium B, comprising the following ingredients by weight: 3.4-3.8% glucose, 3.0% yeast extract FM503, 0.01% MgSO4·7H2O, 0.005% MnSO4·H2O, 0.05% L-cysteine ​​hydrochloride, 0.12% Tween-80, and the balance water.

2. The improved culture medium for high-density fermentation of Lactobacillus acidophilus according to claim 1, characterized in that: The Lactobacillus acidophilus is Lactobacillus acidophilus CCFM137.

3. The improved culture medium for high-density fermentation of Lactobacillus acidophilus according to claim 1, characterized in that: The constant pH is pH 4.5-4.

6.

4. A method for culturing Lactobacillus acidophilus CCFM137 using the improved culture medium according to any one of claims 1 to 3, characterized in that: The main steps include: The seed liquid of Lactobacillus acidophilus CCFM137 was inoculated into the improved culture medium B and fermented anaerobically at 37° C. During the fermentation, a 2 mol / L sodium hydroxide solution was added to maintain the pH value constant at 4.5-4.

6.

5. The method according to claim 4, characterized in that Fermentation time: 10h, stirring speed: 15r / min.

6. The method according to claim 4 or 5, characterized in that: The inoculation amount is 100L of improved culture medium B with 5 to 7L of seed solution.

7. The method according to claim 6, characterized in that The preparation method of the seed solution comprises the following steps: (1) Activation of strains: Streak Lactobacillus acidophilus CCFM137 frozen at -80°C on a solid culture medium plate and culture it anaerobically at 37°C for 48 hours; pick a single colony and inoculate it in MRS culture medium and culture it anaerobically at 37°C for 24 hours, and repeat the inoculation of 5% in the MRS culture medium for more than 3 times to obtain an activated fermentation liquid; (2) Preparation of seed solution: The activated fermentation liquid of Lactobacillus acidophilus CCFM137 in step (1) is inoculated into improved culture medium A. After 3-4 subcultures, the inoculation amount is inoculated into 5-7 L of improved culture medium A at 2%. After anaerobic culture at 37° C. for 9 hours, the seed liquid is obtained.

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