Fermentation method of alcohol decomposition probiotic composition and probiotic composition

Abstract

The invention discloses a fermentation method of an alcohol decomposition probiotic combination and the probiotic combination. The fermentation method comprises the following steps: mixing seed solutions of lactobacillus plantarum, lactobacillus rhamnosus and lactobacillus paracasei, inoculating a fermentation medium with the mixed seed solutions, and fermenting; the fermentation temperature is 28-35 DEG C, and the fermentation time is 40-60 hours. The lactobacillus plantarum, the lactobacillus rhamnosus and the lactobacillus paracasei are creatively subjected to mixed fermentation, the mixed fermentation can generate a synergistic effect, the biomass of a fermentation product is increased in unit time, and a foundation is laid for wide application in industrial production.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, in particular to a fermentation method of an alcohol-decomposing probiotic combination and the technical field of the probiotic combination. [Background technology]

[0002] Alcoholic beverages are an important part of human civilization, especially in China, where the drinking custom is deep-rooted and deeply influenced by the traditional concept of "there must be wine at banquets". Whether it is workplace communication or gatherings with relatives and friends, strong liquor is often a must-have choice to add to the atmosphere. However, drinking a lot of alcohol in a short period of time can cause adverse reactions such as nausea, headache, vomiting and memory loss, while long-term excessive drinking may induce a series of serious health problems, including mild alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis and cirrhosis, and even develop into irreversible alcoholic cirrhosis, alcoholic liver failure, cancer, hypertension, stroke and coronary heart disease, which seriously threaten people's quality of life. In addition, statistics show that traffic accidents and criminal acts caused by drinking have become a major burden on society.

[0003] Currently, the hangover relieving products on the market are mainly divided into chemical drugs (such as tablets or capsules), Chinese herbal extracts (such as candies, powders, capsules or drinks) and microbial fermentations (mainly beverages).

[0004] Plant lactobacillus is a kind of microorganism, plant lactobacillus, anaerobic or facultative anaerobic, strain is straight or curved rod-shaped, single, sometimes in pairs or in chains, optimum pH about 6.5, belongs to homotypic fermentation lactic acid bacteria. The viable count of this bacterium is higher, and can decompose alcohol aldehydes (such as ethanol, acetaldehyde etc.) to produce acid when optimizing fermentation conditions. Therefore plant lactobacillus is an important component of microbial fermentation products for alcohol sobering up, but the acquisition of existing plant lactobacillus is mostly fermentation acquisition, and in the fermentation process, for ensuring the quality of plant lactobacillus, plant lactobacillus is mostly fermented separately, and this kind of fermentation method has low efficiency, slow growth rate, long cycle, and few outputs. [Summary of the invention]

[0005] The purpose of the present invention is to solve the problems in the prior art and to provide a fermentation method and a probiotic combination for alcohol decomposing probiotics. Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei are mixed and fermented. The mixed fermentation produces a synergistic effect and increases the biomass of the fermentation product per unit time.

[0006] To achieve the above object, the present invention proposes a fermentation method of an alcohol-decomposing probiotic combination, wherein the seed liquid of Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei are mixed and inoculated into a fermentation medium for fermentation;

[0007] The fermentation temperature is 28-35°C, the fermentation time is 40-60h, and a mixed fermentation liquid is obtained.

[0008] Preferably, the fermentation medium comprises 15-25 g / L of glucose, 8-12 g / L of peptone, 8-12 g / L of beef extract, 8-12 g / L of yeast extract powder, 1-2.8 g / L of K2HPO4, 4-5.8 g / L of sodium acetate, 0.18-0.26 g / L of magnesium sulfate, 0.04-0.08 g / L of manganese sulfate, 0.7-1.6 mL / L of Tween 80 and water.

[0009] Preferably, the fermentation pH is 4.6-7.5.

[0010] Preferably, the Lactobacillus plantarum, the Lactobacillus rhamnosus and the Lactobacillus paracasei need to be diluted separately before inoculation.

[0011] Preferably, the plant lactobacillus is diluted to (3-7)×10 8 CFU / mL; dilute the Lactobacillus rhamnosus to (5-9)×10 7 CFU / mL; dilute the Lactobacillus paracasei to (3-7)×10 7 CFU / mL.

[0012] Preferably, the seed liquids of the Lactobacillus plantarum, the Lactobacillus rhamnosus and the Lactobacillus paracasei are mixed and inoculated into a fermentation medium for fermentation and stirred at a stirring speed of 40-60 r / min.

[0013] Preferably, the inoculation amount of Lactobacillus plantarum is 1-8%, the inoculation amount of Lactobacillus rhamnosus is 1-6%, and the inoculation amount of Lactobacillus paracasei is 1-6%.

[0014] Preferably, the protective agent is any one or any combination of skimmed milk powder, lactose, trehalose, ascorbic acid, glycerol or sorbitol.

[0015] Preferably, the mixed fermentation liquid is centrifuged to obtain bacterial sludge, and the bacterial sludge is mixed with a protective agent and then freeze-dried to obtain a dry powder.

[0016] Another object of the present invention is to provide a probiotic combination prepared by the fermentation method of any of the above alcohol-decomposing probiotic combinations.

[0017] The invention discloses a fermentation method of an alcohol-decomposing probiotic combination and the beneficial effects of the probiotic combination: the invention creatively ferments Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei, and the mixed fermentation produces a synergistic effect, thereby increasing the biomass of the fermentation product per unit time, and laying a theoretical foundation for its wide application in industrial production; wherein the number of viable bacteria of Lactobacillus plantarum is relatively high, and it can decompose alcohol aldehydes to produce acid under optimized fermentation conditions, and can be used as a main bacterial species for alcohol decomposition; Lactobacillus rhamnosus can play a synergistic role in the mixed fermentation process to increase the yield; Lactobacillus paracasei has antibacterial ability and good cell adhesion characteristics, and the characteristics can enable the probiotics to stably colonize in the gastrointestinal tract; and the three bacteria can also synergize in the human body to improve the alcohol decomposition effect in the human body. The fermentation method of the invention is different from the traditional fermentation method, which has the disadvantages of low yield, poor activity and the like, and optimizes the fermentation process to achieve high yield, high vitality and high efficiency.

[0018] The features and advantages of the present invention will be described in detail through embodiments in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0019] Figure 1 It's OD 600 Schematic diagram of the growth curve of the absorbance test. [Specific implementation method]

[0020] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0021] In the description of the present invention, it should be noted that when an element is referred to as being "fixed to" or "disposed on" another element, it may be directly on the other element or indirectly on the other element. When an element is referred to as being "connected to" another element, it may be directly connected to the other element or indirectly connected to the other element.

[0022] In the description of the present invention, it should be noted that the terms "center", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, or the positions or positional relationships in which the invention product is usually placed when in use, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", "third" and the like are only used to distinguish descriptions, and cannot be understood as indicating or implying relative importance. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "multiple" means two or more, unless otherwise clearly and specifically defined. "Several" means one or more, unless otherwise clearly and specifically defined.

[0023] In the description of the present invention, it is also necessary to explain that, unless otherwise clearly specified and limited, the terms "set", "install", "connect", and "connect" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0024] Embodiment 1:

[0025] The present invention discloses a fermentation method of an alcohol-decomposing probiotic combination. First, a culture medium is prepared. The culture medium ratio in this embodiment is as follows: 18 g / L glucose, 9 g / L peptone, 10 g / L beef extract, 9.5 g / L yeast extract powder, 1.5 g / L K2HPO4, 5.2 g / L sodium acetate, 0.22 g / L magnesium sulfate, 0.06 g / L manganese sulfate, 1.2 mL / L Tween 80, ultrapure water is added to 3 L, pH is adjusted to 6.5, and sterilized at 115° C. for 30 min.

[0026] Then inoculate, dilute the seeds before inoculation, and dilute Lactobacillus plantarum to 5×10 8 CFU / mL, Lactobacillus rhamnosus 7×10 7 CFU / mL, Lactobacillus paracasei 5×10 7 CFU / mL, each strain was inoculated with the above culture medium at a rate of 4%, the stirring speed was 50r / min (stirring effect prevented a large number of bacteria from sinking to the bottom), and the fermentation culture was carried out at 30°C for 50h to obtain the fermentation liquid;

[0027] The fermentation liquid is centrifuged to obtain bacterial sludge, and the bacterial sludge is mixed with a protective agent and freeze-dried to obtain dry powder.

[0028] Lactobacillus plantarum, anaerobic or facultative anaerobic, is a straight or curved rod-shaped bacterium, either alone, in pairs or in chains, with an optimum pH of around 6.5. It is a homofermentative lactic acid bacterium. The number of live cells of this bacterium is relatively high, and it can decompose alcohols and aldehydes (such as ethanol, acetaldehyde, etc.) to produce acid under optimized fermentation conditions.

[0029] Lactobacillus rhamnosus is an anaerobic, acid-resistant, non-spore-forming Gram-positive probiotic. Lactobacillus rhamnosus cannot utilize lactose, but can ferment a variety of monosaccharides (glucose, arabinose, maltose, etc.). Most strains can produce a small amount of soluble ammonia, but do not produce indole and hydrogen sulfide. It has biological characteristics such as acid resistance, bile salt resistance, and resistance to multiple antibiotics. It can play a synergistic role in the mixed fermentation process to increase production.

[0030] Lactobacillus paracasei is a Gram-positive bacterium with a rod-shaped body (0.8-1.0×2.0-4.0 μm), square at both ends, arranged alone or in chains, without flagella, and not moving. It is facultatively anaerobic, with a growth temperature range of 10°C to 40°C, and an optimal growth temperature of 37°C. The G+C content of DNA is 45-47 mol%. The strain has antibacterial ability and good cell adhesion properties, which can enable probiotics to stably colonize in the gastrointestinal tract.

[0031] The fermentation method of the present invention is different from the traditional fermentation method which has the disadvantages of low yield, poor activity and the like, and optimizes the fermentation process to achieve high yield, high activity and high efficiency.

[0032] Comparative Example 1:

[0033] The inoculum volume was 12% and diluted to 2.07 × 10 8 CFU / mL of plant lactobacillus was inoculated into the above culture medium; the stirring speed was 50r / min (the stirring effect prevented the bacteria from sinking to the bottom in large quantities), and the culture was fermented at 30°C for 50h to obtain a fermentation liquid;

[0034] The fermentation liquid is centrifuged to obtain bacterial sludge, and the bacterial sludge is mixed with a protective agent and freeze-dried to obtain dry powder.

[0035] Comparative Example 2:

[0036] The inoculum volume was 12% and diluted to 2.07 × 10 8 CFU / mL of Lactobacillus rhamnosus was inoculated into the above culture medium; the stirring speed was 50 r / min (the stirring effect prevented the bacteria from sinking to the bottom in large quantities), and the culture was fermented at 30° C. for 50 hours to obtain a fermentation liquid;

[0037] The fermentation liquid is centrifuged to obtain bacterial sludge, and the bacterial sludge is mixed with a protective agent and freeze-dried to obtain dry powder.

[0038] Comparative Example 3:

[0039] The inoculum volume was 12% and diluted to 2.07 × 10 8 CFU / mL of Lactobacillus paracasei was inoculated into the above culture medium; the stirring speed was 50 r / min (the stirring effect prevented the bacteria from sinking to the bottom in large quantities), and the culture was fermented at 30° C. for 50 h to obtain a fermentation liquid;

[0040] The fermentation liquid is centrifuged to obtain bacterial sludge, and the bacterial sludge is mixed with a protective agent and freeze-dried to obtain dry powder.

[0041] Bacterial growth test:

[0042] OD 600 Absorbance test: Samples were taken from Example 1 and Comparative Examples 1 to 3 at different time periods during the fermentation process to test OD 600 The absorbance is measured and the growth curve is drawn. The growth curve is shown in the attached figure of the instruction manual. Figure 1 .

[0043] From the attached drawings Figure 1 It can be seen that when the total amount of inoculation is basically close, the growth rate of the mixed flora and the final number of viable bacteria in the final flora of Example 1 are greater than those when Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus paracasei are cultured separately. This proves that when only three strains are mixed and fermented together in the present application, a synergistic effect can be produced, and the biomass of the fermentation product can be increased per unit time, which lays a theoretical foundation for its wide application in industrial production. The mixed fermentation of the three strains produces an effect of 1+1+1 greater than 3.

[0044] Live colony count: dilute the fermentation broth 10 -6 , 10 -7 , 10 -8 Then 100 μL was taken to plate, and each gradient was repeated 3 times, and the number of viable bacteria was counted.

[0045] Among them, the number of viable bacteria (CFU / mL) = the average number of colonies on the same dilution gradient plate × dilution factor × 10. The obtained data is as follows:

[0046] Lactobacillus plantarum Lactobacillus rhamnosus Lactobacillus paracasei Embodiment 1 <![CDATA[6.3*10 10 ]]> <![CDATA[4.8*10 10 ]]> <![CDATA[3.8*10 10 ]]> Comparative Example 1 <![CDATA[4.9*10 10 ]]> / / Comparative Example 2 / <![CDATA[3.3*10 10 ]]> / Comparative Example 3 / / <![CDATA[2.5*10 10 ]]>

[0047] It can be seen from the above table that when Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus paracasei are mixed and fermented, the number of viable bacteria is significantly increased compared to when they are cultured alone. This can further prove that when only three strains of the present application are mixed and fermented together, a synergistic effect can be produced, and the biomass of the fermentation product can be increased per unit time. This has laid a theoretical foundation for its wide application in industrial production, and the mixed fermentation of the three strains produces an effect of 1+1+1 greater than 3.

[0048] Alcohol decomposition test:

[0049] Functional manifestations of hangover: after mice are gavaged with white wine, they will become dizzy, drunk, and even die.

[0050] Among them, the upper manifestations include: excitement, abnormal activities, dragging backwards, etc., but the righting reflex exists.

[0051] Symptoms of drunkenness include: sluggishness, drowsiness, and loss of righting reflex.

[0052] Preparation of experimental mice: Male mice from the same batch were fed normally in the animal laboratory for one week to ensure that the vital signs of the mice were consistent and normal, with an average weight of 22 g.

[0053] Experimental method: Divide the mice into three groups, each with 3 mice. Group 1 was not fed anything, serving as a negative control group; Group 2 was fed water at 5 ml / kg, serving as a placebo control group; Group 3 was prepared by dissolving 1 g of the dry powder obtained by fermentation in this example in 300 ml of water to obtain a solution, and feeding the mice with the solution at 5 ml / kg, serving as a positive control group.

[0054] Five minutes later, the three groups of mice were gavaged with alcohol. In this experiment, Red Star Erguotou with an alcohol content of 43 degrees was used, at a rate of 15 ml / kg. The mental state of the mice, blood alcohol content, and number of deaths were recorded at 10 minutes, 30 minutes, and 50 minutes. The data obtained are as follows:

[0055]

[0056] It can be seen from the results in the above table that compared with other control groups, the mice that took the dry powder prepared in this embodiment had a rapid decrease in blood alcohol content and a rapid recovery in mental state within the same period of time. Therefore, the dry powder of this embodiment has a certain ability to sober up and can be used to assist in sobering up.

[0057] Embodiment 2:

[0058] First, prepare the culture medium. The culture medium ratio in this embodiment is: glucose 15g / L, peptone 8g / L, beef extract 8g / L, yeast extract powder 8g / L, K2HPO41g / L, sodium acetate 4g / L, magnesium sulfate 0.18g / L, manganese sulfate 0.04g / L, Tween 80 0.7mL / L, ultrapure water is added to 3L, pH is adjusted to 4.6, and sterilized at 115°C for 30min; then inoculate, dilute various seeds before inoculation, plant lactobacillus to 3×10 8 CFU / mL, Lactobacillus rhamnosus 5×10 7 CFU / mL Lactobacillus paracasei 4×10 7CFU / mL, inoculated with the above culture medium, stirred at a speed of 50 r / min (stirring to prevent a large amount of bacteria from sinking to the bottom), and fermented at 28°C for 40 h to obtain a fermentation liquid;

[0059] The fermentation liquid is centrifuged to obtain bacterial sludge, and the bacterial sludge is mixed with a protective agent and freeze-dried to obtain dry powder.

[0060] Embodiment three:

[0061] First, prepare the culture medium. The culture medium ratio of this embodiment is: glucose 25g / L, peptone 12g / L, beef extract 12g / L, yeast extract powder 12g / L, K2HPO4 2.8g / L, sodium acetate 5.8g / L, magnesium sulfate 0.26g / L, manganese sulfate 0.08g / L, Tween 80 1.6mL / L, ultrapure water is added to 3L, pH is adjusted to 7.5, and sterilized at 115°C for 30min; then inoculate, dilute various seeds before inoculation, plant lactobacillus to 7×10 8 CFU / mL, Lactobacillus rhamnosus 9×10 7 CFU / mL Lactobacillus paracasei 7×10 7 CFU / mL, inoculated with the above culture medium, stirred at a speed of 50 r / min (stirring to prevent a large amount of bacteria from sinking to the bottom), and fermented at 35°C for 60 h to obtain a fermentation liquid;

[0062] The fermentation liquid is centrifuged to obtain bacterial sludge, and the bacterial sludge is mixed with a protective agent and freeze-dried to obtain dry powder.

[0063] Comparative experiment:

[0064] Prepare the experimental mice again: male mice from the same batch were fed normally in the animal laboratory for one week to ensure that the vital signs of the mice were consistent and normal, with an average weight of 22 g.

[0065] Experimental method: Divide into three groups, each with 3 mice. In the first group, 1 g of the dry powder obtained by fermentation in Example 1 was dissolved in 300 ml of water to obtain a solution, and the solution was fed to mice at a rate of 5 ml / kg, which was used as Example 1; in the second group, 1 g of the dry powder obtained by fermentation in Example 2 was dissolved in 300 ml of water to obtain a solution, and the solution was fed to mice at a rate of 5 ml / kg, which was used as Example 2; in the third group, 1 g of the dry powder obtained by fermentation in Example 3 was dissolved in 300 ml of water to obtain a solution, and the solution was fed to mice at a rate of 5 ml / kg, which was used as Example 3.

[0066] Five minutes later, the three groups of mice were gavaged with alcohol. In this experiment, Red Star Erguotou with an alcohol content of 43 degrees was used, at a rate of 15 ml / kg. The mental state of the mice, blood alcohol content, and number of deaths were recorded at 10 minutes, 30 minutes, and 50 minutes. The data obtained are as follows:

[0067]

[0068]

[0069] As can be seen from the results in the above table, compared with the other embodiment groups, the mice taking the dry powder prepared in Example 1 had a faster reduction in blood alcohol content and a faster recovery of mental state within the same period of time. Therefore, Example 1 has the best hangover-relieving effect.

[0070] The standard parts used in this application document can all be purchased from the market, and the specific connection methods of each part adopt mature conventional means such as bolts, rivets, welding, etc. in the prior art. The electric slide rail slide, cylinder, welding machine, electric telescopic rod and internal components of the controller all adopt conventional models in the prior art, and their internal structures belong to the prior art structure. Workers can complete normal operation of them according to the existing technical manual. In addition, the circuit connection adopts the conventional connection method in the prior art, and no specific description is given here.

[0071] It should be noted that, although the above embodiments have been described in this article, the scope of patent protection of the present invention is not limited thereby. Therefore, based on the innovative concept of the present invention, changes and modifications made to the embodiments described herein, or equivalent structures or equivalent process changes made using the contents of the present specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields are all included in the scope of protection of the patent of the present invention.

Claims

1. A fermentation method of an alcohol-decomposing probiotic combination, characterized in that: The seed liquids of Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus paracasei are mixed and inoculated into a fermentation medium for fermentation; The fermentation temperature is 28-35°C, the fermentation time is 40-60h, and a mixed fermentation liquid is obtained.

2. The fermentation method of the alcohol-decomposing probiotic combination according to claim 1, characterized in that: The fermentation medium comprises 15-25 g / L of glucose, 8-12 g / L of peptone, 8-12 g / L of beef extract, 8-12 g / L of yeast extract powder, 1-2.8 g / L of K2HPO4, 4-5.8 g / L of sodium acetate, 0.18-0.26 g / L of magnesium sulfate, 0.04-0.08 g / L of manganese sulfate, 0.7-1.6 mL / L of Tween 80 and water.

3. The fermentation method of the alcohol-decomposing probiotic combination according to claim 1, characterized in that: The fermentation temperature is 28-35℃ and the fermentation time is 40-60h.

4. The fermentation method of the alcohol-decomposing probiotic combination according to claim 1, characterized in that: Before inoculation, the Lactobacillus plantarum, the Lactobacillus rhamnosus and the Lactobacillus paracasei need to be diluted respectively.

5. The fermentation method of the alcohol-decomposing probiotic combination according to claim 4, characterized in that: Before inoculation, the plant lactobacillus was diluted to (3-7)×10 8 CFU / mL; dilute the Lactobacillus rhamnosus to (5-9)×10 7 CFU / mL; dilute the Lactobacillus paracasei to (3-7)×10 7 CFU / mL.

6. The fermentation method of the alcohol-decomposing probiotic combination according to claim 1, characterized in that: The seed liquids of the plant lactobacillus, the rhamnosus lactobacillus and the paracasei lactobacillus are mixed and inoculated into a fermentation medium for fermentation while maintaining a stirring speed of 40-60 r / min for stirring.

7. The fermentation method of the alcohol-decomposing probiotic combination according to claim 1, characterized in that: The inoculation amount of the Lactobacillus plantarum is 1-8%, the inoculation amount of the Lactobacillus rhamnosus is 1-6%, and the inoculation amount of the Lactobacillus paracasei is 1-6%.

8. The fermentation method of the alcohol-decomposing probiotic combination according to claim 1, characterized in that: The protective agent is any one of skimmed milk powder, lactose, trehalose, ascorbic acid, glycerol or sorbitol, or any combination thereof.

9. The fermentation method of the alcohol-decomposing probiotic combination according to claim 1, characterized in that: The mixed fermentation liquid is centrifuged to obtain bacterial sludge, and the bacterial sludge is mixed with a protective agent and then freeze-dried to obtain dry powder.

10. A probiotic combination, characterized in that: The alcohol-decomposing probiotic combination is prepared by the fermentation method according to any one of claims 1 to 9.

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