Bifidobacterium lactis fermentation medium taking water-soluble component of corn steep liquor dry powder as nitrogen source
By replacing the nitrogen source in traditional culture medium with water-soluble ingredients of corn slurry dry powder, cheap Bifidobacterium lactis fermentation medium is prepared, which solves the problems of high cost, low survival rate and inorganic salt components, and achieves health and economicality fermentation effect of Bifidobacter lactis.
Patent Information
- Application Number
- CN202310850755.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-12
- Publication Date
- 2025-01-14
AI Technical Summary
The existing B. lactobacillus culture medium such as MRS medium is costly, has low survival rate, contains many inorganic salt components that do not conform to the low-salt health concept, and produces irritating odors during high-temperature sterilization, which cannot effectively solve the problem of B. lactobacillus being scaled up. Costs in production and health food requirements.
Use corn slurry dry powder water-soluble ingredients to replace some or all beef soaking powder, yeast soaking powder, and peptone. The water-soluble ingredients are extracted through soaking, ultrasonic, filtering and centrifugation treatment, combining galactose oligosaccharide, yeast peptone and anhydrous sodium acetate. Adjust the pH value to prepare cheap Bifidobacterium lactis fermentation medium.
It reduces the production cost of fermentation of Bifidobacterium lactis, improves the survival rate of the strain and the health of the terminal products, conforms to the concept of low-salt health, and avoids the irritating odor during high-temperature sterilization.
Abstract
Description
Technical Field
[0001] The invention belongs to the field of fermentation engineering, and more specifically relates to a lactic bifidobacterium fermentation culture medium using water-soluble components of corn steep liquor dry powder as a nitrogen source. Background Art
[0002] Bifidobacterium lactis is one of the dominant bacteria in the intestines of humans and many mammals and is a common probiotic colonizing the human intestine. It is an important physiological bacterium in the intestines of humans and animals. It participates in a range of physiological processes, including immunity, nutrition, digestion, and protection, and plays a vital role. Bifidobacterium lactis can rapidly colonize the intestinal mucosa, forming a barrier of beneficial bacteria. A dense biofilm forms on the intestinal mucosa, preventing pathogenic bacteria from colonizing. The acetic and lactic acids produced by Bifidobacterium lactis lower the intestinal pH, inhibiting saprophytic bacteria. Bifidobacterium lactis produces bacteriocin-like proteins with a certain bactericidal effect. Bifidobacterium lactis ferments lactose to produce galactose, a component of cerebrospinal fluid in the brain's nervous system and closely linked to the rapid growth of the infant's brain after birth. This can improve the utilization of dairy products in patients with lactose intolerance.
[0003] Bifidobacterium lactis culture medium currently mostly adopts MRS culture medium, and MRS culture gene adopts nitrogen sources such as beef immersion powder, yeast immersion powder, peptone, etc., which is expensive, and the cost of large-scale production of terminal products is too high, which is not conducive to competition; Phosphate buffer in MRS culture medium easily reduces the survival rate of Bifidobacterium lactis during freeze drying; More inorganic salt components of MRS culture medium are brought into terminal products, which is contrary to the health concept of low salt in modern health food; L-cysteine hydrochloride high temperature sterilization in MRS culture medium produces irritating odor. Having said so many disadvantages of MRS culture medium, is there any solution? The present invention provides an answer to solve the above multiple problems, replaces part or all of beef immersion powder, yeast immersion powder, and peptone in MRS culture medium with corn steep liquor water-soluble components, and makes culture medium by using corn steep liquor water-soluble components, can solve the peculiar taste and smell of terminal products caused by the corn steep liquor macromolecular water-insoluble components that Bifidobacterium lactis cannot absorb, and due to the special nutritional components of corn steep liquor water-soluble components, the scheme does not need to add phosphate buffer, inorganic salts and L-cysteine hydrochloride. Corn steep liquor powder, a byproduct of corn processing, is inexpensive and easy to store and transport. In recent years, domestic manufacturers have obtained licenses to produce food ingredients, providing policy support for the use of the present invention in the food industry. As a cost-effective nitrogen source, corn steep liquor powder has broad prospects for application in various areas of lactic acid fermentation. Summary of the Invention
[0004] The purpose of the present invention is to provide a culture medium with a cheap nitrogen source for the fermentation production of Bifidobacterium lactis.
[0005] The present invention uses the water-soluble components of corn steep liquor powder as a high-quality nitrogen source to prepare the culture medium, and the water-soluble components of corn steep liquor powder are prepared by the following technical scheme: Take the formulated amount of corn steep liquor powder, soak it in 2-4 times the amount of purified water at room temperature for 1 hour, ultrasonicate it for 10 minutes (frequency 40KHZ), filter it with a 120-mesh filter, and then centrifuge it at 5000rpm. Discard the water-insoluble components, i.e. the centrifugal residue, and take the water-soluble clear liquid for use.
[0006] Preparation of fermentation medium: Add the formulated amount of galacto-oligosaccharide, glucose monohydrate, yeast peptone, anhydrous sodium acetate and purified water to the reserved water-soluble clear solution of corn steep liquor, stir evenly, sterilize at 121°C for 15 minutes, adjust the pH to 7.0 with sodium hydroxide after cooling, and set aside.
[0007] The present invention provides a method for fermenting Bifidobacterium lactis, which adopts the following technical solution: A method for fermenting Bifidobacterium lactis, comprising: Inoculating Bifidobacterium lactis strains onto a solid culture medium for activation; Transplanting the activated bacterial strain into a primary liquid culture medium for expansion; The expanded bacterial strain is transplanted into a secondary liquid culture medium for fermentation, and the fermentation liquid is filtered to obtain Bifidobacterium lactis bacterial slurry.
[0008] The formula of the secondary liquid culture medium is 10-20 g / L of galacto-oligosaccharide, 5-10 g / L of glucose monohydrate, 30-40 g / L of corn steep liquor powder, 5-10 g / L of yeast peptone, and 1-3 g / L of anhydrous sodium acetate.
[0009] The optimized formula of the secondary liquid culture medium is 15 g / L galacto-oligosaccharide, 7 g / L glucose monohydrate, 35 g / L corn steep liquor powder, 7 g / L yeast peptone, and 2 g / L anhydrous sodium acetate.
[0010] During the fermentation process, sodium hydroxide is added to the secondary liquid culture medium, and the pH of the secondary liquid culture medium is controlled at 5.5-6.5.
[0011] In a specific embodiment, the method includes: S1. Inoculate the bacterial strain onto solid culture medium at a volume ratio of 1:10, and then activate it in an oxygen-free environment at 37°C for 24 hours. S2, the activated bacteria were placed in a 60cm 2 The solid culture medium was inoculated with 300 mL of liquid culture medium in the same proportion and then transplanted into the first-level liquid culture medium, and then incubated in an oxygen-free environment at 37°C for 12 hours. S3. The expanded bacterial strain was transferred to a secondary liquid culture medium at an inoculum size of 1 vol%, and then fermented in an anaerobic environment at 37° C. for 15-20 hours. During the fermentation process, nitrogen was introduced into the fermentation tank, and 3M sodium hydroxide was added to the secondary liquid culture medium to control the pH of the secondary liquid culture medium at 5.5-6.5. S4, fermentation for more than 15h, take samples of the fermentation liquid and detect OD 600 , when OD 600 If the number of viable bacteria in the fermentation liquid does not increase, it can be judged as the end point of the fermentation and the fermentation is stopped. S5. Centrifuge the fermentation broth obtained in step S4 using a tubular centrifuge.
[0012] The next flow direction of the bacterial sludge is not discussed in this application.
[0013] By adopting the above technical solution, the fermentation and cultivation process of Bifidobacterium lactis is optimized, which is conducive to obtaining an ideal fermentation and cultivation effect of Bifidobacterium lactis.
[0014] In a specific embodiment, in S3, the secondary liquid culture medium is stirred at a speed of 60-100 rpm during fermentation.
[0015] In a specific embodiment, in S3, the pH value of the second liquid culture medium is controlled by adding sodium hydroxide.
[0016] By adopting the above technical solution, sodium hydroxide can effectively adjust the pH value of the secondary liquid culture medium.
[0017] In a specific embodiment, in S5, the culture medium is prepared using water-soluble components of corn slurry powder, and the resulting bacterial slurry culture medium has less residual substances. If the corn slurry powder is directly used to prepare the culture medium without differentiation, the fermentation process can also be completed, but the fiber, macromolecular protein and other water-insoluble substances in the corn slurry powder remain in the bacterial slurry, and the weight of the resulting bacterial slurry is 20%-30% more (including the extra adsorbed water), but the number of viable bacteria obtained is reduced. The adverse effects of the water-insoluble residue of corn slurry powder on subsequent processes are also reflected in the following aspects: (1) the excessive amount of protective agent required for the bacterial slurry increases energy consumption during freeze-drying, increasing costs; (2) the water-insoluble residue of corn slurry powder has a special taste of corn slurry powder, which affects the taste of the freeze-dried product.
[0018] In a specific embodiment, the primary liquid culture medium is prepared using a conventional MRS culture medium formula according to a conventional method. The primary liquid culture medium has a small dosage and a small cost share, and has little impact on the cost of the final product, and the present invention does not provide a detailed description.
[0019] In summary, the present application has at least one of the following beneficial technical effects.
[0020] 1. The present invention uses cheap corn steep liquor powder to replace or partially replace expensive beef extract powder, yeast extract powder, and peptone in the fermentation medium of Bifidobacterium lactis, which greatly reduces the material cost input during the fermentation production of Bifidobacterium lactis, thereby reducing the cost of large-scale industrial production of Bifidobacterium lactis.
[0021] 2. The present application prepares a lactic acid Bifidobacterium fermentation medium through the water-soluble components of corn steep liquor powder. The water-soluble components of corn steep liquor powder contain a large amount of amino acid components, which have a good buffering effect on acid and alkali. The culture medium does not need to add phosphate, which is beneficial to improving the survival rate of live bacteria during subsequent freeze-drying.
[0022] 3. The present application prepares a Bifidobacterium lactis fermentation medium through the water-soluble components of corn steep liquor powder. The water-soluble components of corn steep liquor powder include inorganic salts such as calcium, magnesium, and manganese, which are necessary for the growth of Bifidobacterium lactis. No additional inorganic salts need to be added to the culture medium, and the end product complies with the modern low-salt health concept.
[0023] 4. The present application prepares a Bifidobacterium lactis fermentation medium by using the water-soluble components of corn steep liquor powder. The water-soluble components of corn steep liquor powder provide sufficient small molecule proteins and amino acids, so there is no need to add L-cysteine hydrochloride to the culture medium, and no irritating odor is generated during high-temperature sterilization. DETAILED DESCRIPTION
[0024] The present invention is further described in detail below with reference to the examples. Example 1
[0025] S1. Activation S1.1. Take Bifidobacterium lactis and inoculate it onto a solid slant culture medium at a volume ratio of 1:10. The solid slant culture medium is prepared by conventional methods from the following raw materials: 20g glucose, 5g yeast extract powder, 12g peptone, 2g triammonium citrate, 5g sodium acetate, 2g potassium dihydrogen phosphate, 0.07g magnesium sulfate, 0.5g potassium chloride, 0.5g Tween-80, 20g agar, and 1000g water. S1.2. Isolate the sample from oxygen and incubate at 37°C for 24 hours to activate the bacteria.
[0026] S2. Expansion training S2.1, the colony of Bifidobacterium lactis activated by S1 was placed on a 60cm 2 The solid culture medium was inoculated with 300 mL of liquid culture medium and then transferred to 100 mL of the first-stage liquid culture medium and mixed evenly. The first-stage liquid culture medium was prepared using conventional MRS medium formula and conventional methods. S2.2. Isolate oxygen; incubate the liquid culture medium at 37°C for 12 hours to further activate and expand the bacteria.
[0027] S3, Fermentation S3.1. Transplant the cultured Bifidobacterium lactis strain cultured in S2 into a secondary liquid culture medium at an inoculum volume of 1 vol% and mix well. The secondary liquid culture medium is a water-soluble culture medium made of corn steep liquor powder, which is prepared by conventional methods from the following raw materials: 15 g / L galacto-oligosaccharide, 7 g / L glucose monohydrate, 35 g / L corn steep liquor powder, 7 g / L yeast peptone, and 2 g / L anhydrous sodium acetate. The corn steep liquor powder is pre-treated before being used to prepare the culture medium, and its water-soluble components are extracted. S3.2. Place the liquid culture medium obtained in S3.1 in a fermenter and ferment at 37°C in an anaerobic environment at a stirring speed of 80 rpm for 15 hours. During this period, the pH of the liquid culture medium is controlled at 6.0 using 3M sodium hydroxide.
[0028] S4, fermentation for more than 15h, take samples of the fermentation liquid and detect OD 600 , when OD 600 If it no longer increases, it can be judged as the fermentation end point and fermentation is stopped.
[0029] S5. Centrifuge the fermentation broth obtained in step S4 using a tubular centrifuge. Example 2
[0030] The operation steps of Example 2 are the same as those of Example 1. When preparing the secondary liquid culture medium, corn steep liquor powder is added as a feed and the medium is pre-treated by water dissolution and centrifugation.
[0031] Specifically: the secondary liquid culture medium is prepared from the following raw materials according to a conventional method: 15 g / L galacto-oligosaccharide, 7 g / L glucose monohydrate, 45 g / L corn steep liquor powder, 7 g / L yeast peptone, and 2 g / L anhydrous sodium acetate. Example 3
[0032] The operation steps of Example 3 are the same as those of Example 1, except that the amount of corn steep liquor powder added is reduced during the preparation of the secondary liquid culture medium, and the medium is pre-treated by water-soluble centrifugation.
[0033] Specifically: the secondary liquid culture medium is prepared from the following raw materials according to a conventional method: 15 g / L galacto-oligosaccharide, 7 g / L glucose monohydrate, 25 g / L corn steep liquor powder, 7 g / L yeast peptone, and 2 g / L anhydrous sodium acetate. Example 4
[0034] The operating steps of Example 4 are the same as those of Example 1, except that phosphate buffered saline is added during the preparation of the secondary liquid culture medium.
[0035] Specifically: The secondary liquid culture medium is prepared from the following raw materials according to a conventional method: 15 g / L galacto-oligosaccharide, 7 g / L glucose monohydrate, 35 g / L corn steep liquor powder, 7 g / L yeast peptone, 2 g / L anhydrous sodium acetate, 2 g / L diammonium hydrogen citrate, and 2 g / L potassium dihydrogen phosphate. Example 5
[0036] The operating steps of Example 5 are the same as those of Example 1, except that magnesium and manganese inorganic salts are added during the preparation of the secondary liquid culture medium.
[0037] Specifically: The secondary liquid culture medium is prepared from the following raw materials according to a conventional method: 15 g / L galacto-oligosaccharide, 7 g / L glucose monohydrate, 35 g / L corn steep liquor powder, 7 g / L yeast peptone, 2 g / L anhydrous sodium acetate, 2 g / L diammonium hydrogen citrate, and 2 g / L potassium dihydrogen phosphate. Comparative Example 1
[0038] Comparative Example 1 and Example 1 have the same operating steps, except that: the corn steep liquor powder is not pretreated when preparing the secondary liquid culture medium, and the culture medium contains water-soluble components and water-insoluble components of the corn steep liquor powder. Comparative Example 2
[0039] The operation steps of Comparative Example 2 were the same as those of Example 1, except that the secondary liquid culture medium was replaced with ordinary MRS culture medium.
[0040] Specifically: The secondary liquid culture medium adopts the ordinary MRS culture medium formula and is prepared by the following raw materials according to the conventional method: beef extract 15 g / L, glucose 15 g / L, lactose 5 g / L, peptone 10 g / L, diammonium hydrogen citrate 2 g / L, potassium dihydrogen phosphate 2 g / L, manganese sulfate 0.05 g / L, magnesium sulfate 0.1 g / L, sodium acetate 2 g / L, and Tween-80 1 g / L.
[0041] Ordinary MRS culture medium can ensure the normal growth of Bifidobacterium lactis, but the formula is not optimized according to the growth characteristics of Bifidobacterium lactis.
[0042] Performance testing
[0043] Take the fermentation end point bacterial solution of Bifidobacterium lactis obtained in Example 1-5 and Comparative Example 1-2 and measure OD 600 The test results are listed in Table 1.
[0044] Absorbance (OD) Determination: Transfer 200 μL of each bacterial solution to a 96-well microtiter plate and measure the absorbance at 600 nm using a UV spectrophotometer. A higher absorbance indicates a higher bacterial density, indicating a better fermentation of Bifidobacterium lactis.
[0045] Table 1 OD of bacterial suspension obtained in Examples 1-5 and Comparative Examples 1-2 600 value project <![CDATA[0D 600 Value]]> Example 1 1.58 Example 2 1.60 Example 3 1.25 Example 4 1.55 Example 5 1.52 Comparative Example 1 1.65 Comparative Example 2 1.50
[0046] See Table 1, OD 600 It is mainly used for the detection of the fermentation end point. In the examples and comparative examples, we can use it to understand the proliferation of Bifidobacterium lactis. 600 The value is lower than 1.25, and the OD of other examples and comparative examples is 600 The values are all greater than 1.5 and are relatively close. According to experience and the microplate reader used in the experiment, it is shown that the growth of Bifidobacterium lactis during the fermentation process of Example 1, Implementation 2, Example 4, Example 5, Comparative Example 1 and Comparative Example 2 is good.
[0047] Live bacteria detection
[0048] The bacterial liquid obtained from the fermentation end point of Bifidobacterium lactis obtained in Examples 1-5 and Comparative Examples 1 and 2 was used to detect the viable bacteria of Bifidobacterium lactis. The detection results are shown in Table 2.
[0049] Table 2 The number of viable bacteria in the bacterial solution obtained in Examples 1-5 and Comparative Examples 1-2 project Viable CFU / ml Example 1 <![CDATA[1.4×10 10 ]]> Example 2 <![CDATA[1.3×10 10 ]]> Example 3 <![CDATA[7×10 9 ]]> Example 4 <![CDATA[1.3×10 10 ]]> Example 5 <![CDATA[1.2×10 10 ]]> Comparative Example 1 <![CDATA[1.2×10 10 ]]> Comparative Example 2 <![CDATA[9×10 9 ]]>
[0050] See Table 2, Example 3 viable count combined with OD 600Value situation, Bifidobacterium lactis grows poorly during fermentation, and the corn steep liquor powder addition in the culture medium of embodiment 3 is too little to hinder the proliferation of Bifidobacterium lactis; Comparing embodiment 1 and embodiment 2, we correspondingly increase the corn steep liquor powder addition in the culture medium in embodiment 2, embodiment 2 viable count and embodiment 2 viable count same level, from economic point of view, it is more appropriate to use the corn steep liquor powder amount in the culture medium of embodiment 1. Phosphate buffer salt was added in embodiment 4, but the viable count as a result is at the same level as embodiment 1, and experimental result confirms that corn steep liquor powder water-soluble component culture medium can not add phosphate buffer salt. With the same aspect, we added magnesium, manganese inorganic salt in embodiment 5, but the viable count as a result is at the same level as embodiment 1, and experimental result confirms that corn steep liquor powder water-soluble component culture medium can not add magnesium, manganese inorganic salt. Comparative example 1 is a culture medium scheme that does not distinguish corn steep liquor water-soluble component and water-insoluble component, also is our working culture medium before culture medium is improved, although Bifidobacterium lactis also can normal growth, but compare fermentation broth viable count with embodiment 1 almost the same, but just can compare and find bacterium mud more than embodiment 1 after follow-up centrifugation, the viable count of bacterium mud is compared with embodiment 1, and its shortcoming we have made explanation in the foregoing text.Contrast 2 adopts common MRS culture medium formula, because test is not optimized for Bifidobacterium lactis characteristics, its fermentation result is slightly inferior to embodiment 1 comparison, and result explanation corn steep liquor water-soluble component culture medium proliferation Bifidobacterium lactis is feasible.
[0051] in conclusion The present invention provides a Bifidobacterium lactis fermentation medium using a water-soluble component of corn steep liquor powder as a nitrogen source. The fermentation medium can effectively support the growth and metabolism of Bifidobacterium lactis, has low cost and good feasibility. Through the application of the present invention, the goal of sustainable utilization of resources and increased yield can be achieved in Bifidobacterium lactis production.
[0052] The embodiments and comparative examples are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.
Claims
1. A fermentation medium for Bifidobacterium lactis using water-soluble components of corn steep liquor powder as a main nitrogen source, characterized in that: Culture medium formula: galacto-oligosaccharide 10-20 g / L, glucose monohydrate 5-10 g / L, corn steep liquor powder 30-40 g / L, yeast peptone 5-10 g / L, anhydrous sodium acetate 1-3 g / L.
2. The fermentation medium of Bifidobacterium lactis using corn steep liquor powder water-soluble components as main nitrogen source according to claim 1, characterized in that: Culture medium formula: galacto-oligosaccharide 15 g / L, glucose monohydrate 7 g / L, corn steep liquor powder 35 g / L, yeast peptone 7 g / L, anhydrous sodium acetate 2 g / L.
3. The fermentation medium of Bifidobacterium lactis using corn steep liquor powder water-soluble components as main nitrogen source according to claim 1, characterized in that: The culture medium formula uses the water-soluble components of corn slurry powder as the main nitrogen source. The water-soluble cost of corn slurry powder is prepared by using corn slurry powder as the main raw material. The process is to soak the corn slurry powder with 2-4 times the amount of purified water at room temperature for 1 hour, ultrasonicate for 10 minutes (frequency 40KHZ), centrifuge (5000rpm), take the water-soluble clear liquid to prepare the culture medium, and discard the water-insoluble components, i.e. the centrifugal residue.
4. The corn syrup dry powder pretreatment process according to claim 3, characterized in that: The culture medium is made of water-soluble components of corn syrup powder. The water-soluble components of corn syrup powder mainly include small molecule proteins, amino acids, vitamins, calcium, magnesium, etc., which provide the above nutrients for the growth of Bifidobacterium lactis; the water-insoluble components mainly include large molecule proteins and cellulose, which cannot be absorbed and utilized by the growth of Bifidobacterium lactis. The weight of the bacterial sludge will increase when it is collected, and the corresponding number of live bacteria will be passively diluted and decreased due to the increase in weight; removing the water-insoluble components can also remove the special taste of corn syrup powder and bring it into the final product.
5. The corn steep liquor dry powder pretreatment process according to claim 3 and the corn steep liquor dry powder water-soluble component preparation culture medium according to claim 4, characterized in that: Corn syrup powder is water-soluble and contains inorganic salts, so the culture medium does not need to add additional sodium, magnesium, manganese and other inorganic salts; it contains a large number of small molecule proteins and amino acids that form a good acid-base buffer, so the culture medium does not need to add phosphate as an acid-base buffer.
6. The corn steep liquor dry powder pretreatment process according to claim 3 and the corn steep liquor dry powder water-soluble component preparation culture medium according to claim 4, characterized in that: The culture medium uses the water-soluble components of corn syrup powder as a nitrogen source, and the properties and odor of the end product are similar to those of the end product of MRS culture medium fermentation. The end product has no corn syrup powder insoluble residues and no special odor of corn syrup powder.