Lactobacillus inoculant and preparation method thereof

Abstract

The invention discloses a lactobacillus inoculant preparation method, which comprises: adding a freeze-drying protective agent to a lactobacillus fermentation broth, and stirring to obtain a uniformly mixed slurry, the freeze-drying protective agent comprising 8-10 parts by weight of pomelo peel powder and 0-2 parts by weight of skimmed milk powder; and freeze-drying the uniformly mixed slurry to obtain the lactobacillus inoculant. The freeze-drying protective agent disclosed by the invention is strong in water absorption, and can omit a centrifugal process when being applied to a process of preparing a lactobacillus fungicide, so that the inactivation of lactobacillus cells caused by centrifugal operation is reduced, and the freeze-drying survival rate reaches 85% or above.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a lactobacillus agent and a preparation method thereof. BACKGROUND

[0002] Lactobacillus is an important probiotic and is widely used in food, health products and medicine. To prepare a high-activity lactobacillus agent, the bacterial cells are usually separated from the fermentation broth, concentrated, and then freeze-dried. At present, centrifugation technology is widely used in the separation and concentration process of bacterial cells. High-speed centrifugation is used to reduce water content and increase bacterial concentration, so as to reduce the energy consumption in the freeze-drying process.

[0003] However, the centrifugation process inevitably causes mechanical damage to the bacterial cells, resulting in the death of part of the bacterial cells. The number of viable bacteria after centrifugation generally decreases by 1-2 orders of magnitude, which significantly reduces the survival rate of the bacterial cells. In addition, in order to maintain the cell viability and stability of lactobacillus and facilitate the transportation and storage of the product, vacuum freeze-drying technology is often used to sublimate ice. In the subsequent freeze-drying process, the bacterial cells are subjected to low temperature and dehydration stress, which further reduces the overall freeze-drying survival rate.

[0004] In the prior art, in order to reduce the damage caused by centrifugation, the centrifugation conditions are usually optimized, such as reducing the rotation speed, shortening the time or using a milder separation method. However, the effect is limited and the cost may be increased. In order to simplify the production process and reduce the death of bacterial cells, it is necessary to consider omitting or replacing the centrifugation process in the production of the bacterial agent to improve the number of viable bacteria in the bacterial agent product. Therefore, how to avoid the damage to the bacterial cells caused by the centrifugation step and improve the freeze-drying survival rate of the lactobacillus agent is a problem to be solved in the current technical field. SUMMARY

[0005] The main purpose of the present application is to provide a preparation method of a lactobacillus agent, which aims to solve the technical problem that the centrifugation step and the freeze-drying process in the existing preparation method of the lactobacillus agent will cause the loss of viable bacteria, resulting in a low survival rate of the bacterial cells.

[0006] In order to achieve the above-mentioned purpose, the present application provides a preparation method of a lactobacillus agent, which comprises the following steps: adding a freeze-drying protective agent to the lactobacillus fermentation broth, and stirring to obtain a mixed slurry, wherein the freeze-drying protective agent comprises 8-10 parts by weight of pomelo peel powder and 0-2 parts by weight of nutrients; freeze-drying the mixed slurry to obtain the lactobacillus agent.

[0007] The application provides a preparation method of lactobacillus agent, which utilizes pomelo peel powder and nutrients to be compounded as a freeze-drying protective agent.

[0008] The freeze-drying protective agent combined with the pomelo peel powder and the nutrients in the application can produce obvious synergistic effect, the pomelo peel powder contains some soluble oligosaccharides and insoluble polysaccharides (mainly cellulose), wherein, the small molecular oligosaccharides can enter and dissolve in the bacterial cells, thereby reducing the freezing point of the cell sap inside the cells, increasing the osmotic pressure of the system, and preventing freezing; the large molecular cellulose is attached to the cell surface through the polyhydroxy structure to form a physical barrier, thereby protecting the cells and reducing the damage of low temperature to the cells.

[0009] The pomelo peel powder is cheap and easy to obtain, realizes the reuse of pomelo processing by-products, and has a lower cost than other types of freeze-drying protective agents, and has strong water absorption, which is beneficial to saving the energy consumption of the subsequent freeze-drying step.

[0010] The nutrients of the application can provide nutrients for bacterial cells, including lactose, protein, inorganic salt and other nutrients. The nutrients can be lactose powder and other nutrients that can provide carbohydrates, and are matched with protein sources, including whey protein or soy protein and other sources.

[0011] Preferably, the freeze-drying protective agent is used after irradiation sterilization.

[0012] Further, the ratio of the freeze-drying protective agent to the lactobacillus fermentation broth is (0.1-0.2g):1mL. The addition ratio of the freeze-drying protective agent to the lactobacillus fermentation broth is preferably in the above range; more preferably, the ratio of the freeze-drying protective agent to the lactobacillus fermentation broth is 0.15g:1mL, and the freeze-drying effect is the best, which can reduce the damage of ice crystals, but the addition amount higher than 20% is not conducive to the growth of the bacterial cells.

[0013] Further, the weight ratio of the pomelo peel powder to the nutrients is 8:2, and the ratio of the freeze-drying protective agent to the lactobacillus fermentation broth is 0.15g:1mL. The inventors have found through a large number of experiments that the addition amount of the pomelo peel powder and the nutrients in the above range is sufficient to absorb the free water in the lactobacillus fermentation broth system, and the effect is the best; if the addition amount of the pomelo peel powder is insufficient, the lactobacillus fermentation broth and the bacterial cells therein cannot form a muddy material, which is not convenient for subsequent freeze-drying operation, and the drying time is prolonged, thereby increasing the energy consumption.

[0014] Further, the preparation method of the pomelo peel powder comprises the following steps: removing the surface oil-containing layer of the pomelo peel to obtain white mesocarp, and then drying, crushing and sieving to obtain the pomelo peel powder. The inventors have found that if the pomelo peel is not treated and the oil-containing layer is not removed, and the pomelo peel is directly crushed and sieved through a 100-mesh sieve, the oil will not only affect the taste of the pomelo peel powder and the use of the bacterial agent due to its own smell, but also affect the drying and crushing process, resulting in the formation of clumps and affecting the preparation of the pomelo peel powder. Moreover, the presence of oil will change the permeability of the cell membrane, thereby affecting the survival rate of the bacterial cells during freezing. Therefore, the use of the pomelo peel powder in the preparation method of the lactobacillus bacterial agent has an unexpected effect.

[0015] Further, the preparation method more specifically comprises the following steps: Strain activation: inoculating lactobacillus into a liquid culture medium and activating to obtain a seed liquid; Expansion culture: inoculating the seed liquid into a new culture medium for expansion culture to obtain a lactobacillus fermentation broth; Adding a freeze-drying protective agent: adding a freeze-drying protective agent to the lactobacillus fermentation broth, and stirring to obtain a uniformly mixed slurry; Freeze-drying: pre-freezing the uniformly mixed slurry, and then freeze-drying to obtain the lactobacillus bacterial agent.

[0016] Preferably, before the step of adding a freeze-drying protective agent, the lactobacillus fermentation broth is filtered to remove possible particulate impurities.

[0017] Further, in the strain activation step, the inoculation amount of the strain is 1×10 7 ~3×10 7 CFU / mL, the activation time is 24 h, and the activation temperature is 37℃.

[0018] Further, in the expansion culture step, the inoculation amount of the seed liquid is 5% of the mass of the new culture medium, and the culture time is 24 h.

[0019] Further, the freeze-drying step comprises the following steps: pre-freezing the uniformly mixed slurry at-80℃ for 12 h, and then freeze-drying in an environment with a cold trap temperature of-70℃ and a vacuum degree of 0 Pa for 48 h.

[0020] Further, the lactobacillus includes at least one of lactobacillus rhamnosus, lactobacillus bulgaricus and lactobacillus plantarum; and the nutrient includes at least one of skimmed milk powder, lactose powder, whey powder, whey protein powder and soybean protein isolate powder. It is easy to understand that the pomelo peel powder alone can be used as a freeze-drying protective agent, and the use of skimmed milk powder and pomelo peel powder has better effects, and the pomelo peel powder can be combined with the combination of lactose powder, whey powder, whey protein powder and soybean protein isolate powder for compounding, so as to ensure that the types of carbon source, protein and inorganic salt and other nutrients are comprehensive.

[0021] The application further provides a lactobacillus agent prepared by the preparation method.

[0022] Beneficial effects: The preparation method of the lactobacillus agent provided by the application uses the freeze-drying protective agent of the combination of pomelo peel powder and nutrients. Since the pomelo peel powder has strong water absorption capacity, when added to the bacterial culture medium, the whole culture medium can be directly changed into a mud-like substance, so that the bacteria are concentrated into bacterial mud, and the bacteria are not harmed, and the activity and the freeze-drying process of the bacteria are not affected. The freeze-drying protective agent can be directly added to the fermentation liquid after expansion, so that the centrifugation process is removed, the cell mortality is reduced, the live bacteria count of the prepared lactobacillus agent is higher, the survival rate of the lactobacillus agent after freeze-drying reaches more than 85%, and the survival rate is 1-2 orders of magnitude higher than that of the traditional method (cultivation, centrifugation, addition of freeze-drying protective agent, freeze-drying, and finished product).

[0023] The freeze-drying protective agent of the combination of pomelo peel powder and nutrients in the application can produce obvious synergistic protection. The pomelo peel powder contains some soluble oligosaccharides and insoluble polysaccharides (mainly cellulose). Among them, the small molecular oligosaccharides can enter and dissolve in the bacterial cells, thereby reducing the freezing point of the cell sap inside the cells, increasing the osmotic pressure of the system, and preventing freezing. The macromolecular cellulose is attached to the cell surface through the polyhydroxy structure to form a physical barrier and protect the cells, thereby reducing the damage of low temperature to the cells. The lactose, protein and inorganic salt in the nutrients also have a certain protective effect on the lactic acid bacteria cells during freeze-drying.

[0024] The pomelo peel powder is cheap and easy to obtain, realizes the reuse of pomelo processing by-products, and has a lower cost than other types of freeze-drying protective agents. Meanwhile, the pomelo peel powder has strong water absorption, which is beneficial to saving the energy consumption of the subsequent freeze-drying step. DETAILED DESCRIPTION

[0025] In the following examples of the application, the experimental methods not specified in the specific conditions are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. The various common chemical reagents used in the examples are all commercially available products.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0027] The terms "comprising" and "having," and any variations thereof, as used in the present application are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of steps or components does not necessarily comprise only those steps or components but can include additional steps or components not expressly listed or inherent to such process, method, article, or apparatus.

[0028] For the purpose of clarity, technical solutions and advantages of the present application, the present application is further described in detail below with specific embodiments. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present application. In addition, in the following description, the description of well-known structures and techniques is omitted to avoid unnecessary confusion of the concept of the present application.

[0029] The present application is further described in detail below with specific embodiments, but the scope of protection of the present application is not limited to the content described.

[0030] Example 1: (1) Initial viable count of Lactobacillus rhamnosus 1g of the bacterial agent sample was added to 9mL of physiological saline and mixed well, diluted by 10 times gradient until the concentration was 10 -8 CFU / mL of the original sample; 0.1mL of the diluted solution was coated on MRS solid medium, and after 48h of constant temperature inversion culture at 37℃, 3 parallel samples were taken for viable count, and the final value was the average of the three samples.

[0031] (2) Preparation of Lactobacillus rhamnosus fermentation broth Lactobacillus rhamnosus was inoculated into MRS broth medium at an inoculum of 3x10 7 CFU / mL, and cultured at 37℃ for 24h as seed liquid; the seed liquid was inoculated into another MRS broth medium at an inoculum of 5.0% (w / w), and cultured for 24h to the early stage of the stable growth period to obtain Lactobacillus rhamnosus fermentation broth.

[0032] (3) Preparation of freeze-drying protectant The surface of the pomelo peel was removed to obtain the white part, which was then dried at 50℃ for 24h, crushed, and sieved through a 100 mesh sieve to obtain pomelo peel powder. 80% pomelo peel powder + 20% skim milk powder were compounded as a freeze-drying protectant, which was sterilized by irradiation before use.

[0033] (4) Preparation of Lactobacillus rhamnosus inoculant: Take three groups of Lactobacillus rhamnosus fermentation broth in a sterile environment, add freeze-drying protectant to each group of Lactobacillus rhamnosus fermentation broth, the ratio of freeze-drying protectant to Lactobacillus rhamnosus fermentation broth is 0.15g:1mL, stir and mix evenly to obtain a muddy mixed slurry; (5) Freeze-drying: first pre-freeze the mixed slurry at -80℃ for 12h, then freeze-dry in a vacuum freeze-dryer with a cold trap temperature of -70℃ and a vacuum degree of 0Pa for 48h to obtain Lactobacillus rhamnosus inoculant.

[0034] Example 2: The difference from Example 1 is that the strain is Lactobacillus plantarum, the inoculum is 1×10 7 CFU / mL, the freeze-drying protectant is 100% pomelo peel powder, the ratio of freeze-drying protectant to Lactobacillus fermentation broth is 0.1g:1mL, and the remaining operation steps are consistent with Example 1.

[0035] Example 3: The difference from Example 1 is that the strain is Lactobacillus bulgaricus, the inoculum is 2×10 7 CFU / mL, the freeze-drying protectant is 90% pomelo peel powder and 10% skim milk powder, the ratio of freeze-drying protectant to Lactobacillus fermentation broth is 0.2g:1mL, and the remaining operation steps are consistent with Example 1.

[0036] Comparative Example 1 The difference from Example 1 is that the components of the freeze-drying protectant are different, and the preparation process has an additional centrifugation step.

[0037] Specifically: Take three groups of fermentation broth in a sterile environment, centrifuge at 15000rpm, 4℃ for 10min, collect the bacterial mud and weigh. Add traditional protectant to the three groups of bacterial mud, the protectant composition is 50% trehalose, 20% skim milk powder, 5% ascorbic acid and 25% lactose, the mass ratio of bacterial mud to protectant is 1:2, mix the bacterial mud and protectant evenly; pre-freeze in a-80℃ refrigerator for 12h, then freeze-dry in a vacuum freeze-dryer for 48h to obtain Lactobacillus rhamnosus inoculant.

[0038] Comparative Example 2 The difference from Example 1 is that the ratio of freeze-drying protectant to Lactobacillus fermentation broth is 0.3g:1mL, and the remaining operation steps are consistent with Example 1.

[0039] Comparative Example 3 The difference from Example 1 is that the composition of the freeze-drying protectant is 80% microcrystalline cellulose and 20% skim milk powder, and the remaining operation steps are consistent with Example 1.

[0040] Experiment 1, determination of viable bacterial count of inoculant Take each example and the comparative example of the three groups of lactobacillus agent 0.1 g dissolved in 9.9 mL of sterile normal saline, vortex, gradient dilution, select 10 8 、10 9 、10 10 、10 11 The four gradients, 0.1 mL of bacteria liquid was taken and coated on the solid plate, two plates were made for each gradient, and incubated at 37℃ for 24h, and finally the colony count was carried out, and the total number of viable bacteria was calculated. The experimental results were taken as average value.

[0041] The survival rate of lactobacillus in the agent was calculated according to the following formula:

[0042] In the formula, SR——the survival rate of lactobacillus agent; N m ——the unit number of viable bacteria of the agent after freeze-drying, CFU / g; N v ——the unit number of viable bacteria of the bacteria liquid before freeze-drying, CFU / mL; m——the mass of the agent after freeze-drying, g; V——the volume of the bacteria liquid before freeze-drying, mL.

[0043] The experimental results are as follows:

[0044] Experiment two, comparison of the storage stability of the agent: Under the condition of 45% relative humidity, the lactobacillus agents prepared in example 1 and comparative example 1 were stored at 25℃, 4℃ and-20℃ respectively, the initial total number of viable bacteria of the sample was determined, and the total number of viable bacteria after 30 and 60 days of storage was determined, and the storage stability of the agent was compared.

[0045] The experimental results are as follows:

[0046] From the above experimental results, it can be seen that examples 1-3 do not go through the centrifugation step, and the unit number of viable bacteria is more than one order of magnitude higher than that of comparative example 1; and in comparative example 2, the excessive use of freeze-drying protectant results in too sticky bacteria mud which is difficult to stir uniformly, and the proportion in the finished product is too large, the number of viable bacteria and the survival rate decrease obviously; and in comparative example 3, the microcrystalline cellulose only contains simple cellulose without small molecular sugar, which cannot enter the cell to increase the osmotic pressure and bind the water molecules in the cell, resulting in the water molecules in the cell being easy to freeze at low temperature and the cell being damaged, finally resulting in the decrease of the number of viable bacteria and the survival rate.

[0047] In addition, the bacterial agent prepared in Example 1 also has better storage stability and long-term stability than that of Comparative Example 1 during storage.

[0048] The above merely describes preferred embodiments of the present application, and is not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process conversion, or direct or indirect application in other related technical fields, made by using the content of the present application, are also included in the patent protection scope of the present application.

Claims

1. A method for preparing a Lactobacillus agent, characterized by, The method comprises the following steps: adding a freeze-drying protective agent to the lactobacillus fermentation liquor, and stirring to obtain a mixed slurry; freezing and drying the mixed slurry to obtain the lactobacillus agent.

2. The method of claim 1, wherein the Lactobacillus agent is prepared by, The ratio of the freeze-drying protective agent to the lactobacillus fermentation liquor is (0.1-0.2 g) : 1 mL.

3. The method of claim 2, wherein the Lactobacillus agent is prepared by, The ratio of the grapefruit peel powder to the nutrient substance is 8:2, and the ratio of the freeze-drying protective agent to the lactobacillus fermentation liquor is 0.15 g: 1 mL.

4. The method of claim 1, wherein the Lactobacillus agent is prepared by, The preparation method of the grapefruit peel powder comprises the following steps:

5. The method for preparing the Lactobacillus inoculant according to claim 1, characterized in that, The preparation method more specifically comprises the following steps: activating the lactobacillus by inoculating it into a liquid culture medium to obtain a seed liquid; expanding the culture by inoculating the seed liquid into a new culture medium to obtain a lactobacillus fermentation liquor; adding a freeze-drying protective agent to the lactobacillus fermentation liquor, and stirring to obtain a mixed slurry; freezing and drying the mixed slurry to obtain the lactobacillus agent.

6. The method of claim 5, wherein the Lactobacillus agent is prepared by, In the strain activation step, the inoculum size is 1×10⁻⁶. 7 ~3×10 7 CFU / mL, activation time 24h, activation temperature 37℃.

7. The method of claim 5, wherein the Lactobacillus agent is prepared by, In the step of expanding the culture, the inoculation amount of the seed liquid is 5% of the mass of the new culture medium, and the culture time is 24 h.

8. The method of preparing a Lactobacillus agent according to claim 1 or 5, wherein the step of freeze-drying comprises: The mixed slurry is first pre-frozen at-80℃ for 12 h, and then freeze-dried in an environment with a cold trap temperature of-70℃ and a vacuum degree of 0 Pa for 48 h.

9. The method of claim 1, wherein the Lactobacillus agent is prepared by, The lactobacillus comprises at least one of Lactobacillus rhamnosus, Lactobacillus bulgaricus and Lactobacillus plantarum; and the nutrient substance comprises at least one of skimmed milk powder, lactose powder, whey powder, whey protein powder and soybean protein isolate powder.

10. A lactobacillus agent prepared by the preparation method according to any one of claims 1-9.

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