Bifidobacterium culture medium and culture and preservation method based on symbiotic nutrition method
By optimizing the ratio of Bifidobacterium culture medium and using symbiotic nutrition method, the problem of low growth and survival rate of Bifidobacterium during culture and preservation is solved, and the effect of improving growth rate, yield and survival rate is achieved.
Patent Information
- Application Number
- CN202411803531.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-07
AI Technical Summary
Bifidobacterium has low growth rate, poor yield during culture, and low survival rate during storage, resulting in a short shelf life.
Provide a Bifidobacterium culture medium, including peanut meal, sucrose, yeast extract, Cigu powder, loofah slurry and onion water extract, etc., by optimizing the raw materials and dosage ratio of the culture medium, the cost is reduced and the nutritional supply is improved. At the same time, a culture method based on symbiotic nutrition method was adopted to promote the growth and survival of Bifidobacterium, Essentialis, and Pilocona lactic acid.
It improves the growth rate and yield of Bifidobacterium, enhances its adaptability to environmental factors, extends the shelf life, and significantly improves the survival rate of Bifidobacterium.
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Figure CN119570672A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of probiotics, and in particular relates to a bifidobacterium culture medium and a culture and preservation method based on a symbiotic nutrition method. Background Art
[0002] Bifidobacterium was originally discovered and named by French scientists. It was identified as a Gram-positive bacterium and is one of the main bacterial groups in the human intestinal microbial flora. Bifidobacterium has a rich enzyme system, rich in glucosidase, phosphoprotein phosphatase and combined bile acid hydrolase, which can promote the decomposition of substances into components that are easy for the human body to absorb. It also has physiological functions such as inhibiting the growth of intestinal pathogens, improving protein metabolism, providing vitamins, lowering cholesterol, and improving immune function. From birth to death, bifidobacteria accompany a person's life. When the human body is aging or in an unfavorable state such as illness, the bifidobacteria in the human body will decrease or even disappear. Therefore, as an excellent ecological strain, bifidobacteria have been developed into microecological preparations and are widely used in medical treatment, health care and food. However, because of the strict anaerobic characteristics of bifidobacteria, the culture conditions and nutrients are relatively strict, so that its culture results are not ideal. Therefore, it is urgent to provide a bifidobacterium culture method that can increase the growth rate of bifidobacteria during cultivation, increase the yield of bifidobacteria, and the survival rate during storage. Summary of the invention
[0003] In view of this, the object of the present invention is to provide a culture medium that can promote the growth and reproduction of bifidobacteria, improve the survival rate of the strains, and extend the shelf life.
[0004] Another object of the present invention is to provide an application of the culture medium in culturing and preserving bifidobacteria.
[0005] Another object of the present invention is to provide a method for culturing bifidobacteria based on symbiotic nutrition.
[0006] Another object of the present invention is to provide a fermentation liquid obtained by culturing the method.
[0007] Another object of the present invention is to provide a bifidobacterium freeze-dried powder and a preparation method thereof.
[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0009] The invention provides a culture medium, which comprises the following raw materials in 1L: 20-30g of peanut meal, 15-20g of sucrose, 5-10g of yeast extract, 10-15g of arrowhead powder, 5-10g of loofah slurry, 5-10g of onion water extract, 0.5-1g of sodium chloride, 2-3g of dipotassium hydrogen phosphate, 1-2g of potassium dihydrogen phosphate, 0.2-0.5g of magnesium sulfate, 0.05-0.1g of manganese chloride, and the balance is water.
[0010] Preferably, the pH of the culture medium is 6-7.
[0011] The present invention also provides an application of the culture medium in culturing and preserving bifidobacteria.
[0012] Preferably, the bifidobacterium is Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis or Bifidobacterium bifidum.
[0013] The present invention also provides a bifidobacterium culture method based on the symbiotic nutrition method, the culture method comprising the following steps: inoculating bifidobacteria, Leuconostoc mesenteroides and Pediococcus acidilactici into the culture medium and conducting closed culture.
[0014] Preferably, the ratio of the inoculated live bacteria of Bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici is 1-5:1-5:1-5.
[0015] Preferably, the culture conditions are: initial temperature 28-34° C. for 1-3 h; temperature raised to 35-40° C. for 16-30 h.
[0016] The present invention also provides a fermentation liquid obtained by culturing using the culturing method.
[0017] The present invention also provides a bifidobacterium freeze-dried powder, which is prepared from the fermentation liquid.
[0018] The present invention also provides a method for preparing the freeze-dried powder, which comprises the following steps: centrifuging the fermentation liquid, adding a protective agent, freeze-drying, and storing.
[0019] Beneficial effects of the present invention:
[0020] The culture medium of the present invention replaces raw materials with high costs such as beef extract and peptone with raw materials such as peanut meal and sucrose, and adds raw materials such as arrowhead powder, loofah pulp, onion water extract, etc., and optimizes the raw materials and dosage ratio of the culture medium, thereby not only reducing the cost of the culture medium, but also optimizing the nutrient supply of the culture medium, making the nutrient components of the culture medium more comprehensive and balanced, and the active ingredients therein can also enhance the adaptability of bifidobacteria to environmental factors, help bifidobacteria maintain physiological balance in cells, reduce the influence of environmental pressure on the growth of bifidobacteria, ensure the stability of growth, and improve the survival rate of bifidobacteria during subsequent storage.
[0021] The bifidobacterium cultivation method based on the symbiotic nutrition method of the present invention utilizes the synergistic symbiosis of bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici to promote the growth and reproduction of bifidobacterium; and by optimizing the raw materials and dosage ratio of the culture medium, not only can the different nutritional requirements of the three bacteria in the symbiotic cultivation process be met, the biologically active substances contained in the culture medium interact with each other, which is helpful to regulate the relationship between the three bacteria and enhance the symbiotic cooperation between them, but also the adaptability of the three bacteria to environmental factors can be enhanced. In the symbiotic cultivation process, the bacteria can be helped to maintain the physiological balance in the cells and reduce the influence of environmental pressure on the growth of the bacteria, thereby ensuring the stability of the symbiotic flora and the smooth progress of the cultivation process, improving the survival rate of the strains and extending the shelf life. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The viable counts of Bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici at different culture times in Example 4 are shown. DETAILED DESCRIPTION
[0023] The invention provides a culture medium, which preferably comprises the following raw materials in 1L: 20-30g of peanut meal, 15-20g of sucrose, 5-10g of yeast extract, 10-15g of arrowhead powder, 5-10g of loofah slurry, 5-10g of onion water extract, 0.5-1g of sodium chloride, 2-3g of dipotassium hydrogen phosphate, 1-2g of potassium dihydrogen phosphate, 0.2-0.5g of magnesium sulfate, 0.05-0.1g of manganese chloride, and the balance is water.
[0024] In the present invention, the arrowhead powder is the powder of the bulb of the arrowhead plant of the Alismataceae family, which can be purchased through conventional channels or prepared by conventional methods; as an optional embodiment, the arrowhead is dried and crushed to obtain; the drying and crushing methods are not particularly limited and can be conventionally selected according to actual needs. The sponge gourd slurry can be prepared by conventional methods; for example, the sponge gourd is peeled, cut into pieces, crushed and filtered, and the filtrate is collected. The preparation method of the onion water extract can be conventionally selected according to actual needs; as an optional embodiment, the onion is washed, dried, and crushed to obtain onion powder; the onion powder is mixed with water for leaching; the residue is removed by filtering and the filtrate is collected; the drying and crushing methods can be conventionally selected according to actual needs; the mass ratio of the onion powder to water is preferably 1:5-15, more preferably 1:8-12, and more preferably 1:10; the extraction time is preferably 8-16h, more preferably 10-14h, and more preferably 12h; after filtering to remove the residue, it is preferably filtered again with a 0.22μm filter membrane, and the collected filtrate is the onion water extract.
[0025] In the present invention, based on 1L of culture medium, the weight of the peanut meal is further preferably 22-27g, more preferably 25g; the weight of the sucrose is further preferably 16-19g, more preferably 18g; the weight of the yeast extract is further preferably 6-9g, more preferably 8g; the weight of the arrowhead powder is further preferably 11-13g, more preferably 12g; the weight of the sponge gourd slurry is further preferably 6-9g, more preferably 8g; the weight of the onion water extract is further preferably 6-9g, more preferably 8g; the weight of the sodium chloride is further preferably 0.6-0.9g, more preferably 0.8g; the weight of the dipotassium hydrogen phosphate is further preferably 2.2-2.7g, more preferably 2.5g; the weight of the potassium dihydrogen phosphate is further preferably 1.2-1.7g, more preferably 1.5g; the weight of the magnesium sulfate is further preferably 0.25-0.4g, more preferably 0.3g; the weight of the manganese chloride is further preferably 0.06-0.09g, more preferably 0.08g.
[0026] In the present invention, the preparation method of the culture medium can be conventionally selected according to actual needs, for example, the above raw materials are mixed according to weight ratio, sterilized, and cooled; the sterilization method can be conventionally selected according to actual needs. The pH of the culture medium is preferably 6 to 7, more preferably 6.2 to 6.7, and more preferably 6.5.
[0027] In the culture medium of the present invention, sucrose is used as the main carbon source, peanut meal and yeast extract are used as the main nitrogen sources, and arrowhead contains rich protein, carbohydrates, vitamins, minerals and other nutrients. In addition to being able to supplement the types of carbon sources and nitrogen sources and provide energy for bacterial growth metabolism, the various nutrients are also helpful in maintaining the normal structure and physiological function of bacterial cells and promoting bacterial growth and reproduction; the sponge gourd slurry contains a variety of amino acids, vitamins and minerals, which provides more comprehensive nutritional support for bacterial growth and helps to improve the growth rate and metabolic activity of bacteria. At the same time, the sponge gourd slurry contains biologically active substances, which can regulate the physical and chemical properties of the culture medium and the microbial growth. growth environment and enhance the adaptability of bacteria to the environment; onion water extract contains a variety of bioactive substances, which can be used as growth factors to stimulate bacterial growth, increase bacterial biomass and metabolic activity, and the oligosaccharides are not only prebiotics, but can also be used as a carbon source, which is conducive to bacterial growth and metabolism; dipotassium hydrogen phosphate and potassium dihydrogen phosphate can be used as buffers to maintain the pH stability of the culture medium, ensure that bacteria grow in a suitable acid-base environment, and provide mineral nutrients such as phosphorus; sodium chloride helps maintain the osmotic pressure balance of the culture medium; manganese chloride and magnesium sulfate provide bacteria with essential mineral elements such as manganese and magnesium, which helps maintain the normal physiological functions and enzyme activities of microorganisms. The invention replaces raw materials with high costs such as beef extract and peptone with raw materials such as peanut meal and sucrose, and adds raw materials such as arrowhead powder, loofah pulp, onion water extract, etc., and optimizes the raw materials and dosage ratio of the culture medium, thereby not only reducing the cost of the culture medium, but also optimizing the nutrient supply of the culture medium, making the nutrient components of the culture medium more comprehensive and balanced, and the active ingredients therein can also enhance the adaptability of bacteria to environmental factors, help bacteria maintain physiological balance in cells, reduce the influence of environmental pressure on bacterial growth, ensure growth stability, and improve the survival rate of strains during subsequent storage.
[0028] The present invention also provides an application of the culture medium in culturing and preserving bifidobacteria.
[0029] In the present invention, the type of bifidobacterium can be conventionally selected according to actual needs, preferably including Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis or Bifidobacterium bifidum, more preferably Bifidobacterium longum or Bifidobacterium animalis, and more preferably Bifidobacterium longum.
[0030] The culture medium of the present invention is used to cultivate and preserve bifidobacteria, which can not only meet the nutritional needs of bifidobacteria, promote their growth and reproduction and product formation process, increase growth rate and yield, but also enhance their adaptability to environmental factors, thereby improving their survival rate during subsequent storage.
[0031] The present invention also provides a bifidobacterium culture method based on the symbiotic nutrition method, the culture method comprising the following steps: inoculating bifidobacteria, Leuconostoc mesenteroides and Pediococcus acidilactici into the culture medium and conducting closed culture.
[0032] In the present invention, the Leuconostoc mesenteroides is preferably Leuconostoc mesenteroides CICC 22264; the Pediococcus acidilactici is preferably Pediococcus acidilactici CICC 10344.
[0033] In the present invention, the culture method preferably further comprises preparing seed solutions of Bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici respectively, and inoculating the seed solutions into the culture medium for closed culture. The method for preparing the seed solution is not particularly limited and can be conventionally selected according to actual needs. The viable bacteria concentration of the seed solution is preferably 1×10 5 ~1×10 7 cfu / mL, more preferably 5×10 5 ~5×10 6 cfu / mL, more preferably 1×10 6 cfu / mL; the inoculation amount of the seed solution of bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici can be conventionally selected according to actual needs. As an implementable embodiment, the total inoculation amount of the seed solution of bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici is 3% to 8% of the volume of the liquid culture medium, more preferably 4% to 6%, and more preferably 5%; the live bacteria ratio of the bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici is preferably 1 to 5:1 to 5:1 to 5, more preferably 2 to 4:3 to 5:1 to 2, and more preferably 3:5:2.
[0034] In the present invention, the culture is a closed culture, and there is no need to deliberately create an anaerobic environment. The culture conditions are preferably: an initial temperature of 28 to 34°C for 1 to 3 hours; a temperature rise to 35 to 40°C for 16 to 30 hours; an initial temperature is further preferably 29 to 32°C, more preferably 30°C; an initial culture time is further preferably 1.5 to 2.5 hours, more preferably 2 hours; a temperature rise is further preferably 36 to 38°C, more preferably 37°C; a temperature rise culture time is further preferably 18 to 28 hours, more preferably 24 hours.
[0035] The bifidobacterium cultivation method based on the symbiotic nutrition method of the present invention utilizes the synergistic symbiosis of bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici to promote the growth and reproduction of bifidobacterium; and by optimizing the raw materials and dosage ratio of the culture medium, not only can the different nutritional requirements of the three bacteria in the symbiotic cultivation process be met, the biologically active substances contained in the culture medium interact with each other, which is helpful to regulate the relationship between the three bacteria and enhance the symbiotic cooperation between them, but also the adaptability of the three bacteria to environmental factors can be enhanced. In the symbiotic cultivation process, the bacteria can be helped to maintain the physiological balance in the cells and reduce the influence of environmental pressure on the growth of the bacteria, thereby ensuring the stability of the symbiotic flora and the smooth progress of the cultivation process.
[0036] The present invention also provides a fermentation liquid obtained by culturing using the culturing method.
[0037] The present invention also provides a bifidobacterium freeze-dried powder, which is prepared from the fermentation liquid.
[0038] In the present invention, the preparation method of the freeze-dried powder preferably includes the following steps: centrifuging the fermentation broth, adding a protective agent, freeze-drying, and storing. The speed and time of the centrifugation can be conventionally selected according to actual needs, for example, centrifuging at 8000r / min for 30min to collect the bacteria; the protective agent can be selected from conventional protective agents in the art, including but not limited to sucrose, maltose, mannitol, sodium glutamate, skimmed milk powder, trehalose; the freeze-drying method can be conventionally selected according to actual needs. The freeze-dried powder is preferably stored frozen at -80°C, or refrigerated at 2-8°C, or can be stored at room temperature.
[0039] The bifidobacterium freeze-dried powder of the present invention has a high survival rate of bifidobacteria during storage. Even if stored at room temperature, the 180-day survival rate can still reach 21.53%, which can significantly improve the survival rate of bifidobacteria and extend the shelf life.
[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0041] In the following embodiments, unless otherwise specified, all of them are conventional methods.
[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0043] In the following examples, Bifidobacterium longum CICC 6202, Leuconostoc mesenteroides CICC 22264 and Pediococcus acidilactici CICC 10344 were purchased from China Industrial Microbiological Culture Collection Center; Bifidobacterium animalis CGMCC 1.1852 was purchased from Shanghai Collection Biotechnology Center.
[0044] Example 1
[0045] A culture medium, the culture medium components are: 25g / L peanut meal, 18g / L sucrose, 8g / L yeast extract, 12g / L arrowhead powder, 8g / L loofah slurry, 8g / L onion water extract, 0.8g / L sodium chloride, 2.5g / L dipotassium hydrogen phosphate, 1.5g / L potassium dihydrogen phosphate, 0.3g / L magnesium sulfate, 0.08g / L manganese chloride, and water is added to make the volume to 1L.
[0046] The arrowhead powder is obtained by drying and crushing the arrowhead;
[0047] The sponge gourd slurry is prepared by peeling and cutting the sponge gourd into pieces, crushing the pieces and filtering the pieces, and the filtrate is the sponge gourd slurry;
[0048] The onion water extract is prepared by washing, drying and crushing onions to obtain onion powder; mixing the onion powder with water in a mass ratio of 1:10, placing the mixture on a magnetic stirrer and stirring for 12 hours; filtering to remove residues, and filtering again using a 0.22 μm filter membrane, wherein the filtrate is the onion water extract.
[0049] Example 2
[0050] A culture medium, the culture medium components are: 30g / L peanut meal, 20g / L sucrose, 5g / L yeast extract, 10g / L arrowhead powder, 5g / L loofah slurry, 5g / L onion water extract, 0.5g / L sodium chloride, 3g / L dipotassium hydrogen phosphate, 2g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate, 0.1g / L manganese chloride, and water is added to make the volume to 1L.
[0051] The arrowhead powder is obtained by drying and crushing the arrowhead;
[0052] The sponge gourd slurry is prepared by peeling and cutting the sponge gourd into pieces, crushing the pieces and filtering the pieces, and the filtrate is the sponge gourd slurry;
[0053] The onion water extract is prepared by washing, drying and crushing onions to obtain onion powder; mixing the onion powder with water in a mass ratio of 1:5, placing the mixture on a magnetic stirrer and stirring for 16 hours; filtering to remove residues, and filtering again using a 0.22 μm filter membrane, wherein the filtrate is the onion water extract.
[0054] Example 3
[0055] A culture medium, the culture medium components are: 20g / L peanut meal, 15g / L sucrose, 10g / L yeast extract, 15g / L arrowhead powder, 10g / L loofah slurry, 10g / L onion water extract, 1g / L sodium chloride, 2g / L dipotassium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 0.2g / L magnesium sulfate, 0.05g / L manganese chloride, and water is added to make the volume to 1L.
[0056] The arrowhead powder is obtained by drying and crushing the arrowhead;
[0057] The sponge gourd slurry is prepared by peeling and cutting the sponge gourd into pieces, crushing the pieces and filtering the pieces, and the filtrate is the sponge gourd slurry;
[0058] The onion water extract is prepared by washing, drying and crushing onions to obtain onion powder; mixing the onion powder with water in a mass ratio of 1:15, placing the mixture on a magnetic stirrer and stirring for 8 hours; filtering to remove residues, and filtering again using a 0.22 μm filter membrane, wherein the filtrate is the onion water extract.
[0059] Comparative Example 1
[0060] A culture medium, the culture medium components are: 25g / L peanut meal, 18g / L sucrose, 8g / L yeast extract, 0.8g / L sodium chloride, 2.5g / L dipotassium hydrogen phosphate, 1.5g / L potassium dihydrogen phosphate, 0.3g / L magnesium sulfate, 0.08g / L manganese chloride, and water is added to make the volume to 1L.
[0061] The preparation methods of the arrowhead powder, loofah slurry and onion water extract are the same as those in Example 1.
[0062] Comparative Example 2
[0063] A culture medium, the culture medium components are: 25g / L peanut meal, 18g / L sucrose, 8g / L yeast extract, 8g / L loofah pulp, 8g / L onion water extract, 0.8g / L sodium chloride, 2.5g / L dipotassium hydrogen phosphate, 1.5g / L potassium dihydrogen phosphate, 0.3g / L magnesium sulfate, 0.08g / L manganese chloride, and water is added to make the volume to 1L.
[0064] The preparation methods of the arrowhead powder, loofah slurry and onion water extract are the same as those in Example 1.
[0065] Comparative Example 3
[0066] A culture medium, the culture medium components are: 25g / L peanut meal, 18g / L sucrose, 8g / L yeast extract, 12g / L arrowhead powder, 8g / L onion water extract, 0.8g / L sodium chloride, 2.5g / L dipotassium hydrogen phosphate, 1.5g / L potassium dihydrogen phosphate, 0.3g / L magnesium sulfate, 0.08g / L manganese chloride, and water is added to make the volume to 1L.
[0067] The preparation methods of the arrowhead powder, loofah slurry and onion water extract are the same as those in Example 1.
[0068] Comparative Example 4
[0069] A culture medium, the culture medium components are: 25g / L peanut meal, 18g / L sucrose, 8g / L yeast extract, 12g / L arrowhead powder, 8g / L loofah slurry, 0.8g / L sodium chloride, 2.5g / L dipotassium hydrogen phosphate, 1.5g / L potassium dihydrogen phosphate, 0.3g / L magnesium sulfate, 0.08g / L manganese chloride, and water is added to make the volume to 1L.
[0070] The preparation methods of the arrowhead powder, loofah slurry and onion water extract are the same as those in Example 1.
[0071] Example 4
[0072] A bifidobacterium cultivation method based on symbiotic nutrition:
[0073] The Bifidobacterium was Bifidobacterium longum CICC 6202, the Leuconostoc mesenteroides was Leuconostoc mesenteroides CICC 22264, and the Pediococcus acidilactici was Pediococcus acidilactici CICC 10344.
[0074] The seed solutions of Bifidobacterium longum, Leuconostoc mesenteroides, and Pediococcus acidilactici were prepared respectively, and the live bacterial concentration of the seed solutions was 1×10 6 cfu / mL; the seed solutions of Bifidobacterium longum, Leuconostoc mesenteroides and Pediococcus acidilactici were inoculated into the culture medium of Example 1 at an inoculum size of 1.5%, 2.5% and 1% (v / v), respectively, and the initial pH of the culture medium was adjusted to 6.5, and the culture was closed. The culture conditions were: initial temperature of 30°C for 2h, and then the temperature was raised to 37°C for 24h.
[0075] Example 5
[0076] A bifidobacterium cultivation method based on symbiotic nutrition:
[0077] The difference from Example 4 is that the culture medium of Example 1 is replaced by the culture medium of Example 2, and the other methods remain unchanged.
[0078] Example 6
[0079] A bifidobacterium cultivation method based on symbiotic nutrition:
[0080] The difference from Example 4 is that the culture medium of Example 1 is replaced by the culture medium of Example 3, and the other methods remain unchanged.
[0081] Example 7
[0082] A bifidobacterium cultivation method based on symbiotic nutrition:
[0083] The difference from Example 4 is that Bifidobacterium animalis CGMCC 1.1852 is used to replace Bifidobacterium longum CICC6202, and the other methods remain unchanged.
[0084] Comparative Example 5
[0085] A bifidobacterium cultivation method based on symbiotic nutrition:
[0086] The difference from Example 4 is that the culture medium of Example 1 is replaced by the culture medium of Comparative Example 1, and the other methods remain unchanged.
[0087] Comparative Example 6
[0088] A bifidobacterium cultivation method based on symbiotic nutrition:
[0089] The difference from Example 4 is that the culture medium of Example 1 is replaced by the culture medium of Comparative Example 2, and the other methods remain unchanged.
[0090] Comparative Example 7
[0091] A bifidobacterium cultivation method based on symbiotic nutrition:
[0092] The difference from Example 4 is that the culture medium of Example 1 is replaced by the culture medium of Comparative Example 3, and the other methods remain unchanged.
[0093] Comparative Example 8
[0094] A bifidobacterium cultivation method based on symbiotic nutrition:
[0095] The difference from Example 4 is that the culture medium of Example 1 is replaced by the culture medium of Comparative Example 4, and the other methods remain unchanged.
[0096] Test Example 1
[0097] The viable counts of the three bacteria in the culture system of Example 4 were measured at 0 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h and 26 h after the start of culture. The results are as follows: Figure 1 shown.
[0098] Depend on Figure 1 It can be seen that there is no obvious inhibitory effect on the growth of the three strains. During the 0-2h culture, Leuconostoc mesenteroides grew rapidly and entered a stable growth period after 12h, with the highest viable count reaching 5.56×10 10 cfu / mL; during the 0-2h period of culture, Bifidobacterium and Pediococcus acidilactici grew slowly, and then entered a rapid growth period. After 12h, the number of viable bifidobacteria was higher than that of the other two bacteria. After 20h, Bifidobacterium entered a stable growth period, with the highest number of viable bacteria reaching 1.65×10 11 cfu / mL, after 24 hours, Pediococcus acidilactici entered the stable growth period, and the maximum number of viable bacteria reached 1.34×10 11cfu / mL. This indicates that the bifidobacterium cultivation method based on the symbiotic nutrition method of the present invention promotes the growth and proliferation of each other through the synergistic symbiosis among bifidobacteria, Leuconostoc mesenteroides and Pediococcus acidilactici, thereby promoting the rapid growth of bifidobacteria and increasing the number of viable bifidobacteria.
[0099] Test Example 2
[0100] After the culture was completed, the number of viable bacteria of each species in the culture systems of Examples 4-7 and Comparative Examples 5-8 was measured respectively. The results are shown in Table 1.
[0101] Table 1 Viable bacteria count (cfu / mL)
[0102] Group Bifidobacterium Leuconostoc mesenteroides Pediococcus acidilactici Example 4 <![CDATA[1.63×10 11 ]]> <![CDATA[5.52×10 10 ]]> <![CDATA[1.34×10 11 ]]> Example 5 <![CDATA[8.33×10 10 ]]> <![CDATA[2.19×10 9 ]]> <![CDATA[7.41×10 10 ]]> Example 6 <![CDATA[1.07×10 11 ]]> <![CDATA[3.87×10 10 ]]> <![CDATA[9.79×10 10 ]]> Example 7 <![CDATA[5.71×10 10 ]]> <![CDATA[1.20×10 10 ]]> <![CDATA[3.57×10 10 ]]> Comparative Example 5 <![CDATA[4.39×10 6 ]]> <![CDATA[3.62×10 7 ]]> <![CDATA[5.33×10 7 ]]> Comparative Example 6 <![CDATA[3.82×10 9 ]]> <![CDATA[1.82×10 8 ]]> <![CDATA[5.25×10 9 ]]> Comparative Example 7 <![CDATA[6.24×10 9 ]]> <![CDATA[5.69×10 8 ]]> <![CDATA[2.36×10 9 ]]> Comparative Example 8 <![CDATA[1.17×10 9 ]]> <![CDATA[1.86×10 9 ]]> <![CDATA[4.61×10 9 ]]>
[0103] It can be seen from the results in Table 1 that the culture method of the present invention can significantly promote the growth and reproduction of bifidobacteria. After the culture is completed, the number of viable bifidobacteria reaches 5.71×10 10 ~1.63×10 11 cfu / mL; when arrowhead powder, loofah slurry and onion water extract were discarded from the culture medium at the same time (Comparative Example 5), or one of the components was discarded (Comparative Examples 6-8), the number of live bacteria of the three bacteria decreased significantly, indicating that arrowhead powder, loofah slurry and onion water extract can enrich nutrients, provide amino acids, vitamins, minerals and other nutrients, provide more comprehensive nutritional support for Bifidobacterium, Enterobacter mesenteroides and Pediococcus acidilactici, and create a relatively stable and favorable environment for the growth of the three bacteria, which is helpful to maintain the balance of the symbiotic flora, enhance the symbiotic relationship, and promote the synergistic growth of the three bacteria.
[0104] Test Example 3
[0105] The fermentation broth obtained after culturing in Example 4 and Comparative Examples 5-8 was centrifuged at 8000 r / min for 30 min, sucrose and sodium glutamate were added as protective agents, and freeze-dried to obtain freeze-dried powder. The freeze-dried powder was stored at 25° C. The number of viable bifidobacteria in the freeze-dried powder was detected at 0 d, 30 d, 60 d, 90 d, and 180 d, respectively. The results are shown in Table 2.
[0106] Table 2 The number of viable bifidobacteria in freeze-dried powder at different storage times (cfu / mL)
[0107] Group 0d 30d 60d 90d 180d 180-day survival rate Example 4 <![CDATA[1.63×10 11 ]]> <![CDATA[1.52×10 11 ]]> <![CDATA[1.31×10 11 ]]> <![CDATA[8.9×10 10 ]]> <![CDATA[3.51×10 10 ]]> 21.53％ Comparative Example 5 <![CDATA[4.39×10 6 ]]> <![CDATA[3.21×10 6 ]]> <![CDATA[1.90×10 6 ]]> <![CDATA[1.13×10 6 ]]> <![CDATA[1.26×10 5 ]]> 2.87％ Comparative Example 6 <![CDATA[3.82×10 9 ]]> <![CDATA[3.43×10 9 ]]> <![CDATA[2.64×10 9 ]]> <![CDATA[1.75×10 9 ]]> <![CDATA[4.34×10 8 ]]> 11.36％ Comparative Example 7 <![CDATA[6.24×10 9 ]]> <![CDATA[5.67×10 9 ]]> <![CDATA[4.97×10 9 ]]> <![CDATA[3.45×10 9 ]]> <![CDATA[6.91×10 8 ]]> 11.07％ Comparative Example 8 <![CDATA[1.17×10 9 ]]> <![CDATA[1.09×10 9 ]]> <![CDATA[8.23×10 8 ]]> <![CDATA[5.44×10 8 ]]> <![CDATA[1.54×10 8 ]]> 13.16％
[0108] It can be seen from the results in Table 2 that the freeze-dried powder prepared by the method of the present invention can still achieve a survival rate of 21.53% of bifidobacteria when stored at room temperature (25° C.) for 180 days, which is significantly improved compared with the freeze-dried powder prepared by Comparative Examples 5-8.
[0109] The above results show that the symbiotic culture of bifidobacteria using the culture medium of the present invention can not only promote the proliferation of bifidobacteria and increase the yield of bifidobacteria, but also effectively improve the survival rate of bifidobacteria during the storage process.
[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A culture medium, characterized in that Based on 1L, the culture medium includes the following raw materials: 20-30g peanut meal, 15-20g sucrose, 5-10g yeast extract, 10-15g arrowhead powder, 5-10g loofah slurry, 5-10g onion water extract, 0.5-1g sodium chloride, 2-3g dipotassium hydrogen phosphate, 1-2g potassium dihydrogen phosphate, 0.2-0.5g magnesium sulfate, 0.05-0.1g manganese chloride, and the balance is water.
2. The culture medium according to claim 1, characterized in that The pH of the culture medium is 6-7.
3. Use of the culture medium according to claim 1 or 2 in culturing and preserving bifidobacteria.
4. The use according to claim 3, characterized in that: The bifidobacterium is Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium animalis or Bifidobacterium bifidum.
5. A method for culturing bifidobacteria based on symbiotic nutrition, characterized in that: The culture method comprises the following steps: inoculating Bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici into the culture medium according to claim 1 or 2, and conducting closed culture.
6. The culture method according to claim 5, characterized in that: The ratio of the inoculated live bacteria of the bifidobacterium, Leuconostoc mesenteroides and Pediococcus acidilactici is 1-5:1-5:1-5.
7. The culture method according to claim 5, characterized in that The culture conditions are as follows: initial temperature of 28-34° C. for 1-3 h; temperature raised to 35-40° C. for 16-30 h.
8. A fermentation liquid obtained by the culturing method according to any one of claims 5 to 7.
9. A freeze-dried powder of Bifidobacterium, characterized in that: The freeze-dried powder is prepared from the fermentation broth according to claim 8.
10. The method for preparing the freeze-dried powder according to claim 9, characterized in that: The preparation method comprises the following steps: centrifuging the fermentation liquid according to claim 8, adding a protective agent, freeze-drying, and storing.