A preparation method and application of probiotic culture medium

Abstract

The present invention discloses a preparation method and application of a probiotic culture medium, wherein the preparation method of the probiotic culture medium comprises the following steps: homogenizing rice wine residue to obtain a homogenate; performing wall-breaking treatment on the homogenate and then extracting to obtain pretreated rice wine residue; adding ultrapure water to the pretreated rice wine residue, sterilizing and centrifuging, collecting the supernatant, and obtaining a rice wine residue extract, which is a probiotic culture medium. The present invention uses rice wine residue as raw material, and prepares a rice wine residue extract after homogenization, wall-breaking, extraction, sterilization, and centrifugation. The extract is used as a culture medium for probiotic fermentation and culture, which can provide necessary carbon sources, nitrogen sources, and rich nutrients for the growth of probiotics, can be suitable for the growth of a variety of probiotics, has good culture effect, and has the advantages of low raw material cost and wide source, and can also improve the utilization rate of rice wine residue, and realize the resource recycling of rice wine residue.

Description

Technical Field

[0001] The invention relates to the technical field of probiotic preparations, and in particular to a preparation method of a probiotic culture medium and application thereof. Background Art

[0002] Currently, the commonly used method of fermenting probiotics is to use MRS culture medium. MRS culture medium is usually composed of more than a dozen raw materials: peptone, beef powder, yeast powder, glucose, Tween-80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, manganese sulfate, agar powder, and distilled water. The preparation is relatively troublesome, and directly purchasing bottled MRS culture medium is expensive, at around 250 yuan per bottle, resulting in the problem of complicated process or high cost of fermenting probiotics. Summary of the invention

[0003] The main purpose of the present invention is to provide a preparation method of a probiotic culture medium and its application, aiming to provide a culture medium suitable for probiotic fermentation culture with simple preparation process and low cost.

[0004] To achieve the above object, the present invention provides a method for preparing a probiotic culture medium, comprising the following steps:

[0005] Homogenize the rice wine residue to obtain a homogenate;

[0006] The homogenate is subjected to cell wall breaking treatment and then leaching to obtain pre-treated rice wine residue;

[0007] Ultrapure water is added to the pretreated rice wine residue, and the mixture is sterilized and centrifuged to collect the supernatant to obtain a rice wine residue extract, which is a probiotic culture medium.

[0008] Optionally, the step of homogenizing the rice wine residue to obtain a homogenate comprises:

[0009] Place the rice wine residue in a homogenizer and homogenize it at a speed of 10000-14000 r / min for 3-8 minutes to obtain a homogenate.

[0010] Optionally, the step of subjecting the homogenate to cell wall breaking treatment and then leaching to obtain pre-treated rice wine residue comprises:

[0011] The homogenate is subjected to a cell wall breaking treatment at an ultrasonic power of 100 to 300 W for 15 to 20 minutes, and then heated and extracted in a water bath at 70 to 80° C. for 30 to 35 minutes.

[0012] Optionally, the step of adding ultrapure water to the pretreated rice wine residue, sterilizing and centrifuging, collecting the supernatant, and obtaining a rice wine residue extract, which is a probiotic culture medium, comprises:

[0013] Ultrapure water is added to the pretreated rice wine residue, sterilized at 120-125° C. for 20-25 min, and then centrifuged at a speed of 4000-6000 r / min for 10-15 min; wherein the mass of the pretreated rice wine residue added to every 100 mL of the ultrapure water is 30-40 g.

[0014] Furthermore, the present invention also provides a probiotic fermentation culture method, comprising the following steps:

[0015] After the probiotics are revived, they are cultured in MRS solid medium and then made into probiotic seed liquid;

[0016] adding the probiotic seed liquid into a probiotic culture medium for fermentation culture;

[0017] Wherein, the probiotic culture medium is prepared by the preparation method of the probiotic culture medium as described above.

[0018] Optionally, the probiotic seed solution is added to a probiotic culture medium for fermentation culture:

[0019] The inoculation amount of the probiotics in the probiotic culture medium is 2-4%; and / or,

[0020] The culture temperature of the fermentation culture is 31-36° C., the shaking culture speed is 100-200 r / min, and the culture time is 22-28 h.

[0021] Optionally, the probiotics include at least one of Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium lactis, and Lactobacillus paracasei.

[0022] Optionally, the probiotics include Lactobacillus plantarum and Lactobacillus reuteri, and the mass ratio of Lactobacillus plantarum to Lactobacillus reuteri is 0.5:1.

[0023] Furthermore, the present invention also provides a method for preparing a probiotic freeze-dried powder, comprising the following steps:

[0024] After the probiotics are revived, they are cultured in MRS solid medium and then made into probiotic seed liquid;

[0025] Adding the probiotic seed liquid into a probiotic culture medium for fermentation to obtain a fermentation liquid;

[0026] Adding rice wine residue after extracting the rice wine residue extract to the fermentation liquid, stirring evenly, and then performing vacuum freeze drying to obtain probiotic freeze-dried powder;

[0027] Wherein, the probiotic culture medium is prepared by the preparation method of the probiotic culture medium as described above.

[0028] Optionally, in the step of adding rice wine residue obtained by extracting rice wine residue extract to the fermentation liquid, stirring evenly, and then performing vacuum freeze drying to obtain probiotic freeze-dried powder:

[0029] The mass of the rice wine residue after extracting the rice wine residue extract is 20-40% of the mass of the fermentation liquid; and / or,

[0030] The freeze-dried thickness is 0.6 to 2.2 cm; and / or,

[0031] The vacuum freeze drying temperature is -45 to -35°C, the vacuum degree is 20 to 80 Pa, and the freeze drying time is 12 to 24 hours.

[0032] In addition, the present invention also provides a probiotic fermented solid beverage, comprising probiotic freeze-dried powder, wherein the probiotic freeze-dried powder is prepared by the preparation method of the probiotic freeze-dried powder as described above.

[0033] In the technical solution provided by the present invention, rice wine residue is used as raw material, and after homogenization, wall breaking, extraction, sterilization and centrifugation, a rice wine residue extract is prepared, which is used as a culture medium for probiotic fermentation and culture, which can provide the necessary carbon source, nitrogen source and rich nutrients for the growth of probiotics, is suitable for the growth of various probiotics, has a good culture effect, and has the advantages of low raw material cost and wide source. It can also improve the utilization rate of rice wine residue and realize the resource recycling of rice wine residue. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 A schematic flow chart of an embodiment of a method for preparing a probiotic culture medium provided by the present invention;

[0036] Figure 2 A schematic diagram of a process of an embodiment of a probiotic fermentation culture method provided by the present invention;

[0037] Figure 3 The present invention is a schematic flow chart of an embodiment of a method for preparing freeze-dried probiotic powder provided by the present invention.

[0038] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0039] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0040] In order to reduce the cost of probiotic fermentation culture, the present invention provides a method for preparing a probiotic culture medium, using rice wine residue as a raw material to prepare a culture medium suitable for probiotic fermentation culture. Figure 1 FIG. 1 is an example of a method for preparing a probiotic culture medium provided by the present invention. Figure 1 As shown, in this embodiment, the preparation method of the probiotic culture medium comprises the following steps:

[0041] Step S10, homogenizing the rice wine residue to obtain a homogenate;

[0042] Step S20, subjecting the homogenate to cell wall breaking treatment and then leaching to obtain pre-treated rice wine residue;

[0043] Step S30, adding ultrapure water to the pretreated rice wine residue, sterilizing and centrifuging, collecting the supernatant, and obtaining the rice wine residue extract, which is the probiotic culture medium.

[0044] First, the rice wine residue is homogenized to obtain a homogenate. The rice wine residue can be the residue left after the rice wine is brewed (i.e., the lees, which is usually the waste in the rice wine production process) or can be obtained by brewing glutinous rice as the raw material. The specific brewing process can refer to the prior art, for example, it can be: glutinous rice → soaking → filtering water → washing → steaming rice → cooling → mixing koji, adding water → filling the cylinder → fermentation → squeezing → clarifying → rice wine, and finally filtering the brewed rice wine to obtain the rice wine residue. Further, in a specific embodiment of the present invention, the homogenization treatment method is: placing the rice wine residue in a homogenizer, homogenizing at a speed of 10000-14000r / min for 3-8min to obtain a homogenate.

[0045] Then, the homogenate is subjected to a cell wall breaking treatment, followed by leaching to obtain pre-treated rice wine residue. In a specific embodiment of the present invention, the cell wall breaking treatment is performed by subjecting the homogenate to a cell wall breaking treatment at an ultrasonic power of 100 to 300 W for 15 to 20 minutes, and the leaching is performed by heating the homogenate in a water bath at 70 to 80° C. for 30 to 35 minutes.

[0046] Next, ultrapure water is added to the pretreated rice wine residue, and after uniform mixing, the mixture is sterilized and centrifuged, and the supernatant is collected to obtain a rice wine residue extract, that is, a probiotic culture medium is prepared. In a specific embodiment of the present invention, when ultrapure water is added to the pretreated rice wine residue, the addition ratio is 30 to 40 g of the mass of the pretreated rice wine residue added to every 100 mL of the ultrapure water; the sterilization is high temperature sterilization, specifically sterilization at 120 to 125° C. for 20 to 25 minutes; the centrifugal speed is 4000 to 6000 r / min, and the centrifugal time is 10 to 15 minutes.

[0047] Rice wine residue is the residue left after glutinous rice is fermented. It still retains the granular shape of rice and is rich in nutrients. It contains a variety of sugars, B vitamins, minerals, and a variety of amino acids. It also has the functions of strengthening the spleen and stomach, relaxing tendons and tendons, relieving pain and removing blood stasis, etc. It also contains the carbon source, nitrogen source, inorganic salts and growth factors required for microbial growth, and is a good raw material for microbial cultivation. However, some alcohol remains in rice wine residue, which will inhibit the growth and reproduction of microorganisms and is not suitable for direct cultivation of probiotics. Therefore, it currently only has some use value in functional foods. The annual output of rice wine is about 3.1 billion liters, and the output of wine residue is about 1 million tons. In the processing process, the utilization rate of rice wine residue is low and the waste is large. Its value has not been effectively explored, and it is generally considered to be highly polluting because the discharge of rice wine residue may have a negative impact on the ecosystem.

[0048] The inventor has found through research that the starch and protein contained in glutinous rice or rice are degraded to varying degrees after the rice wine fermentation process in the early stage, forming a large number of nutrients that can be directly used by microorganisms. The rice wine residue extract is used to cultivate probiotics. As long as the nutrition and proliferation environment suitable for the growth of probiotics can be adjusted, it can be suitable for the growth of multiple probiotics. In the technical scheme provided by the present invention, the rice wine residue is used as the raw material, and the rice wine residue extract is made after homogenization, wall breaking, extraction, sterilization, and centrifugation. It is used as a culture medium for probiotic fermentation and culture, which can provide the necessary carbon source, nitrogen source and rich nutrients for the growth of probiotics, can be suitable for the growth of multiple probiotics, and has good culture effect, and has the advantages of low raw material cost and wide source, suitable for mass production, and can also improve the utilization rate of rice wine residue, realize the resource recycling of rice wine residue, solve the environmental pollution problem caused by the abandonment of rice wine residue, and improve the production profit of rice wine factory.

[0049] Based on the preparation method of the probiotic culture medium provided by the present invention, the present invention also provides a probiotic fermentation culture method, using the probiotic culture medium to carry out probiotic fermentation culture, Figure 2 FIG. 1 is an example of fermentation culture of probiotics provided by the present invention. Figure 2 As shown, in this embodiment, the fermentation and cultivation method of probiotics comprises the following steps:

[0050] Step S100a, reviving the probiotics and culturing them in MRS solid culture medium to prepare a probiotic seed solution;

[0051] Step S200a, adding the probiotic seed liquid into the probiotic culture medium for fermentation culture.

[0052] First, take a probiotic capsule in a clean bench, pour the live bacteria powder in the capsule into 500mL sterilized saline, shake and shake for 10min to obtain a probiotic suspension. Then weigh 46.4g of MRS agar, heat and boil in 1000mL distilled water until completely dissolved, then dispense into 500mL conical flasks, sterilize at 121°C for 15min, and make MRS solid culture medium for standby use. Then, use an inoculation loop to pick the seed liquid and streak it on the MRS solid culture medium. After culturing 2 to 3 generations, pick a single colony in 10mL saline, vortex and shake to make a probiotic seed liquid. Finally, the probiotic seed liquid is inoculated into the probiotic culture medium (i.e., rice wine residue extract) obtained by the method provided above of the present invention, and fermentation culture can be carried out.

[0053] The fermentation and culturing method of probiotics provided by the present invention uses rice wine residue extract as a culture medium for probiotics, can provide necessary carbon sources, nitrogen sources and rich nutrients and a suitable growth environment for the proliferation of probiotics, has good culturing effect and low cost.

[0054] When using rice wine residue extract as a probiotic culture medium to ferment and culture probiotics, the inoculation amount of the rice wine residue extract will lead to different carbon and nitrogen source contents during the fermentation and culture process, which has a certain impact on the growth of the strain. After experimental verification, as a preferred embodiment of the present invention, the inoculation amount of the probiotics in the probiotic culture medium is 2-4%, and more preferably 3%.

[0055] In addition, the culture conditions during the fermentation culture, such as the fermentation temperature, also have a certain influence on the growth of the strain. After experimental verification, as a preferred embodiment of the present invention, the culture conditions of the fermentation culture are: the culture temperature is 31-36°C, the shaking culture speed is 100-200r / min, and the culture time is 22-28h. Further, the culture temperature is preferably 33°C, the shaking culture speed is preferably 150r / min, and the culture time is preferably 24h.

[0056] The probiotic fermentation culture method provided by the present invention is suitable for culturing a variety of probiotics, including at least one of Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium lactis, and Lactobacillus paracasei. The probiotics can be any one of the above probiotics or a mixture of two or more thereof, and all have good culture effects.

[0057] As a preferred embodiment of the fermentation and culturing method of probiotics provided by the present invention, the probiotics include Lactobacillus plantarum and Lactobacillus reuteri, and the mass ratio of Lactobacillus plantarum to Lactobacillus reuteri is 0.5:1.

[0058] Based on the preparation method of the probiotic culture medium provided by the present invention, the present invention further provides a preparation method of probiotic freeze-dried powder, wherein the probiotic culture medium prepared by the method provided by the present invention is used to ferment and culture the probiotics, and then the rice wine residue after the rice wine residue extract is extracted is used as a freeze-drying protective agent for the probiotics. Figure 3 The figure shows the preparation method of the probiotic freeze-dried powder provided by the present invention. Figure 3 As shown, the preparation method of the probiotic freeze-dried powder comprises the following steps:

[0059] Step S100b, reviving the probiotics and culturing them in MRS solid culture medium to prepare probiotic seed liquid;

[0060] Step S200b, adding the probiotic seed liquid into the probiotic culture medium for fermentation to obtain a fermentation liquid;

[0061] Step S300b, adding the rice wine residue after extracting the rice wine residue extract to the fermentation liquid, stirring evenly, and then performing vacuum freeze drying to obtain probiotic freeze-dried powder.

[0062] First, take a probiotic capsule in a clean bench, wherein the probiotics include at least one of Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium lactis, and Lactobacillus paracasei, and the live bacteria powder in the capsule is poured into 500mL sterilized physiological saline, and shaken for 10min to obtain a probiotic suspension. Weigh 46.4g of MRS agar, heat and boil in 1000mL distilled water until completely dissolved, and then dispense into 500mL conical flasks, sterilize at 121°C for 15min, and make MRS solid culture medium for standby use. Then, use an inoculation loop to pick seed liquid and streak it on the MRS solid culture medium. After culturing 2 to 3 generations, pick a single colony in 10mL physiological saline, and vortex and shake to make a probiotic seed liquid. Then, the probiotic seed liquid is inoculated into the probiotic culture medium (i.e., rice wine residue extract) obtained by the method provided above of the present invention, and fermentation and culture are performed to obtain a fermented liquid. Finally, a certain amount of rice wine residue after extracting the rice wine residue extract (that is, the rice wine residue residue after centrifugation and collection of the supernatant in the above step S30 of the present invention) is added to the fermentation liquid, and then vacuum freeze-dried to obtain probiotic freeze-dried powder.

[0063] Skim milk powder, glycerol, trehalose, ascorbic acid and other protective agents are commonly used during probiotic freeze-drying. Skim milk powder is the most commonly used freeze-drying protective agent during freeze-drying, but it is expensive, and the price of 100g skim milk powder is about 150 yuan, which is not conducive to reducing the production cost of probiotic freeze-dried powder. In addition, during freeze-drying, there are many kinds of microorganisms, and the contained proteins are also different, so different microorganisms need different protective agents. When the freeze-drying protective agent formula is selected, a large number of experiments need to be carried out, and the present invention has been found through research that rice wine residue contains abundant protein, sugar, amino acid, etc., when the addition ratio is suitable, it can be directly used as a protective agent during probiotic freeze-drying, and the composition is safe and the survival rate of strains is high after freeze-drying.

[0064] The preparation method of the probiotic freeze-dried powder provided by the present invention, after using rice wine residue extract as a culture medium to ferment and culture probiotics, the rice wine residue residue after extracting the rice wine residue extract is used as a freeze-dried protective agent to prepare the probiotic freeze-dried powder, on the one hand, the use of rice wine residue extract as a probiotic culture medium has the advantages of good culture effect and low cost; on the other hand, the rice wine residue residue is used as a freeze-dried protective agent, and the rich protein in the rice wine residue can form a layer of protein film outside the bacterium when freeze-dried, which has a protective effect on cells and macromolecules. At the same time, the addition of rice wine residue will also play a homogenizing role during freeze-drying, and improve the survival rate during freeze-drying, so the use of rice wine residue as a probiotic freeze-dried protective agent has the advantages of good bacteria preservation effect and high strain survival rate. In addition, compared with commonly used protective agents such as skim milk powder, glycerol, trehalose, ascorbic acid, etc., the use of rice wine residue as a probiotic freeze-dried protective agent can also reduce the production cost of probiotic freeze-dried powder, and further improve the utilization rate of rice wine residue, and improve the environmental problems caused by rice wine residue discharge.

[0065] When using rice wine residue extract as a probiotic culture medium to ferment and culture probiotics, the inoculation amount of the rice wine residue extract will lead to different carbon and nitrogen source contents during the fermentation and culture process, which has a certain effect on the growth of the strain. After experimental verification, as a preferred embodiment of the present invention, the inoculation amount of the probiotics in the probiotic culture medium is 2-4%, and more preferably 3%. In addition, the culture conditions during the fermentation and culture, such as the fermentation temperature, also have a certain effect on the growth of the strain. After experimental verification, as a preferred embodiment of the present invention, the culture conditions of the fermentation and culture are: the culture temperature is 31-36°C, and more preferably 33°C; the culture speed is 100-200r / min, and more preferably 150r / min; the culture time is 22-28h, and more preferably 24h.

[0066] Furthermore, when rice wine dregs are used as freeze-drying protective agents, the amount of addition has a great influence on the survival rate of the strains in the freeze-dried powder. After experimental verification, as a preferred embodiment of the present invention, the amount of rice wine dregs added is: the mass of the rice wine dregs after extracting the rice wine dregs extract is 20-40% of the mass of the fermentation liquid, and more preferably 30%.

[0067] In addition, when freeze-drying the fermentation broth, the freeze-drying thickness is also an important factor, which can easily affect the survival rate of the strain after freeze-drying. After experimental verification, as a preferred embodiment of the present invention, the freeze-drying thickness is 0.6 to 2.2 cm.

[0068] In addition, in a specific embodiment of the present invention, the process parameters of the vacuum freeze-drying are set as follows: the temperature of the vacuum freeze-drying is -45 to -35°C, preferably -40°C; the vacuum degree is 20 to 80 Pa, and the freeze-drying time is 12 to 24 hours.

[0069] The probiotic freeze-dried powder prepared by the method provided by the present invention contains at least one probiotic selected from the group consisting of Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium lactis and Lactobacillus paracasei. After being ingested by a human body, the probiotic freeze-dried powder can regulate the intestinal flora of the human body, balance the intestinal flora and prevent intestinal diseases. The probiotic freeze-dried powder has a high content of live bacteria and a good quick-dissolving effect, and is more conducive to the absorption of nutrients therein by the human body.

[0070] The probiotic freeze-dried powder obtained by the method provided above in the present invention can be used to prepare functional foods containing probiotics, such as probiotic fermented solid beverages, etc. Therefore, the present invention further proposes a probiotic fermented solid beverage, and the probiotic fermented solid beverage includes the probiotic freeze-dried powder obtained by the above method. When the probiotic freeze-dried powder obtained by the method provided above in the present invention is used as a probiotic fermented solid beverage, it has the advantages of high content of live bacteria, good instant effect, can regulate the intestinal flora of the human body, maintain the balance of the intestinal flora, and has low ingredients and good flavor. In addition, in some embodiments of the probiotic fermented beverage provided by the present invention, in addition to the probiotic freeze-dried powder, the probiotic fermented solid beverage may also include, for example, sweeteners, fruit flavor additives, etc., to enrich the flavor of the probiotic fermented solid beverage and adapt to different market demands.

[0071] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0072] Example 1

[0073] (1) Brewing old rice wine: glutinous rice → soak → filter → wash → steam → cool → mix with koji, add water → put into vat → ferment → press → clarify → make rice wine → filter → make rice wine residue.

[0074] (2) Preparation of rice wine residue extract: The rice wine residue was placed in a tissue crusher and homogenized at a speed of 12000 r / min for 5 min to obtain a homogenate; the homogenate was then transferred to a beaker and subjected to a wall breaking treatment at an ultrasonic power of 200 W for 15 min, followed by heating and leaching in a water bath at 75°C for 30 min to obtain pretreated rice wine residue; then, 30 g of the pretreated rice wine residue was taken, 100 mL of ultrapure water was added, the mixture was mixed evenly, and then sterilized at 121°C for 20 min. After sterilization, the mixture was centrifuged at a speed of 5000 r / min for 10 min, and the supernatant was collected to obtain a rice wine residue extract.

[0075] Example 2

[0076] (1) Brewing old rice wine: glutinous rice → soak → filter → wash → steam → cool → mix with koji, add water → put into vat → ferment → press → clarify → make rice wine → filter → make rice wine residue.

[0077] (2) Preparation of rice wine residue extract: The rice wine residue was placed in a tissue crusher and homogenized at a speed of 10,000 r / min for 8 minutes to obtain a homogenate; the homogenate was then transferred to a beaker and subjected to a wall breaking treatment at an ultrasonic power of 100 W for 18 minutes, followed by heating and leaching in a water bath at 70°C for 35 minutes to obtain pretreated rice wine residue; then, 35 g of the pretreated rice wine residue was taken, 100 mL of ultrapure water was added, the mixture was mixed evenly, and then sterilized at 120°C for 22 minutes. After sterilization, the mixture was centrifuged at a speed of 4,000 r / min for 12 minutes, and the supernatant was collected to obtain a rice wine residue extract.

[0078] Example 3

[0079] (1) Brewing old rice wine: glutinous rice → soak → filter → wash → steam → cool → mix with koji, add water → put into vat → ferment → press → clarify → make rice wine → filter → make rice wine residue.

[0080] (2) Preparation of rice wine residue extract: The rice wine residue was placed in a tissue crusher and homogenized at a speed of 14000 r / min for 3 min to obtain a homogenate; the homogenate was then transferred to a beaker and subjected to a wall breaking treatment at an ultrasonic power of 300 W for 20 min, followed by heating and leaching in a water bath at 80°C for 33 min to obtain the pretreated rice wine residue; then, 40 g of the pretreated rice wine residue was taken, 100 mL of ultrapure water was added, the mixture was mixed evenly, and then sterilized at 125°C for 25 min. After sterilization, the mixture was centrifuged at a speed of 6000 r / min for 15 min, and the supernatant was collected to obtain the rice wine residue extract.

[0081] Example 4

[0082] (1) Preparation of strain seed liquid: Take Lactobacillus plantarum and Lactobacillus reuteri capsules in a clean bench, pour the live bacterial powder in the capsules into 500 mL of sterile saline, and shake for 10 minutes to obtain a probiotic suspension; then weigh 46.4 g of MRS agar, heat and boil it in 1000 mL of distilled water until it is completely dissolved, and then divide it into 500 mL conical flasks, sterilize it at 121° C. for 15 minutes, and prepare MRS solid culture medium for use; then, use an inoculation loop to pick up the seed liquid and streak it on the MRS solid culture medium. After culturing for 2 to 3 generations, pick a single colony and put it in 10 mL of saline, vortex and shake to prepare Lactobacillus plantarum seed liquid and Lactobacillus reuteri seed liquid, respectively.

[0083] (2) The prepared Lactobacillus plantarum seed liquid and Lactobacillus reuteri seed liquid were inoculated into the rice wine residue extract prepared in Example 1, the mass ratio of Lactobacillus plantarum to Lactobacillus reuteri was controlled to be 0.5:1, the total inoculum amount of probiotics was 3%, and the shaking fermentation culture was carried out at 33° C. and 150 r / min for 24 h.

[0084] Example 5

[0085] (1) Preparation of strain seed liquid: Take a Lactobacillus reuteri capsule in a clean bench, pour the live bacteria powder in the capsule into 500 mL of sterile saline, and shake for 10 minutes to obtain a probiotic suspension; then weigh 46.4 g of MRS agar, heat and boil it in 1000 mL of distilled water until it is completely dissolved, and then divide it into 500 mL conical flasks, sterilize it at 121° C. for 15 minutes, and prepare MRS solid culture medium for use; then, use an inoculation loop to pick up the seed liquid and streak it on the MRS solid culture medium. After culturing for 2 to 3 generations, pick a single colony and put it in 10 mL of saline, vortex and shake to prepare Lactobacillus reuteri seed liquid.

[0086] (2) The Lactobacillus reuteri seed solution prepared in step (1) was inoculated into the rice wine residue extract prepared in Example 2 at an inoculation amount of 3%, and a shaking fermentation culture was carried out at 36° C. and 150 r / min for 24 h.

[0087] Example 6

[0088] (1) Preparation of strain seed liquid: Take Lactobacillus plantarum and Lactobacillus reuteri capsules in a clean bench, pour the live bacterial powder in the capsules into 500 mL of sterile saline, and shake for 10 minutes to obtain a probiotic suspension; then weigh 46.4 g of MRS agar, heat and boil it in 1000 mL of distilled water until it is completely dissolved, and then divide it into 500 mL conical flasks, sterilize it at 121° C. for 15 minutes, and prepare MRS solid culture medium for use; then, use an inoculation loop to pick up the seed liquid and streak it on the MRS solid culture medium. After culturing for 2 to 3 generations, pick a single colony and put it in 10 mL of saline, vortex and shake to prepare Lactobacillus plantarum seed liquid and Lactobacillus reuteri seed liquid, respectively.

[0089] (2) The Lactobacillus plantarum seed solution and the Lactobacillus reuteri seed solution obtained in step (1) were inoculated into the rice wine residue extract obtained in Example 3, the mass ratio of Lactobacillus plantarum to Lactobacillus reuteri was controlled to be 0.5:1, the total inoculum amount of probiotics was 3%, and the shaking fermentation culture was carried out at 31° C. and 150 r / min for 24 h.

[0090] Example 7

[0091] (1) Preparation of strain seed liquid: Take a Lactobacillus plantarum capsule in a clean bench, pour the live bacteria powder in the capsule into 500 mL of sterile saline, and shake for 10 min to obtain a probiotic suspension; then weigh 46.4 g of MRS agar, heat and boil it in 1000 mL of distilled water until it is completely dissolved, and then divide it into 500 mL conical flasks, sterilize it at 121° C. for 15 min, and prepare MRS solid culture medium for use; then, use an inoculation loop to pick up the seed liquid and streak it on the MRS solid culture medium. After culturing for 2 to 3 generations, pick a single colony and put it in 10 mL of saline, vortex and shake to prepare Lactobacillus plantarum seed liquid.

[0092] (2) The Lactobacillus plantarum seed solution prepared in step (1) was inoculated into the rice wine residue extract prepared in Example 1, with a total probiotic inoculation amount of 2%, and a shaking fermentation culture was carried out at 33° C. and 100 r / min for 28 h.

[0093] Example 8

[0094] (1) Preparation of strain seed liquid: Take Lactobacillus plantarum and Lactobacillus reuteri capsules in a clean bench, pour the live bacterial powder in the capsules into 500 mL of sterile saline, and shake for 10 minutes to obtain a probiotic suspension; then weigh 46.4 g of MRS agar, heat and boil it in 1000 mL of distilled water until it is completely dissolved, and then divide it into 500 mL conical flasks, sterilize it at 121° C. for 15 minutes, and prepare MRS solid culture medium for use; then, use an inoculation loop to pick up the seed liquid and streak it on the MRS solid culture medium. After culturing for 2 to 3 generations, pick a single colony and put it in 10 mL of saline, vortex and shake to prepare Lactobacillus plantarum seed liquid and Lactobacillus reuteri seed liquid, respectively.

[0095] (2) The Lactobacillus plantarum seed solution and the Lactobacillus reuteri seed solution obtained in step (1) were inoculated into the rice wine residue extract obtained in Example 1, the mass ratio of Lactobacillus plantarum to Lactobacillus reuteri was controlled to be 0.5:1, the total inoculum amount of probiotics was 4%, and the fermentation culture was carried out in a shaking table at 33° C. and 200 r / min for 22 h.

[0096] Example 9

[0097] The fermentation liquid obtained by fermentation culture in Example 4 was taken out, and rice wine residue accounting for 30% of the mass of the fermentation liquid (the rice wine residue residue after the rice wine residue extract was centrifuged and collected in Example 1) was added thereto, and the freeze-dried thickness was adjusted to 1.4 cm, and then placed in a vacuum freeze dryer, and the parameter conditions were set as a cold hydrazine temperature of -40°C, a vacuum degree of 20-80 Pa, and a freeze-drying time of 12 h. After the freeze-drying was completed, the probiotic freeze-dried powder was obtained.

[0098] Example 10

[0099] The fermentation liquid obtained by fermentation culture in Example 5 was taken out, and rice wine residue accounting for 20% of the mass of the fermentation liquid (the rice wine residue residue after the rice wine residue extract was centrifuged and collected in Example 2) was added thereto, and the freeze-drying thickness was adjusted to 0.6 cm, and then placed in a vacuum freeze dryer, and the parameter conditions were set as a cold hydrazine temperature of -40°C, a vacuum degree of 20-80 Pa, and a freeze-drying time of 12 h. After the freeze-drying was completed, the probiotic freeze-dried powder was obtained.

[0100] Embodiment 11

[0101] The fermentation liquid obtained by fermentation culture in Example 6 was taken out, and rice wine residue accounting for 40% of the mass of the fermentation liquid (the rice wine residue residue after the rice wine residue extract was centrifuged and collected in Example 3) was added thereto, and the freeze-drying thickness was adjusted to 2.2 cm, and then placed in a vacuum freeze dryer, and the parameter conditions were set as a cold hydrazine temperature of -40°C, a vacuum degree of 20-80 Pa, and a freeze-drying time of 12 h. After the freeze-drying was completed, the probiotic freeze-dried powder was obtained.

[0102] Example 12

[0103] The fermentation liquid obtained by fermentation culture in Example 7 was taken out, and rice wine residue accounting for 25% of the mass of the fermentation liquid (the rice wine residue residue after the rice wine residue extract was centrifuged and collected in Example 1) was added thereto, and the freeze-drying thickness was adjusted to 1.0 cm, and then placed in a vacuum freeze dryer, and the parameter conditions were set as a cold hydrazine temperature of -45°C, a vacuum degree of 20-80 Pa, and a freeze-drying time of 18 h. After the freeze-drying was completed, the probiotic freeze-dried powder was obtained.

[0104] Embodiment 13

[0105] The fermentation liquid obtained by fermentation culture in Example 8 was taken out, and rice wine residue accounting for 35% of the mass of the fermentation liquid was added thereto (the rice wine residue residue after the rice wine residue extract was centrifuged and collected in Example 1), the freeze-drying thickness was adjusted to 1.8 cm, and then placed in a vacuum freeze dryer, and the parameter conditions were set as a cold hydrazine temperature of -35°C, a vacuum degree of 20-80 Pa, and a freeze-drying time of 24 h. After the freeze-drying was completed, the probiotic freeze-dried powder was obtained.

[0106] Comparative Example 1

[0107] The steps are the same as those in Example 4, except that the culture medium for probiotic fermentation culture is replaced by MRS culture medium from rice wine residue extract.

[0108] Comparative Example 2

[0109] The steps are the same as those in Example 9, except that the culture medium for probiotic fermentation culture is replaced by MRS culture medium from rice wine residue extract, and the lyophilization protective agent is replaced by skim milk powder from rice wine residue.

[0110] The OD value and pH value of the fermentation broth obtained by fermentation culture in Examples 4-8 and Comparative Example 1 were measured respectively, and the measurement results are shown in Table 1. The strain survival rate of the probiotic freeze-dried powder prepared in Examples 9-13 and Comparative Example 2 was tested respectively, and the measurement results are shown in Table 2 below.

[0111] Table 1 OD value and pH value of the fermentation broth in Examples 4-8 and Comparative Example 1

[0112] Comparative Example 1 Example 4 Example 5 Example 6 Example 7 Example 8 OD value 1.541 1.145 0.597 1.114 0.752 0.878 pH 3.99 3.43 3.79 3.45 3.71 3.68

[0113] Table 2 Survival rate of probiotic freeze-dried powder in Examples 9-13 and Comparative Example 2

[0114]

[0115] It can be concluded from the data in Table 1 and Table 2 that the rice wine residue extract obtained in the embodiment of the present invention has the ability to culture and preserve strains as a culture medium for probiotic fermentation culture. Under different concentrations of rice wine residue extract and different culture conditions (different culture temperatures), the cultured bacterial cell density is different, and the pH value is also different. The larger the bacterial cell density, the more acid is produced, and the lower the pH value. Embodiment 4 can better culture strains, but there is still a slight gap in the proliferation rate relative to the MRS culture medium. At the same time, the addition amount of rice wine residue is different, and the freeze-drying effect is also different. When adding 30% rice wine residue, the freeze-drying effect is the best and the survival rate is the highest. When the freeze-drying thickness is adjusted to 1.4cm, the survival rate has a certain increase. In terms of freeze-drying effect and strain survival rate, the difference between the results obtained by adding rice wine residue in the bacterial solution and skim milk powder is not large, and the freeze-drying effect of rice wine residue for probiotics is very obvious.

[0116] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for preparing a probiotic freeze-dried powder, characterized in that: The following steps are involved: Homogenize the rice wine residue to obtain a homogenate; The homogenate is subjected to a wall-breaking treatment at an ultrasonic power of 100-300 W for 15-20 min, and then heated and extracted in a water bath at 70-80° C. for 30-35 min to obtain pretreated rice wine residue; Add ultrapure water to the pretreated rice wine residue, sterilize at 120-125° C. for 20-25 min, then centrifuge at a speed of 4000-6000 r / min for 10-15 min, collect the supernatant, and obtain the rice wine residue extract, which is the probiotic culture medium; After the probiotics are revived, they are cultured in MRS solid medium and then made into probiotic seed liquid; adding the probiotic seed liquid into a probiotic culture medium for fermentation to obtain a fermentation liquid; Adding rice wine residue after extracting the rice wine residue extract to the fermentation liquid, stirring evenly, and then performing vacuum freeze drying to obtain probiotic freeze-dried powder; Wherein, the mass of the pretreated rice wine residue added to every 100 mL of the ultrapure water is 30-40 g; The inoculation amount of the probiotics in the probiotic culture medium is 2-4%; The fermentation culture has a culture temperature of 31-36°C, a shaker culture speed of 100-200 r / min, and a culture time of 22-28 h; The probiotics are a combination of Lactobacillus plantarum and Lactobacillus reuteri, and the mass ratio of Lactobacillus plantarum to Lactobacillus reuteri is 0.5:1; The mass of the rice wine residue after the rice wine residue extract is extracted is 30% of the mass of the fermentation liquid.

2. The method for preparing the probiotic freeze-dried powder according to claim 1, characterized in that: The step of adding the rice wine residue after extracting the rice wine residue extract to the fermentation liquid, stirring evenly, and then performing vacuum freeze drying to obtain the probiotic freeze-dried powder: The freeze-dried thickness is 0.6 to 2.2 cm; and / or, The vacuum freeze drying temperature is -45~-35°C, the vacuum degree is 20~80pa, and the freeze drying time is 12~24h.

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