CN111235074A - Composite functional microbial inoculum of saccharomyces cerevisiae - Google Patents
Composite functional microbial inoculum of saccharomyces cerevisiae Download PDFInfo
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- CN111235074A CN111235074A CN202010239575.4A CN202010239575A CN111235074A CN 111235074 A CN111235074 A CN 111235074A CN 202010239575 A CN202010239575 A CN 202010239575A CN 111235074 A CN111235074 A CN 111235074A
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- saccharomyces cerevisiae
- lactobacillus casei
- fermentation
- microbial inoculum
- composite functional
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 89
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 title claims abstract description 89
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 20
- 239000002131 composite material Substances 0.000 title claims abstract description 16
- 244000199866 Lactobacillus casei Species 0.000 claims abstract description 67
- 235000013958 Lactobacillus casei Nutrition 0.000 claims abstract description 67
- 229940017800 lactobacillus casei Drugs 0.000 claims abstract description 67
- 238000000855 fermentation Methods 0.000 claims abstract description 62
- 230000004151 fermentation Effects 0.000 claims abstract description 39
- 238000004321 preservation Methods 0.000 claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 19
- 238000010564 aerobic fermentation Methods 0.000 claims description 16
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- 229940029339 inulin Drugs 0.000 claims description 7
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 7
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/14—Yeasts or derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/18—Baker's yeast; Brewer's yeast
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/125—Casei
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
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- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
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- Zoology (AREA)
- Biotechnology (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a saccharomyces cerevisiae composite functional microbial inoculum combination, which has the function of remarkably improving the immunity, is beneficial to the auxiliary treatment and prevention of viral diseases, can be directly applied to food fermentation, can be prepared into pure probiotic products or food, and has wide application range and high acceptability. The composite functional microbial inoculum combination of the saccharomyces cerevisiae is characterized in that: comprises Saccharomyces cerevisiae DJ-012 and Lactobacillus casei LC-N235. The preservation date of the saccharomyces cerevisiae DJ-012 is as follows: the preservation number is CGMCC No.6200 at 6, 8 and 6 months in 2012. The preservation date of the lactobacillus casei LC-N235 is as follows: day 5/10 2012, deposit number: CCTCC M2012157.
Description
Technical Field
The invention relates to a composite functional microbial inoculum of saccharomyces cerevisiae, belonging to the technical field of microorganisms.
Background
In the process of fighting against viruses and bacteria, immunity plays a vital role. It is an important system for the human body to recognize and destroy viruses and bacteria, and is also called the second line of defense of the body.
In the face of the novel coronavirus, all people are generally susceptible. The old with low immunity needs to pay attention to protection, and the young people with few diseases need to take protection measures. For people with strong immunity, the novel coronavirus also has great threat.
For a new virus, no antibody is formed in human body, and the common people need to rely on the autoimmune system for resisting. The elderly and other people with weak constitution or basic diseases, such as patients with chronic obstructive pulmonary disease, asthma, tracheitis, and diabetes, need to be vigilant.
Probiotics are active microorganisms which are beneficial to a host and change the composition of flora at a certain part of the host by colonizing in a human body. The health of the intestinal tract is kept by promoting the absorption of nutrients by regulating the immune function of the host mucous membrane and the system or by regulating the balance of flora in the intestinal tract, so that single microorganisms or mixed microorganisms with definite compositions which are beneficial to the health are generated. At present, researches show that the yeast has good probiotic characteristics of regulating intestinal balance, promoting feed conversion, improving the immune function of organisms and the like, and is mostly used as a feed additive for livestock and poultry breeding. The yeast of most research includes Saccharomyces, Torulopsis, Candida, Willemm, Pichia, Saccharomyces, Torulopsis, Schoeckera, Rhodotorula, Schizosaccharomyces pombe, and Botrytis. Lactic acid bacteria are a generic term for a class of non-spore, gram-positive bacteria in which the main product of the fermentation sugars is lactic acid. Lactic acid bacteria are the most common probiotic bacteria, have been widely used and are considered safe for humans and animals.
The invention aims to develop a saccharomyces cerevisiae composite functional microbial inoculum which generates a culture with the function of improving the immunity through the synergistic effect of specific saccharomyces cerevisiae and lactobacillus.
Disclosure of Invention
The invention aims to develop a composite functional microbial inoculum of saccharomyces cerevisiae, which has the function of remarkably improving immunity, is beneficial to the auxiliary treatment and prevention of viral diseases, can be directly applied to food fermentation, can be prepared into pure probiotic products or foods, and has wide application range and high acceptability.
Chinese patent 2012102058101 discloses Saccharomyces cerevisiae DJ-012, which is deposited in China general microbiological culture Collection center, address: the microbial research institute of China academy of sciences, No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation date is 100101, 6 months and 8 days in 2012, and the preservation number is CGMCC No. 6200. The known performance of the strain is that the hexose and pentose in the fermentation liquor can be effectively utilized to produce alcohol, so that the saccharides in the fermentation raw materials are fully utilized, the raw material utilization rate and the alcohol yield are obviously improved, and the production cost is reduced.
The reference saccharomyces cerevisiae used in the embodiment of the invention has the following latin name: saccharomyces cerevisiae, available from Guangdong province center for the preservation of microbial strains, accession number: GIM2.200, main use: producing arginase, brewing alcohol, histone deacetylase, nicotinic acid and the like.
In research and development, the applicant finds that generally speaking, the fermentation liquor of Saccharomyces cerevisiae does not have the function of remarkably improving the immunity, but the fermentation liquor of Saccharomyces cerevisiae DJ-012 has the function of remarkably improving the immunity.
Chinese patent 2012101725644 discloses a strain of Lactobacillus casei (Lactobacillus casei) LC-N235, which has been preserved in China center for type culture Collection CCTCC at 5/10/2012 with the preservation number: CCTCC M2012157. The strain can effectively utilize cheap corn flour saccharification liquid to ferment and produce L-lactic acid, and the saccharic acid conversion rate is high. In a 5L fermentation tank, the yield of L-lactic acid reaches 173g/L, which is improved by 46.6 percent compared with the original fermentation of the original spawn.
The research and development team of the applicant further optimizes, and finds that the lactobacillus casei (Lactobacillus casei) LC-N235 and the Saccharomyces cerevisiae (Saccharomyces cerevisiae) DJ-012 co-ferment under a specific culture condition, so that the effect of improving the immunity of the Saccharomyces cerevisiae (Saccharomyces cerevisiae) DJ-012 fermentation liquor can be obviously improved. However, the lactobacillus casei (lactobacillus casei) LC-N235 fermentation broth itself has no significant immunity enhancing effect, so it is presumed that the lactobacillus casei (lactobacillus casei) LC-N235 produces some active substances stimulating the saccharomyces cerevisiae during the co-fermentation process. Other lactobacillus casei were tested and did not have this significant effect.
Examples the control lactobacillus casei exemplified is commercially available from lactobacillus casei, strain deposit No.: CICC 6117.
The invention can directly adopt a one-step co-fermentation method, preferably a sectional fermentation method, firstly carries out anaerobic fermentation on lactobacillus casei (Lactobacillus casei) LC-N235, the activity and the proliferation rate of the lactobacillus casei are ensured by the anaerobic fermentation, and the substances of the culture medium are pretreated. Then inoculating Saccharomyces cerevisiae DJ-012 for aerobic fermentation, wherein, along with the generation of a certain degree of alcohol and the disadvantage of oxygen, Lactobacillus casei LC-N235 becomes weak flora in fermentation, and Saccharomyces cerevisiae DJ-012 becomes dominant flora. By controlling the sugar content of the culture medium, the alcohol concentration can be controlled, and products with high or low alcohol content can be prepared.
The invention discovers that the fermentation liquor of Saccharomyces cerevisiae DJ-012 has the function of improving immunity for the first time, and discovers that lactobacillus casei (Lactobacillus casei) LC-N235 and co-fermentation thereof can further obviously improve immunity for the first time.
The technical scheme of the invention is as follows:
a composite functional microbial inoculum combination of saccharomyces cerevisiae is characterized in that:
comprises Saccharomyces cerevisiae DJ-012 and Lactobacillus casei LC-N235.
The preservation date of the saccharomyces cerevisiae DJ-012 is as follows: the preservation number is CGMCC No.6200 at 6, 8 and 6 months in 2012.
The preservation date of the lactobacillus casei LC-N235 is as follows: day 5/10 2012, deposit number: CCTCC M2012157.
Preferably, the complex microbial inoculum combination can be used for co-fermenting substrates or fermenting substrates in a segmented mode.
The segmented fermentation method comprises the following steps: firstly, carrying out anaerobic fermentation on lactobacillus casei (Lactobacillus casei) LC-N235, wherein the activity and the proliferation rate of the lactobacillus casei are ensured by the anaerobic fermentation, and the substances of a culture medium are pretreated. Then inoculating Saccharomyces cerevisiae DJ-012 for aerobic fermentation.
Preferably, the weight portions of the lactobacillus casei are 1 portion of saccharomyces cerevisiae DJ-012 and 1 portion of lactobacillus casei LC-N235.
Preparing a seed solution of the strain:
saccharomyces cerevisiae DJ-012: the preserved saccharomyces cerevisiae strain is weighed and inoculated into a yeast solid culture medium for 24 hours. Selecting single colony with good growth condition in the plate, inoculating into yeast liquid culture medium, activating, culturing for 24 hr, and quantifying to 5 × 107(CFU/ml) yeast seed solution. The culture conditions are as follows: 30 ℃ and 200 r/min.
Yeast solid medium: 1% Yeast Extract, 2% Peptone (Peptone), 2% dextrose (glucose), 2% agar powder.
Yeast liquid medium: 1% Yeast Extract, 2% Peptone (Peptone), 2% dextrose (glucose).
Lactobacillus casei LC-N235: inoculating a lactobacillus strain into MRS solid culture medium, streaking, and performing anaerobic culture at 37 ℃ for 24 h. Selecting single colony with better growth in the plate, inoculating the single colony in MRS liquid culture medium for activation, and quantitatively obtaining 5 x 10 after anaerobic culture7(CFU/ml) Lactobacillus casei seed liquid.
The combined fermentation method by using the complex functional microbial inoculum comprises the following steps:
(1) preparing a special culture medium, comprising the following steps: 3% of glucose, 2% of xylose, 0.5% of inulin, 1% of whole milk powder, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate and a proper amount of Tween 80; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;
(2) anaerobic fermentation of lactobacillus casei:
inoculating activated lactobacillus casei LC-N235 seed liquid according to the inoculation amount of 2%, and performing anaerobic fermentation for 12h at the fermentation temperature of 30 ℃;
(3) carrying out aerobic fermentation on saccharomyces cerevisiae:
and (3) after the lactobacillus casei is fermented in the step (2), inoculating the activated saccharomyces cerevisiae DJ-012 seed liquid according to the inoculation amount of 2%, and performing aerobic fermentation for 36h at the fermentation temperature of 30 ℃ at the rotation speed of 150 r/min.
(4) Heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.
The invention has the advantages that:
the invention innovatively provides a novel saccharomyces cerevisiae composite functional microbial inoculum combination which can obviously improve the immunity. The invention can directly adopt a one-step co-fermentation method, preferably a sectional fermentation method, firstly carries out anaerobic fermentation on lactobacillus casei (Lactobacillus casei) LC-N235, the activity and the proliferation rate of the lactobacillus casei are ensured by the anaerobic fermentation, and the substances of the culture medium are pretreated. Then inoculating Saccharomyces cerevisiae DJ-012 for aerobic fermentation, wherein, along with the generation of a certain degree of alcohol and the disadvantage of oxygen, Lactobacillus casei LC-N235 becomes weak flora in fermentation, and Saccharomyces cerevisiae DJ-012 becomes dominant flora. The alcohol concentration can be controlled by controlling the sugar content of the culture medium, and the product with high or low alcohol content can be prepared, so that the application range is wide.
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.
The inoculation amount of the invention is v/v.
Example 1:
preparing a seed solution of the strain:
saccharomyces cerevisiae DJ-012: the preserved saccharomyces cerevisiae strain is weighed and inoculated into a yeast solid culture medium for 24 hours. Selecting single colony with good growth condition in the plate, inoculating into yeast liquid culture medium, activating, culturing for 24 hr, and quantifying to 5 × 107(CFU/ml) yeast seed solution. The culture conditions are as follows: 30 ℃ and 200 r/min.
Saccharomyces cerevisiae GIM 2.200: the preserved saccharomyces cerevisiae strain is weighed and inoculated into a yeast solid culture medium for 24 hours. Selecting single colony with good growth condition in the plate, inoculating into yeast liquid culture medium, activating, culturing for 24 hr, and quantifying to 5 × 107(CFU/ml) yeast seed solution. The culture conditions are as follows: 30 ℃ and 200 r/min.
Yeast solid medium: 1% Yeast Extract, 2% Peptone (Peptone), 2% dextrose (glucose), 2% agar powder.
Yeast liquid medium: 1% Yeast Extract, 2% Peptone (Peptone), 2% dextrose (glucose).
Lactobacillus casei LC-N235: inoculating a lactobacillus strain into MRS solid culture medium, streaking, and performing anaerobic culture at 37 ℃ for 24 h. Selecting single colony with better growth in the plate, inoculating the single colony into MRS liquid culture medium for activation, and quantitatively culturing the single colony into 5 x 107(CFU/ml) Lactobacillus casei seed liquid.
Lactobacillus casei cic 6117: inoculating a lactobacillus strain into MRS solid culture medium, streaking, and performing anaerobic culture at 37 ℃ for 24 h. Selecting single colony with better growth in the plate, inoculating the single colony into MRS liquid culture medium for activation, and quantitatively culturing the single colony into 5 x 107(CFU/ml) Lactobacillus casei seed liquid.
Example 2:
the combined fermentation method with the composite functional microbial inoculum comprises the following steps:
(1) preparing a special culture medium, comprising the following steps: 3% of glucose, 2% of xylose, 0.5% of inulin, 1% of whole milk powder, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate and a proper amount of Tween 80; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;
(2) anaerobic fermentation of lactobacillus casei:
inoculating activated lactobacillus casei LC-N235 seed liquid according to the inoculation amount of 2%, and performing anaerobic fermentation for 12h at the fermentation temperature of 30 ℃;
(3) carrying out aerobic fermentation on saccharomyces cerevisiae:
and (3) after the lactobacillus casei is fermented in the step (2), inoculating the activated saccharomyces cerevisiae DJ-012 seed liquid according to the inoculation amount of 2%, and performing aerobic fermentation for 36h at the fermentation temperature of 30 ℃ at the rotation speed of 150 r/min.
(4) Heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.
Control 1, saccharomyces cerevisiae DJ-012 alone:
(1) preparing a special culture medium, comprising the following steps: 3% of glucose, 2% of xylose, 0.5% of inulin, 1% of whole milk powder, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate and a proper amount of Tween 80; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;
(2) carrying out aerobic fermentation on saccharomyces cerevisiae:
after the culture medium is prepared, inoculating the activated saccharomyces cerevisiae DJ-012 seed liquid according to the inoculation amount of 2%, and carrying out aerobic fermentation for 36h at the fermentation temperature of 30 ℃ and at the rotation speed of 150 r/min.
(3) Heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.
Control 2, lactobacillus casei LC-N235:
the combined fermentation method with the composite functional microbial inoculum comprises the following steps:
(1) preparing a special culture medium, comprising the following steps: 3% of glucose, 2% of xylose, 0.5% of inulin, 1% of whole milk powder, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate and a proper amount of Tween 80; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;
(2) anaerobic fermentation of lactobacillus casei:
inoculating activated lactobacillus casei LC-N235 seed liquid according to the inoculation amount of 2%, and performing anaerobic fermentation for 12h at the fermentation temperature of 30 ℃;
(3) heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.
Control 3: only saccharomyces cerevisiae GIM 2.200:
(1) preparing a special culture medium, comprising the following steps: 3% of glucose, 2% of xylose, 0.5% of inulin, 1% of whole milk powder, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate and a proper amount of Tween 80; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;
(2) carrying out aerobic fermentation on saccharomyces cerevisiae:
after the culture medium is prepared, inoculating the activated saccharomyces cerevisiae GIM2.200 seed liquid according to the inoculation amount of 2%, and performing aerobic fermentation for 36h at the fermentation temperature of 30 ℃ and the rotation speed of 150 r/min.
(3) Heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.
Control 4: compounded with lactobacillus casei CICC6117
(1) Preparing a special culture medium, comprising the following steps: 3% of glucose, 2% of xylose, 0.5% of inulin, 1% of whole milk powder, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate and a proper amount of Tween 80; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;
(2) anaerobic fermentation of lactobacillus casei:
inoculating activated lactobacillus casei CICC6117 seed liquid according to the inoculation amount of 2%, and performing anaerobic fermentation for 12h at the fermentation temperature of 30 ℃;
(3) carrying out aerobic fermentation on saccharomyces cerevisiae:
and (3) after the lactobacillus casei is fermented in the step (2), inoculating the activated saccharomyces cerevisiae DJ-012 seed liquid according to the inoculation amount of 2%, and performing aerobic fermentation for 36h at the fermentation temperature of 30 ℃ at the rotation speed of 150 r/min.
(4) Heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.
Example 3:
immunity test
1. Preparation and administration of mouse with low immune function
70 SPF male KM mice with the constitution of 18-22g are randomly divided into 10 mice in the groups of example 1, comparative example 2, comparative example 3, comparative example 4, positive control and negative control, and 7 groups. Mice were acclimated to the laboratory environment for 3 days prior to the experiment. The administration route of each group of mice is as follows:
example 1 group: gavage example 1 supernatant (100mg/kg) D1-21
Comparative example 1 group: gavage comparative example 1 supernatant (100mg/kg) D1-21
Comparative example 2 group: gavage comparative example 2 supernatant (100mg/kg) D1-21
Comparative example 3 group: gavage comparative example 3 supernatant (100mg/kg) D1-21
Comparative example 4 group: gavage comparative example 4 supernatant (100mg/kg) D1-21
Positive control group: distilled water for intragastric administration (100mg/kg) D1-21
Negative control group: distilled water for intragastric administration (100mg/kg) D1-21
On day 17 of gavage (D17), mice of example 1 group, comparative example 2 group, comparative example 3 group, comparative example 4 group, and positive control group were injected with cyclophosphamide (100mg/kg) intraperitoneally for 5 consecutive days; the negative control group mice were injected with the same volume of saline.
2. Spleen index and thymus index detection of each group of mice
After 1 hour of the last administration, the mice were sacrificed, and the thymus and spleen of the mice were isolated, and tissues such as fascia were removed, and the mass was measured. Spleen and thymus indices were calculated according to the following formulas: organ index is 100% organ mass/animal mass. The results are shown in table 1:
TABLE 1 measurement of spleen index and thymus index for different groups
| Thymus index (mg/g) | Spleen index (mg/g) | |
| EXAMPLE 1 group | 1.77±0.20 | 4.25±0.15 |
| Comparative example 1 group | 1.43±0.13 | 3.66±0.33 |
| Comparative example 2 group | 1.31±0.11 | 3.38±0.13 |
| Comparative example 3 group | 1.29±0.11 | 3.26±0.15 |
| Comparative example 4 group | 1.45±0.12 | 3.74±0.14 |
| Positive control group | 1.24±0.14 | 3.16±0.18 |
| Negative control group | 1.83±0.24 | 4.53±0.14 |
The results showed that the negative control group had the highest thymus index and spleen index. The positive control group had the lowest thymus and spleen indices in the cyclophosphamide-induced immunocompromised mouse model. The index of the comparative example 1/4 group is higher than that of the comparative example 2/3 group, which suggests that the fermentation broth of Saccharomyces cerevisiae DJ-012 has a more obvious function of improving immunity, and lactobacillus alone has no effect. The group in the embodiment 1 is obviously higher than other comparative groups and is close to the negative control group, which prompts that the lactobacillus casei (Lactobacillus casei) LC-N235 and the Saccharomyces cerevisiae (Saccharomyces cerevisiae) DJ-012 are co-fermented under specific culture conditions, and the effect of improving the immunity of the Saccharomyces cerevisiae (Saccharomyces cerevisiae) DJ-012 fermentation liquor can be obviously improved.
3. ELISA method for determining IFN-gamma of mouse serum of each group
After 1h of the last administration, mice were sacrificed, the eyeballs were removed to collect blood samples, the supernatant was collected by centrifugation, 3 duplicate wells were set for each treatment group, and blank wells were reserved. Adding serum and a standard substance, reacting at 37 ℃ for 90min, washing the plate for 2 times, adding IFN-gamma biotinylation antibody working solution, reacting at 37 ℃ for 60min, adding an enzyme conjugate working solution, reacting at 37 ℃ for 60min, washing the plate for 3 times, adding an enzyme conjugate working solution (except blank holes), reacting at 37 ℃ for 30min, washing the plate for 3 times, developing TMB (tetramethylbenzidine), measuring an OD (450nm) value, and determining the IFN-gamma level of the serum of the mouse. The results are shown in table 2:
TABLE 2 measurement of IFN-. gamma.serum levels in mice of different groups
| IFN-γ(pg/ml) | |
| EXAMPLE 1 group | 79.21±4.93 |
| Comparative example 1 group | 37.31±3.31 |
| Comparative example 2 group | 23.43±2.11 |
| Comparative example 3 group | 25.87±2.61 |
| Comparative example 4 group | 41.65±3.63 |
| Positive control group | 20.24±2.14 |
| Negative control group | 90.83±7.22 |
IFN-gamma is a heavy and immunoregulation cytokine, and the reduction of the level of the IFN-gamma can cause the immune factor disorder, so that the result of the reduction of the immune function shows that the IFN-gamma in the negative control group is the highest. The positive control group had the lowest IFN-. gamma.in the mouse model with cyclophosphamide-induced hypoimmunity. The IFN-gamma level of the comparative example 1/4 group is higher than that of the comparative example 2/3 group, but lower than that of the example 1 group, and the fact that the Saccharomyces cerevisiae DJ-012 can improve the IFN-gamma of the mice with low immunity is suggested, but the effect of the lactobacillus casei LC-N235 alone is not obvious, but the effect of improving the immunity of the Saccharomyces cerevisiae DJ-012 fermentation liquor can be obviously improved when the Saccharomyces cerevisiae DJ-012 and the lactobacillus casei LC-N235 are co-fermented under specific culture conditions.
Therefore, generally speaking, the fermentation liquid of the Saccharomyces cerevisiae does not have the function of obviously improving the immunity, but the fermentation liquid of the Saccharomyces cerevisiae DJ-012 has the function of obviously improving the immunity. The lactobacillus casei (Lactobacillus casei) LC-N235 and the Saccharomyces cerevisiae (Saccharomyces cerevisiae) DJ-012 are co-fermented under a specific culture condition, so that the effect of improving the immunity of the Saccharomyces cerevisiae (Saccharomyces cerevisiae) DJ-012 fermentation liquor can be obviously improved. However, the lactobacillus casei (lactobacillus casei) LC-N235 fermentation broth itself has no significant immunity enhancing effect, so it is presumed that the lactobacillus casei (lactobacillus casei) LC-N235 produces some active substances stimulating the saccharomyces cerevisiae during the co-fermentation process. Other lactobacillus casei were tested and did not have this significant effect.
It should be understood that the foregoing is only a preferred embodiment of the present invention.
Variations that do not depart from the gist of the invention are intended to be within the scope of the invention.
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 (6)
1. A composite functional microbial inoculum combination of saccharomyces cerevisiae is characterized in that:
comprises Saccharomyces cerevisiae DJ-012 and Lactobacillus casei LC-N235;
the preservation date of the saccharomyces cerevisiae DJ-012 is as follows: 6 months and 8 days in 2012, and the preservation number is CGMCC No. 6200;
the preservation date of the lactobacillus casei LC-N235 is as follows: day 5/10 2012, deposit number: CCTCC M2012157.
2. The saccharomyces cerevisiae composite functional microbial inoculum combination of claim 1, which is characterized in that:
the microbial inoculum combination can co-ferment the substrate and can also ferment the substrate in sections.
3. The saccharomyces cerevisiae composite functional microbial inoculum combination of claim 2, wherein the sectional fermentation method comprises the following steps: firstly, the lactobacillus casei and LC-N235 are subjected to anaerobic fermentation, the activity and the proliferation rate of the lactobacillus casei and LC-N235 are ensured by the anaerobic fermentation, and the substances of a culture medium are pretreated. Then inoculating saccharomyces cerevisiae DJ-012 for aerobic fermentation.
4. The use of the saccharomyces cerevisiae composite functional microbial inoculum combination of any one of claims 1 to 3.
5. The use of claim 4, which is a fermented health food or prepared into a pure microbial inoculum.
6. The method for fermenting by using the saccharomyces cerevisiae composite functional microbial inoculum of claim 1 is characterized by comprising the following steps:
(1) preparing a special culture medium, comprising the following steps: 3% of glucose, 2% of xylose, 0.5% of inulin, 1% of whole milk powder, 1% of peptone, 0.05% of magnesium sulfate heptahydrate, 3% of ground calcium carbonate and a proper amount of Tween 80; heating to 95 deg.C with steam, sterilizing for 30s, sealing, and cooling to room temperature;
(2) anaerobic fermentation of lactobacillus casei:
inoculating activated lactobacillus casei LC-N235 seed liquid according to the inoculation amount of 2%, and performing anaerobic fermentation for 12h at the fermentation temperature of 30 ℃;
(3) carrying out aerobic fermentation on saccharomyces cerevisiae:
and (3) after the lactobacillus casei is fermented in the step (2), inoculating the activated saccharomyces cerevisiae DJ-012 seed liquid according to the inoculation amount of 2%, and performing aerobic fermentation for 36h at the fermentation temperature of 30 ℃ at the rotation speed of 150 r/min.
(4) Heating to 95 deg.C with steam, sterilizing for 1min, centrifuging at 1000r/min for 10min to obtain supernatant.
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