CN110904022A - Bacillus culture medium and application thereof - Google Patents
Bacillus culture medium and application thereof Download PDFInfo
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- CN110904022A CN110904022A CN201911059552.9A CN201911059552A CN110904022A CN 110904022 A CN110904022 A CN 110904022A CN 201911059552 A CN201911059552 A CN 201911059552A CN 110904022 A CN110904022 A CN 110904022A
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- milk
- bacillus
- culture medium
- goat milk
- fermenting
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- 230000009385 viral infection Effects 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
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Abstract
The invention discloses a fermentation medium of bacillus, which is characterized by comprising the following components in percentage by weight: fresh goat milk, sucrose, trehalose, inulin, glycerol, hawthorn powder and ligusticum wallichii extract.
Description
Technical Field
The invention relates to a bacillus culture medium and application thereof, in particular to a bacillus culture medium which can produce curd and has a function of regulating intestines and stomach and application thereof.
Background
Probiotic refers to a live bacterial preparation and a metabolite thereof which can improve the health status of human and animals by improving the ecological balance of host intestinal flora after being ingested to exert beneficial effects. In addition to lactobacillus aerotolerant, lactobacillus plantarum, lactobacillus acidophilus, bifidobacterium infantis, bacillus, yeast and the like, all of which can provide potential health benefits by maintaining a good bacterial flora composition in the intestinal tract, such as improving the body's ability to resist pathogen invasion, maintaining the body's health and enhancing immunity; reducing cholesterol and blood pressure, and relieving lactose intolerance; improving the nutritive value of food and promoting the digestion and absorption of nutrient substances.
The functional dairy product is a dairy product which is added with substances with physiological activity or microorganisms beneficial to human bodies so that the dairy product has specific physiological effects besides basic nutritional functions. The goat milk in China has rich resources and distinct characteristics, and a large number of researches prove that the goat milk has rich contents of protein, amino acid, vitamin, mineral substances and fat, has the component composition which is most similar to that of the breast milk, and internationally enjoys the reputation of 'king in milk'. Compared with cow milk, the goat milk has the following characteristics: the fat and protein particles are small, and are easy to digest and absorb by a human body; contains 8 kinds of amino acids essential for human body; a large amount of immune factors and active polypeptides exist, which is beneficial to improving the immunity; the specific Epidermal Growth Factor (EGF) can promote the repair of skin and mucous membrane, and the superoxide dismutase (SOD) can play the beauty roles of clearing free radicals, resisting aging and the like; the tea is rich in DHA, nucleotide, taurine, cephalin and the like, and can promote the development of cranial nerves and vision of a human body; the vitamin content is high, and particularly the Vc content is obviously higher than that of cow milk; the goat milk belongs to weak alkaline food and is helpful for recovery of nephritis. In addition, the goat milk also has effects of reducing weight and reducing cholesterol
The invention aims to provide a bacillus culture medium capable of producing curd and having a gastrointestinal conditioning function and application thereof.
Disclosure of Invention
The invention aims to provide a bacillus culture medium capable of producing curd and having a gastrointestinal conditioning function and application thereof.
Bacillus subtilis (Bacillus subtilis) is a kind of Bacillus, CAS number 68038-70-0. The single cell is 0.7-0.8 multiplied by 2-3 microns and is uniformly colored. Without capsule, the perigenic flagellum can move. Gram-positive bacteria, spores of 0.6-0.9 multiplied by 1.0-1.5 microns, oval to columnar shape, central or slightly deviated, and the bacteria do not expand after the spores are formed. The colony surface is rough and opaque, and is dirty white or yellowish, and when the colony grows in a liquid culture medium, the skin becomes always formed. Aerobic bacteria. Tryptophan can be decomposed to form indole by using protein, various sugars and starch.
The application of the bacillus subtilis is wide, and researches show that a part of the bacillus subtilis produces chymosin, but the enzyme yield is low, so that the bacillus subtilis is difficult to be used for producing yoghourt generally.
According to the invention, Bacillus subtilis ATCC 7058 is selected for research, the activity of chymosin is very low during normal fermentation, and the activity of the chymosin can be obviously improved by compounding traditional Chinese medicines in the fermentation process, so that pleasant flavor is generated, active peptide generated in the fermentation process can be greatly improved, and the health care effect is greatly improved.
The ligusticum wallichii is a characteristic traditional Chinese medicine in Jiangxi, and the ligusticum wallichii is known to be beneficial to cardiovascular and has a certain antibacterial effect.
The hawthorn (Crataegus pinnatifida Bunge), also called Crataegus pinnatifida Bunge, and Crataegus pinnatifida Bunge with the height of up to 6 m, are special medicinal and fruit tree species in China, have the effects of reducing blood fat, blood pressure, strengthening heart, resisting arrhythmia, and the like, are also good medicines for strengthening spleen, stimulating appetite, promoting digestion, removing stagnation, promoting blood circulation and. The vitexin is a flavonoid compound in hawthorn, is a drug with strong anticancer effect, and the extract of the vitexin has certain effects on inhibiting the growth, proliferation and infiltration and metastasis of cancer cells in vivo.
The invention discovers that the activity of chymosin can be obviously improved by adding the hawthorn into the culture medium and adding the ligusticum wallichii and the astragalus mongholicus extracting solution at a certain time, so that a pleasant flavor is generated, active peptide generated in the fermentation process can be greatly improved, and the health-care effect is greatly improved.
The technical problem to be solved by the invention can be realized by the following technical scheme.
A fermentation medium of bacillus is characterized in that the medium comprises the following components in percentage by weight:
fresh goat milk, sucrose, trehalose, inulin, glycerol, hawthorn powder and ligusticum wallichii extract.
The method for culturing the bacillus by using the fermentation medium is characterized by comprising the following steps:
(1) strain activation
The culture medium used for activating the bacillus is standard MRS broth culture medium; the activation condition is shake cultivation at 37 deg.C for 10h, and the bacterial amount after activation is 1.5 × 108CFU/mL；
(2) Fermented goat milk
Homogenizing fresh goat milk, and preparing a culture medium according to the following proportion: 2000g of fresh goat milk, 20g of sucrose, 20g of trehalose, 5g of inulin, 2g of glycerol and 20-30g of hawthorn powder; after pasteurization at 95 ℃ for 15min, naturally cooling to 45 ℃, inoculating activated Bacillus subtilis ATCC 7058 with the inoculum size of 30mL of the bacterial liquid activated in the step (1); fermenting at 38 deg.C for 3 hr, adding rhizoma Ligustici Chuanxiong extractive solution 2ml, fermenting at 38 deg.C for 1 hr, adding rhizoma Ligustici Chuanxiong extractive solution 1ml again, heating to 40 deg.C, and fermenting for 4 hr until the milk becomes semisolid fermented milk with certain viscosity, and has milk fragrance, slightly sour taste, and pleasant flavor.
Preferably, the Bacillus is Bacillus subtilis ATCC 7058.
Preferably, the preparation method of the ligusticum wallichii extract comprises the following steps: soaking rhizoma Ligustici Chuanxiong decoction pieces in 10 times of water for 10 hr, heating and reflux extracting for 3 times, each for 2 hr, mixing the filtrates.
Preferably, a spray drying step is further provided.
The invention has the advantages that:
the invention discovers that the activity of rennin can be obviously improved by adding hawthorn into a culture medium and adding ligusticum wallichii and astragalus mongholicus extracting solution at a certain time, so that a pleasant flavor is generated, active peptide generated in the fermentation process can be greatly improved, the health care effect is greatly improved, and particularly, the invention has a good nursing effect on intestines and stomach, such as the capability of resisting infection of pathogens such as rotavirus and the like.
Drawings
FIG. 1 shows fermented milk obtained by fermentation in example 1, which has a certain consistency.
Fig. 2 is a control picture of example 5.
Fig. 3 is a picture of RV group of example 5.
FIG. 4 is a picture of RV + milk powder group of example 5.
Detailed Description
The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.
Specific examples of the present invention are described below.
Example 1
A bacillus culture medium capable of producing curd and having a function of regulating intestines and stomach and a fermentation method thereof are disclosed:
(1) strain activation
The culture medium used for activating the Bacillus subtilis ATCC 7058 is standard MRS broth culture medium; the activation condition is shake cultivation at 37 deg.C for 10h, and the bacterial amount after activation is 1.5 × 108CFU/mL；
(2) Fermented goat milk
Homogenizing fresh goat milk, and preparing a culture medium according to the following proportion: 2000g of fresh goat milk, 20g of sucrose, 20g of trehalose, 5g of inulin, 2g of glycerol and 30g of hawthorn powder; after pasteurization at 95 ℃ for 15min, naturally cooling to 45 ℃, inoculating activated Bacillus subtilis ATCC 7058 with the inoculum size of 30mL of the bacterial liquid activated in the step (1); fermenting at 38 deg.C for 3 hr, adding rhizoma Ligustici Chuanxiong extractive solution 2ml, continuing to ferment at 38 deg.C for 1 hr, adding rhizoma Ligustici Chuanxiong extractive solution 1ml again, heating to 40 deg.C, and continuing to ferment for 4 hr until the milk becomes semisolid fermented milk with certain viscosity, and has milk fragrance, slightly sour taste, and pleasant flavor;
the preparation method of the ligusticum wallichii extract comprises the following steps: soaking rhizoma Ligustici Chuanxiong decoction pieces in 10 times of water for 10 hr, heating and reflux extracting for 3 times, each for 2 hr, mixing the filtrates;
fully shaking and shaking the obtained fermented milk uniformly, pouring the fermented milk into a 100mL clean beaker, firstly adjusting the pH of the fermented milk by using a standard hydrochloric acid solution with the concentration of 1mol/L to enable the pH of the fermented milk to be 3.6, centrifuging the fermented milk for 15min under the condition of 5000r/min, and taking supernatant; regulating the pH of the supernatant to 8.3 by using a sodium hydroxide standard solution with the concentration of 1mol/L, centrifuging for 15min under the condition of 5000r/min, and taking the supernatant, wherein the supernatant is the whey sample solution.
(3) Spray drying to obtain milk powder
Uniformly stirring the fermented milk obtained in the step (2) for spray drying, and setting parameters as follows: the air outlet temperature is 90 ℃, the solid content is 45 percent, the feed flow is 3mL/min, and the air inlet volume is 45m3The survival rate of the cells under the conditions is 83.52%, and the water content is 4.7%.
Example 2:
a bacillus culture medium capable of producing curd and having a function of regulating intestines and stomach and a fermentation method thereof are disclosed:
(1) strain activation
The culture medium used for activating the Bacillus subtilis ATCC 7058 is standard MRS broth culture medium; the activation condition is shake cultivation at 37 deg.C for 10h, and the bacterial amount after activation is 1.5 × 108CFU/mL；
(2) Fermented goat milk
Homogenizing fresh goat milk, and preparing a culture medium according to the following proportion: 2000g of fresh goat milk, 20g of sucrose, 20g of trehalose, 5g of inulin, 2g of glycerol and 20g of hawthorn powder; after pasteurization at 95 ℃ for 15min, naturally cooling to 45 ℃, inoculating activated Bacillus subtilis ATCC 7058 with the inoculum size of 30mL of the bacterial liquid activated in the step (1); fermenting at 38 deg.C for 3 hr, adding rhizoma Ligustici Chuanxiong extractive solution 2ml, continuing to ferment at 38 deg.C for 1 hr, adding rhizoma Ligustici Chuanxiong extractive solution 1ml again, heating to 40 deg.C, and continuing to ferment for 4 hr until the milk becomes semisolid fermented milk with certain viscosity, and has milk fragrance, slightly sour taste, and pleasant flavor;
the preparation method of the ligusticum wallichii extract comprises the following steps: soaking rhizoma Ligustici Chuanxiong decoction pieces in 10 times of water for 10 hr, heating and reflux extracting for 3 times, each for 2 hr, mixing the filtrates;
fully shaking and shaking the obtained fermented milk uniformly, pouring the fermented milk into a 100mL clean beaker, firstly adjusting the pH of the fermented milk by using a standard hydrochloric acid solution with the concentration of 1mol/L to enable the pH of the fermented milk to be 3.4, centrifuging the fermented milk for 15min under the condition of 5000r/min, and taking supernatant; regulating the pH of the supernatant to 8.3 by using a sodium hydroxide standard solution with the concentration of 1mol/L, centrifuging for 15min under the condition of 5000r/min, and taking the supernatant, wherein the supernatant is the whey sample solution.
(3) Spray drying to obtain milk powder
Uniformly stirring the fermented milk obtained in the step (2) for spray drying, and setting parameters as follows: the air outlet temperature is 90 ℃, the solid content is 45 percent, the feed flow is 3mL/min, and the air inlet volume is 45m3The survival rate of the cells under the conditions is 82.45 percent, and the water content is 4.6 percent.
Example 3
Comparison 1, no chuanxiong rhizome was added:
(1) strain activation
The culture medium used for activating the Bacillus subtilis ATCC 7058 is standard MRS broth culture medium; the activation condition is shake cultivation at 37 deg.C for 10h, and the bacterial amount after activation is 1.5 × 108CFU/mL；
(2) Fermented goat milk
Homogenizing fresh goat milk, and preparing a culture medium according to the following proportion: 2000g of fresh goat milk, 20g of sucrose, 20g of trehalose, 5g of inulin, 2g of glycerol and 20-30g of hawthorn powder; after pasteurization at 95 ℃ for 15min, naturally cooling to 45 ℃, inoculating activated Bacillus subtilis ATCC 7058 with the inoculum size of 30mL of the bacterial liquid activated in the step (1); fermenting at 40 deg.C for 8 hr,
the consistency of the milk hardly changes.
The fermentation is continued for 2 hours, the thickness of the milk liquid is still unchanged, and the milk liquid has milk fragrance, slight salty taste and no sour taste.
Therefore, the addition of ligusticum wallichii is not added, so that enough rennin can not be generated to coagulate the goat milk, and a pleasant flavor can not be generated.
Fully shaking and shaking the obtained fermented milk uniformly, pouring the fermented milk into a 100mL clean beaker, firstly adjusting the pH of the fermented milk by using a standard hydrochloric acid solution with the concentration of 1mol/L to ensure that the pH of the fermented milk is 3.4-3.6, centrifuging the fermented milk for 15min under the condition of 5000r/min, and taking supernatant; regulating the pH of the supernatant to 8.3 by using a sodium hydroxide standard solution with the concentration of 1mol/L, centrifuging for 15min under the condition of 5000r/min, and taking the supernatant, wherein the supernatant is the whey sample solution.
Comparison 2, no hawthorn was added:
(1) strain activation
The culture medium used for activating the Bacillus subtilis ATCC 7058 is standard MRS broth culture medium; the activation condition is shake cultivation at 37 deg.C for 10h, and the bacterial amount after activation is 1.5 × 108CFU/mL；
(2) Fermented goat milk
Homogenizing fresh goat milk, and preparing a culture medium according to the following proportion: 2000g of fresh goat milk, 20g of cane sugar, 20g of trehalose, 5g of inulin, 2g of glycerol and 20-30g of soybean meal; after pasteurization at 95 ℃ for 15min, naturally cooling to 45 ℃, inoculating activated Bacillus subtilis ATCC 7058 with the inoculum size of 30mL of the bacterial liquid activated in the step (1); fermenting at 38 deg.C for 3 hr, adding rhizoma Ligustici Chuanxiong extractive solution 2ml, fermenting at 38 deg.C for 1 hr, adding rhizoma Ligustici Chuanxiong extractive solution 1ml again, heating to 40 deg.C, and fermenting for 4 hr;
the consistency of the milk hardly changes.
After the fermentation is continued for 2 hours, the milk still has unchanged consistency, milk flavor and almost unchanged taste, and the number of the bacillus subtilis is obviously lower than that of the bacillus subtilis in the example 1-2 under the observation of a microscope, so that the ligusticum wallichii inhibits the propagation of the bacillus subtilis.
Therefore, the chuanxiong rhizome can inhibit the reproduction of bacillus subtilis without adding hawthorn, and can not produce enough rennin to coagulate goat milk and can not produce a pleasant flavor.
In conclusion, the hawthorn and the ligusticum wallichii have synergistic effect, so that the bacillus subtilis can grow normally and generate enough chymosin to obtain the fermented milk product with pleasant flavor.
Fully shaking and shaking the obtained fermented milk uniformly, pouring the fermented milk into a 100mL clean beaker, firstly adjusting the pH of the fermented milk by using a standard hydrochloric acid solution with the concentration of 1mol/L to ensure that the pH of the fermented milk is 3.4-3.6, centrifuging the fermented milk for 15min under the condition of 5000r/min, and taking supernatant; regulating the pH of the supernatant to 8.3 by using a sodium hydroxide standard solution with the concentration of 1mol/L, centrifuging for 15min under the condition of 5000r/min, and taking the supernatant, wherein the supernatant is the whey sample solution.
Example 4 active peptide detection
Taking 2mL whey sample solution and 2mL DPPH solution (0.14 mmol/L dissolved in ethanol), standing at room temperature in a dark place for 30min, and detecting the light absorption value at the wavelength of 517nm, wherein the DPPH free radical clearance is calculated according to the formula (1):
in the formula, A0 is the light absorption value of 2mL deionized water mixed with 2mL DPPH solution; a1 is the light absorption value of 2mL sample mixed with 2mL PPH solution; a2 is the absorbance of a 2mL sample mixed with 2mL ethanol.
The results are shown in the following table.
| Group of | DPPH radical scavenging ratio (%) |
| Example 1 | 85.9 |
| Example 2 | 89.8 |
| Comparative example 1 | 37.6 |
| Comparative example 2 | 22.3 |
Therefore, the invention can prepare more active peptides, and has better antioxidant capacity, thereby having better health care function.
Example 5
Rotavirus infection is a cause of acute gastroenteritis, which is a main cause of infant emergency treatment and death (except respiratory tract infection), and diarrhea, vomiting and fever are main symptoms. If acute diarrhea caused by RV infection cannot be treated in time, serious electrolyte disorder in vivo can be caused, and finally death is caused, and the acute gastroenteritis caused by RV infection is treated by the medicine which is not clinically specific at present.
Caco-2 cells are intestinal epithelial cells cultured in vitro, and the cells are derived from human colon adenocarcinoma cells.
The Caco-2 cells in a good state grow rapidly, are round or oval, are uniformly distributed and grow in an adherent manner, the Caco-2 cells are cultured to grow until the coverage rate reaches about 70%, and are washed for 3-4 times by sterile 1XPBS buffer solution, wherein each time is 2-3 mL. Adding 5 microgram/ml trypsin 200 microliter and adding 30 microliter rotavirus, and then incubating for lh; the cells were washed with PBS buffer several times until no stock culture was present, and then 500. mu.l of 2% DMEM (high-sugar) culture was added.
Caco-2 cells were plated in 6-well plates, 4 plates each. The group was divided into 3 groups, i.e., RV group, RV + milk powder group (concentration of 1 g/ml), control group, and then cultured in an incubator, and sampled at 12 hours.
FIG. 2 shows a control group, FIG. 3 shows an RV group, and FIG. 4 shows an RV + milk powder group.
As can be seen, after inoculation of RV, the rotavirus infected cells have obvious CPE, the RV + milk powder group only has slightly reduced cell density, and the shape is kept relatively normal. Therefore, the probiotic milk powder has a remarkable effect on resisting virus infection.
It is to be understood that the foregoing is only a preferred embodiment of the invention and that modifications, variations and changes may be made in the invention without departing from the spirit or scope of the invention as defined in the appended claims.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (5)
1. A fermentation medium of bacillus is characterized in that the medium comprises the following components in percentage by weight:
fresh goat milk, sucrose, trehalose, inulin, glycerol, hawthorn powder and ligusticum wallichii extract.
2. A method of culturing Bacillus using the fermentation medium of claim 1, characterized by the steps of:
(1) strain activation
The culture medium used for activating the bacillus is standard MRS broth culture medium; the activation condition is shake cultivation at 37 deg.C for 10h, and the bacterial amount after activation is 1.5 × 108CFU/mL；
(2) Fermented goat milk
Homogenizing fresh goat milk, and preparing a culture medium according to the following proportion: 2000g of fresh goat milk, 20g of sucrose, 20g of trehalose, 5g of inulin, 2g of glycerol and 20-30g of hawthorn powder; after pasteurization at 95 ℃ for 15min, naturally cooling to 45 ℃, inoculating activated Bacillus subtilis ATCC 7058 with the inoculum size of 30mL of the bacterial liquid activated in the step (1); fermenting at 38 deg.C for 3 hr, adding rhizoma Ligustici Chuanxiong extractive solution 2ml, fermenting at 38 deg.C for 1 hr, adding rhizoma Ligustici Chuanxiong extractive solution 1ml again, heating to 40 deg.C, and fermenting for 4 hr until the milk becomes semisolid fermented milk with certain viscosity, and has milk fragrance, slightly sour taste, and pleasant flavor.
3. The fermentation medium of bacillus of claim 1, wherein:
the Bacillus is Bacillus subtilis ATCC 7058.
4. The fermentation medium of bacillus of claim 1, wherein:
the preparation method of the ligusticum wallichii extract comprises the following steps: soaking rhizoma Ligustici Chuanxiong decoction pieces in 10 times of water for 10 hr, heating and reflux extracting for 3 times, each for 2 hr, mixing the filtrates.
5. The culture method according to claim 2, wherein:
there is a further spray drying step.
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| Publication number | Priority date | Publication date | Assignee | Title |
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