Multilayer embedding method of lactic acid bacteria

Abstract

The invention relates to the technical field of microbial preparation technology and yoghurt application, and provides a multilayer embedding method of lactic acid bacteria, which comprises the following steps of: fermenting and culturing lactic acid bacteria to obtain fermentation liquor; separating the fermentation liquid to obtain bacterial sludge; mixing the bacterial sludge with a first layer of embedding medium, and drying to obtain a layer of embedded lactic acid bacteria; mixing the first-layer embedded lactic acid bacteria with a second-layer embedding medium to obtain second-layer embedded lactic acid bacteria; and sequentially embedding the third-layer embedding medium, the fourth-layer embedding medium and the fifth-layer embedding medium on the surface of the two-layer embedded lactic acid bacteria to obtain wet crystal ball particles, and cleaning and drying to obtain the lactic acid bacteria crystal ball particles. According to the technical scheme, the problems that lactic acid bacteria are not resistant to gastric acid and bile salt and the taste is changed due to post-acid increase in the yoghourt product in the related technology are solved.

Description

Technical Field

[0001] The present invention relates to the technical fields of microbial agents and yogurt application technologies. Specifically, it relates to a multi-layer embedding method for lactic acid bacteria. Background Art

[0002] As early as in the 1970s of the last century, R & D personnel in Europe, America, Japan and South Korea began to develop new microecological agents to partially or completely replace the use of antibiotics and hoped to improve human immunity. After decades of development, certain achievements have been made in related fields. With the economic development of our country and the continuous deepening of people's understanding of the health system, people have gradually realized that lactic acid bacteria microecological agents have great advantages in regulating the balance of intestinal flora and improving immunity.

[0003] At present, lactic acid bacteria face multiple stresses in the processes of processing, storage and gastrointestinal environment, such as erosion by high temperature, oxygen, gastric acid and bile salts, resulting in insufficient viable bacteria count, reduced survival rate, and failure to reach the minimum dose that can play a health care role, greatly reducing the efficacy of probiotics and directly affecting the product efficacy. To break through this bottleneck, the lactic acid bacteria embedding technology forms a physical barrier through the encapsulation of wall materials to isolate the external adverse environment, becoming the core means to improve the tolerance of lactic acid bacteria.

[0004] The principle of the lactic acid bacteria embedding method is to use natural or synthetic macromolecular edible materials to encapsulate lactic acid bacteria to form a microcapsule structure through certain technical means. The advantage of this technology is that the core material is coated by the wall material and isolated from the external environment, thus playing a role in isolating moisture and oxygen, and at the same time being far away from the erosion of gastric acid and bile. Under appropriate conditions, when the wall material is damaged, the encapsulated bacteria can be released, and finally it can achieve direct colonization in the intestine and truly play a role in improving intestinal health.

[0005] Yogurt, as the product of the fermentation of lactic acid bacteria, is highly favored by consumers because of its special flavor, rich nutrition and good physiological functions, and has become the largest fermented dairy product in our country. Although yogurt products are transported and sold under cold chain, the lactic acid bacteria in them are still growing and multiplying slowly, and the acidity will continue to increase. The excessive accumulation of lactic acid will seriously stimulate the taste and reduce the sensory quality of yogurt. The post-acidification in this process seriously affects the quality of yogurt, shortens the shelf life of yogurt, and further increases the cross-regional sales cost of yogurt. Therefore, it is very necessary to propose a multi-layer embedding method for lactic acid bacteria to achieve the performance of lactic acid bacteria against gastric acid and bile salts and improve the problem of taste change caused by the post-acid growth of lactic acid bacteria in yogurt products. Summary of the Invention

[0006] The present invention proposes a multi-layer embedding method for lactic acid bacteria, which solves the problems in the related technologies that lactic acid bacteria are intolerant to gastric acid and bile salts and the post-acid growth in yogurt products leads to taste change.

[0007] The technical solution of the present invention is as follows: The present invention provides a multi-layer embedding method for lactic acid bacteria, comprising the following steps: S1. After fermentation and cultivation of lactic acid bacteria, a fermentation broth is obtained; S2. The fermentation broth is separated to obtain bacterial sludge; S3. The bacterial sludge is mixed with a first-layer embedding agent, and after drying, lactic acid bacteria embedded in one layer are obtained; the first-layer embedding agent includes skim milk powder, trehalose, water-soluble vitamin E, sodium glutamate, β-cyclodextrin, resistant dextrin, and Tween; S4. The lactic acid bacteria embedded in one layer are mixed with a second-layer embedding agent to obtain lactic acid bacteria embedded in two layers; the second-layer embedding agent includes lecithin, glycerol, and hydrogenated vegetable oil; S5. A third-layer embedding agent, a fourth-layer embedding agent, and a fifth-layer embedding agent are sequentially embedded on the surface of the lactic acid bacteria embedded in two layers to obtain wet crystal ball particles; S6. The wet crystal ball particles are washed and dried to obtain lactic acid bacteria crystal ball particles; The third-layer embedding agent is a pectin solution; the fourth-layer embedding agent is a mixed solution containing sodium ascorbate, sodium alginate, and chitosan; the fifth-layer embedding agent is a mixed solution containing gelatin and pectin.

[0008] As a further technical solution, in step S3, the drying is freeze-drying.

[0009] As a further technical solution, the first-layer embedding agent is composed of the following raw materials in weight percentages: 2% - 20% of skim milk powder, 2% - 20% of trehalose, 0.5% - 5% of water-soluble vitamin E, 0.5% - 5% of sodium glutamate, 1% - 10% of β-cyclodextrin, 1% - 10% of resistant dextrin, 0.1% - 1% of Tween 80, and the balance is water.

[0010] In the present invention, the main functions of skim milk powder, trehalose, etc. in the first-layer embedding agent are to serve as a freeze-drying filler during freeze-drying. Using it as the first-layer protective agent is to make the bacterial cells disperse evenly, facilitating subsequent embedding by the embedding agent. The amino acids and small peptides in skim milk powder can enter the cells, and the hydrogen bonds of trehalose can form a framework structure, playing a protective role for the lactic acid bacteria cells, and synergistically acting with other embedding agents to improve the survival rate of the bacteria when passing through gastric acid and bile salts.

[0011] As a further technical solution, the second-layer embedding agent is composed of the following raw materials in weight percentages: 0.1% - 1% of lecithin, 0.2% - 2% of glycerol, and the balance is hydrogenated vegetable oil.

[0012] In the present invention, the hydrogenated vegetable oil in the second layer of embedding agent plays a role in isolating moisture and oxygen, improving the survival rate of the bacterial cells. Glycerol and lecithin provide the dispersion effect of the bacterial cells in the vegetable oil, and cooperate with other components to improve the survival rate of the lactic acid bacteria microecological preparation product when passing through gastric acid and bile salts.

[0013] As a further technical solution, the third layer of embedding agent is composed of the following raw materials in weight percentages: pectin 1% - 10%, and the balance is water.

[0014] In the present invention, the pectin in the third layer of embedding agent is a natural polysaccharide substance with good film-forming property, which can effectively block the intrusion of external strongly corrosive substances such as gastric acid and bile salts, reduce the direct contact of these substances with lactic acid bacteria, and provide more reliable protection for lactic acid bacteria.

[0015] As a further technical solution, the fourth layer of embedding agent is composed of the following raw materials in weight percentages: sodium ascorbate 0.1% - 5%, sodium alginate 0.1% - 3%, chitosan 3% - 15%, and the balance is water.

[0016] In the present invention, there are many free hydrophilic groups (hydroxyl and carboxyl groups) on the molecular chain of sodium alginate in the fourth layer of embedding agent. Ca 2+ 、Zn 2+ 、Al 3+ etc. can occupy the hydrophilic space in sodium alginate and chelate with the oxygen atoms on the carboxyl functional group to form a three-dimensional network structure gel, embedding the bacterial cells therein; chitosan can block the entry of disaccharides and polysaccharides and the overflow of organic acids such as lactic acid, playing a barrier role.

[0017] As a further technical solution, the fifth layer of embedding agent is composed of the following raw materials in weight percentages: gelatin 5% - 40%, pectin 0.1% - 5%, and the balance is water.

[0018] In the present invention, gelatin in the fifth layer of embedding agent is a natural macromolecular product obtained by partial hydrolysis of collagen, which can form a gel structure under low-temperature conditions. When the environmental pH value is lower than the isoelectric point of gelatin, the surface positive charges and negative charges can interact with each other, making the microcapsule structure more compact.

[0019] As a further technical solution, in step S1, the total number of lactic acid bacteria in the fermentation broth is 3.0×10 9 ~1.0×10 10 cfu / mL.

[0020] In the present invention, the total number of lactic acid bacteria in the fermentation broth is 3.0×10 9 ~1.0×10 10cfu / mL. A sufficient number of lactic acid bacteria is the basis for achieving effective multi-layer embedding. During the subsequent embedding process, a sufficient number of lactic acid bacteria can ensure that there are enough bacterial cells to participate in the mixing and embedding of each layer of embedding agent.

[0021] As a further technical solution, in step S2, the water content of the bacterial sludge is 5% - 50%, preferably 30%.

[0022] As a further technical solution, in step S3, the mass ratio of the bacterial sludge to the first layer of embedding agent is 1:1 - 3.

[0023] As a further technical solution, in step S4, the mass ratio of the second layer of embedding agent to the lactic acid bacteria embedded in one layer is 1 - 3:1.

[0024] As a further technical solution, in step S5, the mass ratio of the third layer of embedding agent to the lactic acid bacteria embedded in one layer, the mass ratio of the fourth layer of embedding agent to the lactic acid bacteria embedded in one layer, and the mass ratio of the fifth layer of embedding agent to the lactic acid bacteria embedded in one layer are each independently 1 - 3:1.

[0025] In the present invention, the mass ratio of the embedding agent to the lactic acid bacteria embedded in one layer is 1 - 3:1. The amount of the embedding agent is sufficient to form a uniform and complete protective barrier on the surface of the bacterial sludge. If the amount of the embedding agent is too small, it may not be able to completely wrap the lactic acid bacteria in the bacterial sludge, resulting in some lactic acid bacteria being exposed and being easily damaged in the subsequent process; if the amount of the embedding agent is too large, although it can achieve encapsulation, it will lead to insufficient bacterial density and may increase the burden of subsequent processes.

[0026] As a further technical solution, step S3 includes the following steps: mixing the bacterial sludge with the first layer of embedding agent, homogenizing to obtain an emulsion, granulating the emulsion, and freeze-drying to obtain lactic acid bacteria embedded in one layer.

[0027] As a further technical solution, the granulation includes the following steps: dropping the emulsion into liquid nitrogen at -196°C for granulation.

[0028] As a further technical solution, the sublimation drying temperature of the freeze-drying is 10°C, the vacuum degree is 0.015 mbar, the analytical drying temperature is 32°C, and the vacuum degree is 0.005 mbar.

[0029] As a further technical solution, in step S3, the water content of the lactic acid bacteria embedded in one layer is 3% - 6%.

[0030] As a further technical solution, in step S4, the mixing is homogeneous mixing.

[0031] As a further technical solution, in step S5, the embedding method of the third layer embedding agent, the fourth layer embedding agent, and the fifth layer embedding agent is the concentric droplet granulation technology.

[0032] As a further technical solution, in step S6, the drying is fluidized bed drying, and the temperature of the drying is 10~20°C, and the humidity is 25%~35%.

[0033] As a further technical solution, in step S6, the diameter of the lactic acid bacteria crystal ball particles is 0.2~0.8 cm.

[0034] The present invention also proposes an application of lactic acid bacteria crystal ball particles prepared by a multi-layer embedding method of lactic acid bacteria in the fields of food and health products.

[0035] The working principle and beneficial effects of the present invention are as follows: In the present invention, the five-layer embedded lactic acid bacteria are successively protected layer by layer with embedding agents of different raw materials from the inside to the outside, forming a more solid and stable multi-layer protection structure, isolating the lactic acid bacteria from the external environment, thereby playing the role of isolating moisture and oxygen, while being far away from the erosion of gastric acid and bile, significantly improving the survival rate of lactic acid bacteria in the gastrointestinal tract, and increasing the number of lactic acid bacteria reaching the intestine; encapsulating the probiotics in yogurt, in the early stage of yogurt storage, the embedding structure can inhibit the over-fermentation of lactic acid bacteria, so that the lactic acid produced by its metabolism cannot enter the yogurt, controlling the rising speed of acidity, and thus not affecting the acidity and taste of yogurt, and is applicable to the development and use of various products; solving the problem of post-acidification of yogurt caused by adding lactic acid bacteria. The embedding method of the present invention also has the advantages of simple production process, low energy consumption, no three wastes, small investment, and easy large-scale production. Specific Embodiments

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present invention.

[0037] In the following examples and comparative examples: Skim milk powder: food grade; Trehalose: average particle size of 80 mesh; Water-soluble vitamin E: vitamin E content of 99%wt; Sodium glutamate: average particle size of 60 mesh; β-cyclodextrin: average particle size of 80 mesh; Resistant dextrin: food grade; Tween 80: model T-80, food grade; Lecithin: Soybean lecithin, food grade; Glycerol: Food grade, with a glycerol content of 99.5 wt%; Hydrogenated vegetable oil: Hydrogenated castor oil, pH 5 - 7; Pectin: Food grade; Sodium ascorbate: Food grade; Sodium alginate: Average particle size of 200 mesh, food grade; Chitosan: Food grade; Gelatin: Food grade.

[0038] Example 1 A multi - layer embedding method for lactic acid bacteria, comprising the following steps: S1. After the lactic acid bacteria are fermented and cultured in a fermenter, a fermentation broth is obtained, and the total number of lactic acid bacteria in the fermentation broth is 3.0×10 9 cfu / mL; S2. After the fermentation broth is centrifuged, a bacterial sludge is obtained, and the moisture content of the bacterial sludge is 30%; S3. The bacterial sludge is mixed with the first - layer embedding agent, and the mass ratio of the bacterial sludge to the first - layer embedding agent is 1:1. The mixture is fully mixed using a homogenizer to obtain an emulsion. The emulsion is dropped into liquid nitrogen at - 196°C for deep - cooling granulation, and then vacuum freeze - dried in a freeze - dryer. The temperature of sublimation drying is set at 10°C, the vacuum degree is 0.015 mbar, the temperature of analytical drying is 32°C, and the vacuum degree is 0.005 mbar. Drying is ended after the product moisture content reaches 3%, obtaining the first - layer embedded lactic acid bacteria; S4. The first - layer embedded lactic acid bacteria are mixed with the second - layer embedding agent, and the mass ratio of the first - layer embedded lactic acid bacteria to the second - layer embedding agent is 1:3. The mixture is fully mixed evenly using a homogenizer to obtain the second - layer embedded lactic acid bacteria; S5. The third - layer embedding agent, the fourth - layer embedding agent, and the fifth - layer embedding agent are successively embedded on the surface of the second - layer embedded lactic acid bacteria using the concentric droplet - granulation technique. The mass ratio of the first - layer embedded lactic acid bacteria to the third - layer embedding agent, the fourth - layer embedding agent, and the fifth - layer embedding agent is all 1:3, obtaining wet crystal - ball particles; S6. The wet crystal - ball particles are washed with water and dried in a fluidized bed. The drying temperature is 10°C and the humidity is 25%. After drying, the lactic acid bacteria crystal - ball particles are obtained; The first - layer embedding agent is composed of the following raw materials by weight percentage: skim milk powder 2%, trehalose 2%, water - soluble vitamin E 0.5%, sodium glutamate 0.5%, β - cyclodextrin 1%, resistant dextrin 1%, Tween 80 0.1%, and the balance is water; The second - layer embedding agent is composed of the following raw materials by weight percentage: lecithin 0.1%, glycerol 0.2%, and the balance is hydrogenated vegetable oil; The third layer of embedding agent is composed of the following raw materials by weight percentage: pectin 1%, and the balance is water; The fourth layer of embedding agent is composed of the following raw materials by weight percentage: sodium ascorbate 0.1%, sodium alginate 0.1%, chitosan 3%, and the balance is water; The fifth layer of embedding agent is composed of the following raw materials by weight percentage: gelatin 5%, pectin 0.1%, and the balance is water.

[0039] Example 2 A multi-layer embedding method for lactic acid bacteria includes the following steps: S1. After the lactic acid bacteria are fermented and cultured in a fermenter, a fermentation broth is obtained, and the total number of lactic acid bacteria in the fermentation broth is 8.0×10 9 cfu / mL; S2. After the fermentation broth is centrifuged, a bacterial sludge is obtained, and the moisture content of the bacterial sludge is 30%; S3. The bacterial sludge is mixed with the first layer of embedding agent, and the mass ratio of the bacterial sludge to the first layer of embedding agent is 1:2. The mixture is fully mixed by a homogenizer to obtain an emulsion. The emulsion is dropped into liquid nitrogen at -196°C for cryogenic granulation, and then vacuum freeze-dried in a freeze-dryer. The temperature of sublimation drying is set at 10°C, the vacuum degree is 0.015 mbar, the temperature of analytical drying is 32°C, and the vacuum degree is 0.005 mbar. The drying is ended after the product moisture content reaches 4.5% to obtain the first-layer embedded lactic acid bacteria; S4. The first-layer embedded lactic acid bacteria are mixed with the second layer of embedding agent, and the mass ratio of the first-layer embedded lactic acid bacteria to the second layer of embedding agent is 1:2. The mixture is fully mixed and homogenized by a homogenizer to obtain the second-layer embedded lactic acid bacteria; S5. The third layer of embedding agent, the fourth layer of embedding agent, and the fifth layer of embedding agent are sequentially embedded on the surface of the second-layer embedded lactic acid bacteria. The mass ratio of the first-layer embedded lactic acid bacteria to the third layer of embedding agent, the fourth layer of embedding agent, and the fifth layer of embedding agent is 1:2. The embedding method is the concentric dropping granulation technique to obtain wet crystal ball particles; S6. The wet crystal ball particles are washed with water and dried in a low-temperature fluidized bed. The drying temperature is 15°C and the humidity is 30%. After drying, the lactic acid bacteria crystal ball particles are obtained; The first layer of embedding agent is composed of the following raw materials by weight percentage: skim milk powder 10%, trehalose 10%, water-soluble vitamin E 3%, sodium glutamate 3%, β-cyclodextrin 5%, resistant dextrin 5%, Tween 80 0.5%, and the balance is water; The second layer of embedding agent is composed of the following raw materials by weight percentage: lecithin 0.5%, glycerol 1%, and the balance is hydrogenated vegetable oil; The third layer of embedding agent is composed of the following raw materials by weight percentage: pectin 5%, and the balance is water; The fourth layer of embedding agent is composed of the following raw materials by weight percentage: sodium ascorbate 3%, sodium alginate 2%, chitosan 8%, and the balance is water; The fifth layer of embedding agent is composed of the following raw materials by weight percentage: gelatin 20%, pectin 3%, and the balance is water.

[0040] Example 3 A multi-layer embedding method for lactic acid bacteria includes the following steps: S1. After the lactic acid bacteria are fermented and cultured in a fermentation tank, a fermentation broth is obtained, and the total number of lactic acid bacteria in the fermentation broth is 1.0×10 10 cfu / mL; S2. After the fermentation broth is centrifuged and separated, a bacterial sludge is obtained, and the moisture content of the bacterial sludge is 30%; S3. The bacterial sludge is mixed with the first layer of embedding agent, and the mass ratio of the bacterial sludge to the first layer of embedding agent is 1:3. The mixture is fully mixed by a homogenizer to obtain an emulsion. The emulsion is dropped into liquid nitrogen at -196°C for deep cooling granulation, and then vacuum freeze-dried in a freeze-dryer. The temperature of sublimation drying is set at 10°C, the vacuum degree is 0.015 mbar, the temperature of analytical drying is 32°C, and the vacuum degree is 0.005 mbar. Drying is terminated after the product moisture content reaches 6%, and the first-layer embedded lactic acid bacteria are obtained; S4. The first-layer embedded lactic acid bacteria are mixed with the second layer of embedding agent, and the mass ratio of the first-layer embedded lactic acid bacteria to the second layer of embedding agent is 1:1. The mixture is fully mixed and homogenized by a homogenizer to obtain the second-layer embedded lactic acid bacteria; S5. The third layer of embedding agent, the fourth layer of embedding agent, and the fifth layer of embedding agent are sequentially embedded on the surface of the second-layer embedded lactic acid bacteria. The mass ratio of the first-layer embedded lactic acid bacteria to the third layer of embedding agent, the fourth layer of embedding agent, and the fifth layer of embedding agent is 1:1. The embedding method is the concentric dropping granulation technology, and wet crystal ball particles are obtained; S6. The wet crystal ball particles are washed with water and dried in a low-temperature fluidized bed. The drying temperature is 20°C and the humidity is 35%. After drying, the lactic acid bacteria crystal ball particles are obtained; The first layer of embedding agent is composed of the following raw materials by weight percentage: skim milk powder 20%, trehalose 20%, water-soluble vitamin E 5%, sodium glutamate 5%, β-cyclodextrin 10%, resistant dextrin 10%, Tween 80 1%, and the balance is water; The second layer of embedding agent is composed of the following raw materials by weight percentage: lecithin 1%, glycerol 2%, and the balance is hydrogenated vegetable oil; The third layer of embedding agent is composed of the following raw materials by weight percentage: pectin 10%, and the balance is water; The fourth layer of embedding agent is composed of the following raw materials by weight percentage: sodium ascorbate 5%, sodium alginate 3%, chitosan 15%, and the balance is water; The fifth layer of embedding agent is composed of the following raw materials by weight percentage: 40% gelatin, 5% pectin, and the balance is water.

[0041] Comparative Example 1 Compared with Example 1, the difference in Comparative Example 1 is that the embedding process of the fifth layer of embedding agent in step S5 is removed.

[0042] Comparative Example 2 Compared with Example 1, the difference in Comparative Example 2 is that the embedding process of the fourth layer of embedding agent in step S5 is removed.

[0043] Comparative Example 3 Compared with Example 1, the difference in Comparative Example 3 is that the embedding process of the third layer of embedding agent in step S5 is removed.

[0044] Comparative Example 4 Compared with Example 1, the difference in Comparative Example 4 is that the raw materials of the fourth layer of embedding agent and the raw materials of the first layer of embedding agent are interchanged.

[0045] Comparative Example 5 Compared with Example 1, the difference in Comparative Example 5 is that the raw materials of the third layer of embedding agent and the raw materials of the first layer of embedding agent are interchanged.

[0046] Comparative Example 6 Compared with Example 1, the difference in Comparative Example 6 is that the raw materials of the third layer of embedding agent and the raw materials of the fourth layer of embedding agent are interchanged.

[0047] Comparative Example 7 Compared with Example 1, the difference in Comparative Example 7 is that the raw materials of the fourth layer of embedding agent and the raw materials of the fifth layer of embedding agent are interchanged.

[0048] Comparative Example 8 Compared with Example 1, the difference in Comparative Example 8 is that chitosan in the raw materials of the fourth layer of embedding agent is removed, and an equal amount of chitosan is added to the raw materials of the fifth layer of embedding agent.

[0049] Comparative Example 9 Compared with Example 1, the difference in Comparative Example 9 is that the hydrogenated vegetable oil in the raw materials of the second layer of embedding agent is exchanged with the gelatin in the raw materials of the fifth layer of embedding agent.

[0050] Comparative Example 10 Compared with Example 1, the difference in Comparative Example 10 is that the pectin in the raw materials of the third layer of embedding agent is exchanged with the sodium alginate in the raw materials of the fourth layer of embedding agent.

[0051] Comparative Example 11 Compared with Example 1, the difference in Comparative Example 11 is that the chitosan in the raw material of the fourth-layer embedding agent is swapped with the gelatin in the raw material of the fifth-layer embedding agent.

[0052] Experimental Example 1 Control group: The control group is the single-layer embedded lactic acid bacteria prepared in Example 1.

[0053] Artificial gastric acid and bile salt tests were conducted on the lactic acid bacteria crystal ball particles prepared in Examples 1 to 3, Comparative Examples 1 to 11, and the control group. The test conditions were: temperature 37°C, relative humidity 65%, 0 h, 2 h, and 4 h. The viable cell count was detected, and the survival rate was calculated. The survival rate refers to the ratio of the detected value of the viable cell count after 2 h and 4 h to the initial value, expressed as a percentage.

[0054] The test results are shown in Tables 1 and 2: Table 1 Detection results of the gastric acid survival rate of the lactic acid bacteria crystal ball particles prepared in Examples 1 to 3, Comparative Examples 1 to 11, and the control group

[0055] Table 2 Detection results of the bile salt survival rate of the lactic acid bacteria crystal ball particles prepared in Examples 1 to 3, Comparative Examples 1 to 11, and the control group

[0056] As can be seen from Tables 1 and 2, the survival rates of Examples 1 to 3 are all higher than those of Comparative Examples 1 to 11, indicating that when the lactic acid bacteria crystal ball particles are co-embedded with five layers of embedding agents, and the embedding order of the five layers of embedding agents from the inside to the outside is the first-layer embedding agent, the second-layer embedding agent, the third-layer embedding agent, the fourth-layer embedding agent, and the fifth-layer embedding agent, each layer and each component play a synergistic role, and the survival rate of the viable bacteria in the lactic acid bacteria crystal ball particles can be better improved.

[0057] Experimental Example 2 Control group: The control group is the single-layer embedded lactic acid bacteria prepared in Example 1.

[0058] The acidity of the yogurt containing the lactic acid bacteria crystal ball particles prepared in Examples 1 to 3, Comparative Examples 1 to 11, and the control group was tested at a temperature of 4°C.

[0059] The test results are shown in Table 3: Table 3 Acidity test of Examples 1 to 3, Comparative Examples 1 to 11, and the control group

[0060] As can be seen from Table 3, the acidity of Examples 1 to 3 is less than that of Comparative Examples 9 to 11, indicating that when the lactic acid bacteria are embedded according to the raw materials of this scheme, the increase in the acidity of the yogurt can be reduced.

[0061] In Comparative Example 9, hydrogenated vegetable oil leaked during the preparation stage. After the vegetable oil and gelatin were interchanged, 5 complete crystal spheres could not be formed, so the experiments on resistance to gastric acid and bile salts could not be carried out, and thus there was no data. The oil layer floated, resulting in abnormal fermentation. The acidity did not increase after reaching 30°T, and the fermentation did not meet the standard. Subsequently, the acidity detection was not carried out on it.

[0062] In Comparative Example 5, the protective effect could not be provided during freeze-drying, resulting in the death of the bacterial cells, so the fermentation did not meet the standard, and the subsequent acidity detection of acidification was not carried out.

[0063] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A multi-layer embedding method for lactic acid bacteria, characterized in that: The following steps are involved: S1, after lactic acid bacteria fermentation culture, obtain fermentation liquid; S2, the fermentation liquid is separated to obtain bacterial sludge; S3, mixing the bacterial mud with a first layer of embedding agent, and obtaining a layer of embedded lactic acid bacteria after drying; the first layer of embedding agent comprises skim milk powder, trehalose, water-soluble vitamin E, sodium glutamate, β-cyclodextrin, resistant dextrin, and Tween; S4, mixing the first layer of embedded lactic acid bacteria with the second layer of embedding agent to obtain a second layer of embedded lactic acid bacteria; the second layer of embedding agent comprises lecithin, glycerol, and hydrogenated vegetable oil; S5, embedding the third layer of embedding agent, the fourth layer of embedding agent, and the fifth layer of embedding agent in sequence on the surface of the second layer of embedding lactic acid bacteria to obtain wet crystal sphere particles; S6, the wet crystal ball particles are washed and dried to obtain lactic acid bacteria crystal ball particles; The third layer embedding agent is a pectin solution; the fourth layer embedding agent is a mixed solution containing sodium ascorbate, sodium alginate and chitosan; and the fifth layer embedding agent is a mixed solution containing gelatin and pectin.

2. The multi-layer embedding method of lactic acid bacteria according to claim 1, characterized in that: The first layer embedding agent is composed of the following raw materials in weight percentage: 2%-20% skim milk powder, 2%-20% trehalose, 0.5%-5% water-soluble vitamin E, 0.5%-5% sodium glutamate, 1%-10% beta-cyclodextrin, 1%-10% resistant dextrin, 0.1%-1% Tween 80, and the balance is water.

3. The multi-layer embedding method of lactic acid bacteria according to claim 1, characterized in that: The second layer embedding agent is composed of the following raw materials in weight percentage: 0.1% to 1% lecithin, 0.2% to 2% glycerol, and the remainder is hydrogenated vegetable oil.

4. The multi-layer embedding method of lactic acid bacteria according to claim 1, characterized in that: The third layer embedding agent is composed of the following raw materials in weight percentage: 1% to 10% pectin, and the balance is water.

5. The multi-layer embedding method of lactic acid bacteria according to claim 1, characterized in that: The fourth layer embedding agent is composed of the following raw materials in percentage by weight: 0.1% to 5% sodium ascorbate, 0.1% to 3% sodium alginate, 3% to 15% chitosan, and the balance is water.

6. The multi-layer embedding method of lactic acid bacteria according to claim 1, characterized in that: The fifth layer embedding agent is composed of the following raw materials in weight percentage: 5% to 40% gelatin, 0.1% to 5% pectin, and the balance is water.

7. The multi-layer embedding method of lactic acid bacteria according to claim 1, characterized in that: In step S1, the total number of lactic acid bacteria in the fermentation broth is 3.0×10 9 ~1.0×10 10 cfu / mL.

8. The multi-layer embedding method of lactic acid bacteria according to claim 1, characterized in that: In step S2, the moisture content of the bacterial sludge is 5% to 50%.

9. The multi-layer embedding method of lactic acid bacteria according to claim 1, characterized in that: In step S6, the drying is fluidized bed drying, the drying temperature is 10-20° C., and the humidity is 25%-35%.

10. Use of the lactic acid bacteria crystal spherical particles prepared by the multi-layer embedding method of lactic acid bacteria according to any one of claims 1 to 9 in the field of food and health care products.

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