A microbial composition and its application in the preparation of food additives

Abstract

The present invention provides a microbial composition and its application in the preparation of food additives, belonging to the technical field of microbial fermentation. The microbial composition described in the present invention includes Bifidobacterium animalis, Enterococcus durans, and Candida tropicalis. The food additive obtained by fermenting yam, selenium-rich black beans, wolfberries, licorice, tangerine peel, and tremella fuciformis with this microbial composition has the effect of helping sleep, can be taken for a long time, and is safe, nutritious, and healthy.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to a microbial composition and its application in the preparation of food additives. Background Art

[0002] At present, the fast-paced life and the blue light interference of electronic devices have disrupted the original biological clock rhythm of the human body, causing sleep disorders. To solve the above problems of sleep disorders, many fields such as medicine and nutrition have carried out explorations. Although traditional drug treatments can relieve insomnia symptoms in the short term, they are often accompanied by side effects such as dizziness, fatigue, and increased dependence, and are not suitable for long-term conditioning.

[0003] Microbial fermentation technology is widely used in the food field and can improve the nutritional components, flavor, and texture of food. By using microbial fermentation raw materials, macromolecules can be converted into small molecules, and at the same time, the utilization rate and biological activity of the nutritional components in the raw materials can be improved. However, the active functions of the products obtained from different microbial fermentation raw materials are different, resulting in uneven active functions of the products obtained by microbial fermentation. At present, there are few studies on combining microbial fermentation technology with the research and development of sleep aid products. Developing a safe, nutritious, healthy, and long-term edible sleep aid product is a technical problem to be solved at the present stage. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a microbial composition and its application in the preparation of food additives. The microbial composition of the present invention can degrade macromolecules in the raw materials into small molecules that help sleep, and the food additive prepared by using this microbial fermentation composition combined with special raw materials has a significant sleep aid effect.

[0005] To solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a microbial composition, which includes Bifidobacterium animalis, Enterococcus durans, and Candida tropicalis; the strain number of Bifidobacterium animalis is CICC 6165, the strain number of Enterococcus durans is CICC 20379, and the strain number of Candida tropicalis is CICC 1779.

[0007] The present invention provides the application of the microbial composition in the preparation of food additives.

[0008] The present invention provides a method for preparing a food additive from the microbial composition, comprising the following steps: mixing 15-25 parts by weight of Chinese yam with water, heating until the Chinese yam is cooked, cooling and then pulping to obtain a Chinese yam slurry; mixing 10-20 parts by weight of selenium-enriched black beans, 8-12 parts by weight of wolfberries, 1-4 parts by weight of liquorice, 5-11 parts by weight of wheat and 4-10 parts by weight of tremella with water, adding a mixed bacterial solution of Bifidobacterium animalis, Enterococcus durans and Candida tropicalis, fermenting, filtering to obtain a filtrate, and obtaining a fermentation broth; mixing the Chinese yam slurry with the fermentation broth, standing for 8-12 h, and drying to obtain the food additive.

[0009] Preferably, the preparation of the mixed bacterial solution comprises the following steps: after activating Bifidobacterium animalis, Enterococcus durans and Candida tropicalis respectively, culturing at 32-37 °C until the bacterial concentration reaches 1×10 8 -6×10 8 cfu / mL of bacterial solution, and mixing in sequence according to the volume ratio of Bifidobacterium animalis bacterial solution, Enterococcus durans bacterial solution and Candida tropicalis bacterial solution of 1:(0.1-3.5):(0.1-3.5).

[0010] Preferably, the temperature of the fermentation is 28-37 °C, and the time of the fermentation is 12-84 h.

[0011] The present invention provides a food additive prepared by the method.

[0012] The present invention provides the use of the food additive prepared by the method or the food additive in the preparation of a sleep aid product.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention first proposes to use a combination of microorganisms, namely Bifidobacterium animalis (CICC 6165), Enterococcus durans (CICC 20379) and Candida tropicalis (CICC 1779), to ferment Chinese yam, selenium-enriched black beans, wolfberries, liquorice, wheat and tremella, degrade macromolecules in the above raw materials into small molecules, and convert selenium elements in selenium-enriched black beans into organic selenium that is more easily absorbed by the body. The additive obtained by fermentation has the functions of nourishing yin and moistening the lungs, regulating the balance of yin and yang and qi and blood, and has a certain effect on improving sleep. Detailed implementation manners

[0015] The present invention provides a microbial composition, which includes Bifidobacterium animalis, Enterococcus durans, and Candida tropicalis. The strain number of Bifidobacterium animalis is CICC 6165, the strain number of Enterococcus durans is CICC 20379, and the strain number of Candida tropicalis is CICC 1779. The Bifidobacterium (CICC6165), Enterococcus durans (CICC 20379), and Candida tropicalis (CICC 1779) in the present invention are all commercially purchased from the China Center for Industrial Culture Collection.

[0016] The present invention also provides a method for preparing a food additive from the microbial composition, which includes the following steps: Mix 15 - 25 parts by weight of Chinese yam with water, heat until the Chinese yam is cooked, cool and then make a pulp to obtain a Chinese yam pulp solution; Mix 10 - 20 parts by weight of selenium - rich black beans, 8 - 12 parts by weight of wolfberries, 1 - 4 parts by weight of liquorice, 5 - 11 parts by weight of wheat, and 4 - 10 parts by weight of tremella with water, add the mixed bacterial solution of Bifidobacterium animalis, Enterococcus durans, and Candida tropicalis, ferment, filter to obtain the filtrate to get a fermentation broth; Mix the Chinese yam pulp solution with the fermentation broth, stand for 8 - 12 h, and dry to obtain the food additive. The fermentation temperature in the present invention is 28 - 37°C, preferably 30 - 35°C; the fermentation time is 12 - 84 h, preferably 24 - 72 h.

[0017] The present invention uses the mixed bacterial solution prepared from Bifidobacterium animalis, Enterococcus durans, and Candida tropicalis to ferment selenium - rich black beans, wolfberries, liquorice, wheat, and tremella simultaneously, which can degrade the macromolecules in the above - mentioned raw materials into small molecules, obtain small - molecule active ingredients helpful for sleep, and at the same time convert the selenium element in the selenium - rich black beans into organic selenium, which is more conducive to the body's absorption and utilization. The purpose of mixing the Chinese yam pulp with the fermentation broth and standing in the present invention is to further react the components in the fermentation broth with the components in the Chinese yam pulp solution, which is beneficial to the release of the active ingredients in the Chinese yam.

[0018] In the present invention, the Chinese yam has the functions of invigorating the spleen and nourishing the stomach, promoting the production of body fluid and benefiting the lungs, and tonifying the kidney and arresting seminal emission; it can improve the intestines, regulate blood sugar and blood pressure, improve hormone levels, enhance immunity, and nourish yin and moisten the lungs. The selenium-rich black beans have the functions of benefiting essence and improving eyesight, nourishing blood and expelling wind, promoting diuresis and detoxifying; they can resist oxidation, enhance immunity and protect the eyes, and especially the selenium element can improve sleep. The wolfberries have the functions of tonifying the liver and kidney and improving eyesight; they can resist fatigue, improve sleep, resist oxidation and delay aging. The licorice has the functions of invigorating the spleen and replenishing qi, relieving cough and resolving phlegm, and alleviating spasm and relieving pain, and is a food homologous to medicine for replenishing qi. The wheat has the functions of nourishing the heart and tranquilizing the mind, strengthening the spleen and regulating the stomach, loosening bowel to relieve constipation, replenishing qi and stopping sweating, and consolidating the exterior and stopping sweating. The tremella has the functions of moistening the lungs and promoting the production of body fluid and nourishing yin and nourishing the stomach; it can improve blood circulation, enhance the vitality of the body and improve the immunity of the respiratory tract. The combination of the above-mentioned raw materials homologous to medicine and food in the present invention can regulate the balance of yin and yang in the body, harmonize qi and blood, enhance the vitality of the body, and regulate the zang-fu organs, enabling the body to be in a healthy state, and then the body is helpful for sleep in the resting state.

[0019] In the present invention, the preparation of the mixed bacterial liquid comprises the following steps: After activating Bifidobacterium animalis, Enterococcus durans and Candida tropicalis respectively, they are cultured at 32 - 37 °C until the bacterial concentration reaches 1×10 8 -6×10 8 cfu / mL of the bacterial liquid, and they are mixed in sequence according to the volume ratio of the Bifidobacterium animalis bacterial liquid, Enterococcus durans bacterial liquid and Candida tropicalis bacterial liquid of 1:0.1 - 3.5:0.1 - 3.5. For the activation of Bifidobacterium animalis, Enterococcus durans and Candida tropicalis in the present invention, they are respectively activated twice with MRS solid medium, and the activation culture conditions are culturing at 32 - 38 °C for 12 - 48 h, preferably culturing at 37 °C for 24 h. After activation in the present invention, the active strains are respectively inoculated into MRS liquid medium and cultured with shaking, and the culture conditions are preferably culturing at 36 °C until the bacterial concentration reaches 4×10 8 cfu / mL. The mixing ratio of the mixed bacterial liquid in the present invention is preferably that the volume ratio of the Bifidobacterium animalis bacterial liquid, Enterococcus durans bacterial liquid and Candida tropicalis bacterial liquid is 1:0.5 - 2.0:0.5 - 2.0. The MRS solid medium and MRS liquid medium in the present invention are media well-known in the art, and can be obtained through commercial channels without special instructions.

[0020] The present invention also provides a food additive prepared by the above-mentioned preparation method. The food additive of the present invention has the effects of conditioning the sub-healthy state of the body, enhancing immunity and assisting sleep.

[0021] The present invention provides the application of the food additive prepared by the method or the food additive in the preparation of a sleep-assisting product. The products of the present invention include but are not limited to foods or health foods.

[0022] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well-known to those skilled in the art.

[0023] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope of protection of the present invention.

[0024] The strain numbers of Bifidobacterium animalis, Enterococcus durans, Candida tropicalis, Saccharomyces cerevisiae (CICC 1251), Bifidobacterium longum (CICC 6193) and Bacillus licheniformis (CICC 10037) used in the following examples were all purchased from the China Center for Industrial Culture Collection. Other raw materials or culture media used, unless otherwise specified, can be purchased through commercial channels.

[0025] Example 1

[0026] 1. Preparation of the mixed bacterial liquid: Bifidobacterium animalis (CICC 6165), Enterococcus durans (CICC 20379) and Candida tropicalis (CICC 1779) were respectively inoculated in MRS solid medium and cultured at 37 °C for 24 h. The strains were activated for two generations, and the strains after two generations of activation were inoculated into MRS liquid medium and cultured with shaking at 36 °C until the bacterial concentration reached 4×10 8 cfu / mL of bacterial liquid, and the bacterial liquids of the above three strains were obtained respectively; the bacterial liquid of Bifidobacterium animalis, the bacterial liquid of Enterococcus durans and the bacterial liquid of Candida tropicalis were mixed in a volume ratio of 1:0.8:1.5 in sequence to obtain the mixed bacterial liquid of the microbial composition.

[0027] 2. The preparation of the food additive includes the following steps:

[0028] (1) Peel the Chinese yam, mix 20 parts by weight of peeled Chinese yam with 40 parts by weight of water, heat to boiling, cook the Chinese yam until cooked, take out the Chinese yam and water and cool, then make a pulp to obtain the Chinese yam pulp.

[0029] (2) Bake and crush the selenium-rich black beans, wolfberries, licorice, wheat and tremella fuciformis until they pass through a 4-mesh sieve, then weigh 15 parts by weight of selenium-rich black bean powder, 10 parts by weight of wolfberry powder, 2.5 parts by weight of licorice powder, 8 parts by weight of wheat flour and 7 parts by weight of tremella fuciformis powder, mix evenly, add 20 times the weight of water of the above materials, mix evenly and soak for 12 h to obtain the material mixture.

[0030] (3) Add the mixed bacterial solution prepared in step 1 above to the material mixture at an inoculation amount of 1% (volume percentage), ferment at 33°C for 48 h, filter through an 80-mesh sieve to obtain the filtrate, and obtain the fermentation broth.

[0031] (4) Mix the yam slurry and the fermentation broth evenly, let it stand for 8 h, and perform low-temperature spray drying to obtain the food additive.

[0032] Example 2

[0033] 1. The preparation of the mixed bacterial solution is the same as that in Example 1.

[0034] 2. The preparation of the food additive includes the following steps:

[0035] (1) Peel the yam, mix 15 parts by weight of yam with 30 parts by weight of water, heat to boiling, cook until the yam is cooked, cool and then make a slurry to obtain the yam slurry.

[0036] (2) Dry and crush the selenium-rich black beans, wolfberries, licorice, wheat, and tremella fuciformis respectively until they pass through a 6-mesh sieve. Weigh 10 parts by weight of selenium-rich black bean powder, 8 parts by weight of wolfberry powder, 1 part by weight of licorice powder, 5 parts by weight of wheat powder, and 4 parts by weight of tremella fuciformis powder, mix evenly, then add 15 times the weight of the above materials in water, mix evenly, and soak for 8 h to obtain the material mixture.

[0037] (3) Add the mixed bacterial solution prepared in step 1 above to the material mixture at an inoculation amount of 0.5% (volume percentage), ferment at 30°C for 24 h, filter through a 90-mesh sieve to obtain the filtrate, and obtain the fermentation broth.

[0038] (4) Mix the yam slurry and the fermentation broth evenly, let it stand for 6 h, and perform spray drying to obtain the food additive.

[0039] Example 3

[0040] 1. The preparation of the mixed bacterial solution is the same as that in Example 1.

[0041] 2. The preparation of the food additive includes the following steps:

[0042] (1) Peel the yam, mix 25 parts by weight of yam with 50 parts by weight of water, heat to boiling, cook until the yam is cooked, take out the yam and water and cool, and then make a slurry to obtain the yam slurry.

[0043] (2) Dry and crush the selenium-rich black beans, wolfberries, licorice, wheat, and tremella fuciformis respectively until they pass through an 8-mesh sieve. Weigh 20 parts by weight of selenium-rich black bean powder, 12 parts by weight of wolfberry powder, 4 parts by weight of licorice powder, 11 parts by weight of wheat powder, and 10 parts by weight of tremella fuciformis powder, mix evenly, then add 30 times the weight of the above materials in water, mix evenly, and soak for 18 h to obtain the material mixture.

[0044] (3) Add the mixed bacterial solution prepared in the above step 1 to the material mixture at an inoculation amount of 1.5% (volume percentage), ferment at 35 °C for 72 h, filter through a 100-mesh sieve to obtain the filtrate, and obtain the fermentation broth;

[0045] (4) Mix the yam slurry and the fermentation broth evenly, let it stand for 12 h, and spray dry to obtain the food additive.

[0046] Comparative Example 1

[0047] The difference from Example 1 is that Candida tropicalis (CICC 1779) is replaced with Saccharomyces cerevisiae (CICC1251), and the remaining steps are the same as those in Example 1.

[0048] Comparative Example 2

[0049] The difference from Example 1 is that Bifidobacterium animalis (CICC 6165) is replaced with Bifidobacterium longum (CICC6193), and the remaining steps are the same as those in Example 1.

[0050] Comparative Example 3

[0051] The difference from Example 1 is that Enterococcus durans (CICC 20379) is replaced with Bacillus licheniformis (CICC10037), and the remaining steps are the same as those in Example 1.

[0052] Comparative Example 4

[0053] The difference from Example 1 is that the yam in step 2 is replaced with taro, and the remaining steps are the same as those in Example 1.

[0054] Comparative Example 5

[0055] The difference from Example 1 is that the wolfberry in step 2 is replaced with polygonatum, and the remaining steps are the same as those in Example 1.

[0056] Comparative Example 6

[0057] The difference from Example 1 is that the wheat in step 2 is replaced with hawthorn, and the remaining steps are the same as those in Example 1.

[0058] Experimental Example 1

[0059] 1. Experimental animals: All animal experiments in this study were carried out in accordance with the "Technical Specifications for the Inspection and Evaluation of Health Foods" (2003 edition), and the implementation plan passed the review and approval of the ethics committee. The SPF mice were fed and adapted in the laboratory conditions for 3 d, and the mice had free access to food and water.

[0060] 2. Grouping, gavage, and observation of mouse status: The mice were randomly divided into 3 groups, namely the normal control group, the model group, and the food additive group of Example 1, with 10 mice in each group. The test substances were administered by gavage at a dose of 0.03 g / kg per day for 30 days. The status of the mice was observed during the experiment. The mice were fasted for 12 h before the last administration, but water was not restricted. One hour after gavage on the 30th day, except for the normal control group, the other groups were intraperitoneally injected with sodium pentobarbital (30 mg / kg) once, and the administration time, the disappearance time of the righting reflex, and the recovery time of the righting reflex were recorded. The sleep latency and sleep duration of the mice were calculated respectively (the time from the injection of sodium pentobarbital to the disappearance of the righting reflex was the sleep latency, and the time from the disappearance of the righting reflex to the reappearance of the righting reflex was the sleep duration). The results are shown in Table 1.

[0061] After observing the status of the mice in each group after 30 days of gavage, the mice in each group had good status. After feeding the food additive prepared in Example 1 of the present invention, there were no adverse reactions within 30 days, and the mental state was good.

[0062] Table 1 Sleep latency and sleep time of mice in each group

[0063]

[0064] As can be seen from Table 1, after the mice were intraperitoneally injected with sodium pentobarbital (30 mg / kg) once, the food additive group of mice was continuously given for 30 days, and the sleep latency was shortened, showing a significant difference compared with the model group. Compared with the model group of mice, the sleep duration of the food additive group of mice was significantly prolonged. It indicates that the food additive prepared in Example 1 can better improve the sleep condition of mice.

[0065] 3. Sampling and detection: Blood was collected from the orbital cavity 24 h after the injection of sodium pentobarbital. The whole blood of the mice obtained by separation was centrifuged at 4°C and 3500 r / min for 8 min, and the supernatant serum was collected. The supernatant serum after centrifugation was used to measure the levels of NOS and 5-HT in the serum using a mouse nitric oxide synthase (NOS) ELISA kit and a mouse 5-hydroxytryptamine (5-HT) ELISA kit respectively. The results are shown in Table 2.

[0066] Table 2 Levels of NOS and 5-HT in the serum of mice in each group

[0067]

[0068] Verify the sleep status of mice by detecting the contents of sleep-promoting factors 5-HT and NOS in mouse serum. As can be seen from Table 2, compared with the normal control group, the contents of 5-HT and NOS in the serum of mice in the model group increased, indicating an increase in the secretion of sleep-promoting factors in the mouse brain and successful modeling; compared with the normal control group, the contents of 5-HT and NOS in the serum of mice in the food additive group increased, indicating that the food additive prepared by the present invention has a sleep-promoting effect.

[0069] Immediately decapitate and sacrifice the mice in each group after blood collection. Immediately dissect and isolate the hippocampal region of the mouse brain on an ice plate. After rinsing with pre-cooled PBS buffer, dry it and weigh the same mass of brain tissue and chop it up. Then put it into a cold tissue grinder, add 12 times the amount of pre-cooled PBS buffer and grind it into a homogenate on ice. Centrifuge at 4°C and 20,000 rpm for 15 min, collect the supernatant to obtain a brain tissue homogenate sample. Determine the contents of GABA, Glu, NE and SOD in the mouse brain tissue according to the instructions of a commercially available ELISA kit. The results are shown in Table 3.

[0070] Table 3 Contents of GABA, Glu, NE, SOD and MDA in the brain tissues of mice in each group

[0071]

[0072] As can be seen from the results in Table 3, the food additive prepared in Example 1 of the present invention can increase the contents of GABA and SOD in the mouse brain tissue and decrease the contents of Glu and NE, indicating that the food additive prepared by the technical solution of the present invention has a significant effect of improving and promoting sleep.

[0073] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a food additive, characterized in that, It includes the following steps: Mix 15 - 25 parts by weight of Chinese yam with water, heat until the Chinese yam is cooked, cool and then make a pulp to obtain a Chinese yam pulp solution; Mix 10 - 20 parts by weight of selenium - rich black beans, 8 - 12 parts by weight of wolfberries, 1 - 4 parts by weight of licorice, 5 - 11 parts by weight of wheat and 4 - 10 parts by weight of tremella with water, add a mixed bacterial solution of Bifidobacterium animalis, Enterococcus durans and Candida tropicalis, ferment, filter to obtain a filtrate, and obtain a fermentation broth; Mix the Chinese yam pulp solution with the fermentation broth, let it stand for 8 - 12 h, and dry to obtain a food additive; The strain number of the Bifidobacterium animalis is CICC6165, the strain number of the Enterococcus durans is CICC20379, and the strain number of the Candida tropicalis is CICC1779; The preparation of the mixed bacterial solution comprises the following steps: After activating Bifidobacterium animalis, Enterococcus durans, and Candida tropicalis respectively, they are cultured at 32-37 °C until the bacterial concentration reaches 1×10 8 -6×10 8 cfu / mL of the bacterial solution, and they are mixed in sequence according to the volume ratio of the Bifidobacterium animalis bacterial solution, Enterococcus durans bacterial solution, and Candida tropicalis bacterial solution being 1:(0.1-3.5):(0.1-3.5); The temperature of the fermentation is 28 - 37 °C, and the fermentation time is 12 - 84 h.

2. A food additive prepared by the method according to claim 1.

3. Use of the food additive prepared by the method according to claim 1 or the food additive according to claim 2 in the preparation of a sleep - aid product.

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