阅读说明：本技术 一种基于w1/o/w2型双重乳液结构的益生菌制剂、制备方法及应用 (Based on W1/O/W2Probiotic preparation with double-emulsion structure, preparation method and application ) 是由 陈义杰 周麒 沈谦 罗依伦 丁爽 陈金玉 郑威 于 2020-05-26 设计创作，主要内容包括：本发明属于生物技术领域,尤其涉及一种基于W<Sub>1</Sub>/O/W<Sub>2</Sub>型双重乳液结构的益生菌制剂、制备方法及应用。制备过程包括：将益生菌菌体与益生菌保护剂混合后形成内水相W<Sub>1</Sub>；将油溶性乳化剂溶于食用油形成油相O,将油相O与内水相W<Sub>1</Sub>初步混合后,经搅拌、低能乳化法或高能乳化法乳化,得到油包水型初乳W<Sub>1</Sub>/O；以亲水性乳化剂为外水相W<Sub>2</Sub>,向初乳W<Sub>1</Sub>/O中加入W<Sub>2</Sub>,经搅拌、低能乳化法或高能乳化法乳化,得到W<Sub>1</Sub>/O/W<Sub>2</Sub>型双重乳液,低温保存,即得到益生菌制剂。本发明采用双重乳液体系对益生菌进行抗冻保护包埋,使得益生菌在冷冻贮藏和冻融过程中保持活性,降低外部环境对益生菌的影响,提高益生菌在产品货架期内的冻融和贮藏稳定性。(The invention belongs to the technical field of biology, and particularly relates to a W-based material 1 /O/W 2 Probiotic preparation with double emulsion structure, preparation method and application. The preparation process comprises the following steps: mixing probiotic bacteria and probiotic protective agent to form internal water phase W 1 (ii) a Dissolving oil soluble emulsifier in edible materialForming oil phase O from the oil, and mixing the oil phase O with the internal water phase W 1 After preliminary mixing, emulsifying by stirring, low energy emulsification method or high energy emulsification method to obtain water-in-oil type primary emulsion W 1 O; using hydrophilic emulsifier as external water phase W 2 To the primary emulsion W 1 W is added to the solution 2 Stirring, emulsifying by low energy emulsification method or high energy emulsification method to obtain W 1 /O/W 2 And (4) storing the double emulsion at low temperature to obtain the probiotic preparation. The invention adopts a double emulsion system to carry out anti-freezing protection embedding on the probiotics, so that the probiotics keep activity in the processes of freezing storage and freezing-thawing, the influence of the external environment on the probiotics is reduced, and the freezing-thawing and storage stability of the probiotics in the shelf life of products is improved.)

1. Based on W₁/O/W₂The preparation method of the probiotic preparation with the double-emulsion structure is characterized by comprising the following steps:

s1: mixing probiotic bacteria and probiotic bacteria protective agent, and vortexing to uniformly disperse the mixture to form an internal water phase W₁；

S2: dissolving oil soluble emulsifier in edible oil to form oil phase O, and mixing oil phase O with internal water phase W₁Mixing, stirring, emulsifying by low energy emulsification method or high energy emulsification method to obtain water-in-oil type colostrum W₁/O；

S3: using hydrophilic emulsifier as external water phase W₂To the colostrum W₁Adding external water phase W into the solution₂Stirring, emulsifying by low energy emulsification method or high energy emulsification method to obtain W₁/O/W₂And (4) storing the double emulsion at low temperature to obtain the probiotic preparation.

2. A W-based alloy according to claim 1₁/O/W₂The preparation method of the probiotic preparation with the double-emulsion structure is characterized by comprising the following steps: internal aqueous phase W in step S1₁The loading concentration of the probiotics is more than or equal to 5 log/mL;

the probiotic protecting agent includes but is not limited to at least one of saccharides, proteins, alcohols, inorganic salts, antioxidants, polymers and complexes; the mass percentage of the protective agent in the internal water phase is 0.5-25 wt%;

the probiotic protecting agent comprises at least one of but not limited to glycerol, skim milk powder, ascorbic acid, whey protein isolate, trehalose, sucrose, fructose, maltose, lactose, dextran, gelatin, peptone, methyl cellulose, sodium glutamate, xylitol and sodium dodecyl sulfate.

3. A W-based alloy according to claim 1₁/O/W₂The preparation method of the probiotic preparation with the double-emulsion structure is characterized by comprising the following steps: the low-energy emulsification method in the step S2 includes, but is not limited to, a phase transition method, a spontaneous emulsification method, a membrane emulsification method; said high energyEmulsification methods include, but are not limited to, high shear, high pressure homogeneous emulsification, microfluidization emulsification.

4. A W-based device according to claim 3₁/O/W₂The preparation method of the probiotic preparation with the double-emulsion structure is characterized by comprising the following steps: the addition amount of the oil-soluble emulsifier is 1-15% of the mass of the oil phase O; the oil phase O and the internal water phase W₁The mass ratio of (A) to (B) is 1-5: 1;

the oil-soluble emulsifier includes but is not limited to at least one of oil-soluble small molecule emulsifier, oil-soluble protein emulsifier, oil-soluble polymer and complex;

the oil-soluble emulsifier includes, but is not limited to, at least one of polyglycerol ricinoleate (PGPR), span 20, span 60, span 65, span 80, span 85, ethylene glycol fatty acid ester, propylene glycol monostearate, glyceryl monostearate, and zein.

5. A W-based alloy according to claim 1₁/O/W₂The preparation method of the probiotic preparation with the double-emulsion structure is characterized by comprising the following steps: the low-energy emulsification method in the step S3 includes, but is not limited to, a phase transition method, a spontaneous emulsification method, a membrane emulsification method; the high energy emulsification method includes but is not limited to high speed shearing, high pressure homogeneous emulsification, micro jet emulsification.

6. A W-based alloy according to claim 1₁/O/W₂The preparation method of the probiotic preparation with the double-emulsion structure is characterized by comprising the following steps: outer water phase W₂In the preparation method, the mass percent of the emulsifier is 0.1-15 wt%; outer water phase W₂With colostrum W₁The volume ratio of the oxygen to the oxygen is 1-5: 1;

the hydrophilic emulsifier includes, but is not limited to, at least one of a small molecule emulsifier, a polysaccharide emulsifier, a polypeptide emulsifier, a protein emulsifier, a polymer, and a complex.

The hydrophilic emulsifier includes but is not limited to at least one of tween 80, pectin, whey protein isolate, whey protein concentrate, casein.

7. Based on W₁/O/W₂The probiotic preparation with the double-emulsion structure is characterized in that: prepared by the preparation method of any one of claims 1 to 6.

8. W-based according to claim 7₁/O/W₂The application of the probiotic preparation with the double-emulsion structure in probiotic products.

9. Use according to claim 8, characterized in that: the probiotic product comprises a low temperature product.

10. Use according to claim 9, characterized in that: the low-temperature product comprises any one of ice cream, ice cream bar, low-temperature yoghourt, cheese, low-temperature beverage and low-temperature animal health care products.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a W-based material₁/O/W₂Probiotic preparation with double emulsion structure, preparation method and application.

Background

Probiotics is a kind of active microorganisms beneficial to a host, is a general term for active beneficial microorganisms which are planted in the intestinal tract and the reproductive system of a human body and can produce exact health efficacy so as to improve the microbial ecological balance of the host and play beneficial roles. Numerous studies have shown that probiotics possess a variety of physiological activities, such as: regulating intestinal health, improving in vivo nutrient synthesis and bioavailability, lowering cholesterol level, lowering blood pressure, relieving tumor, preventing intestinal cancer, and improving immunity.

Among products prepared from probiotics, which are also known as microecologics, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus rhamnosus (Lactobacillus rhamnous), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Bifidobacterium animalis (Bifidobacterium), Bifidobacterium bifidum (Bifidobacterium bifidum), Bifidobacterium adolescentis (Bifidobacterium adolescentis), and Streptococcus thermophilus (Streptococcus thermophilus) are representative bacteria. Researches show that the minimum intake of the probiotics can be fully exerted only by 6-7 log/mL, so that the probiotics product is required to be stable in the storage and organism digestion processes.

However, most probiotics are facultative anaerobic, are easily affected by oxygen during processing and storage, and have reduced storage stability under the influence of environmental pH and temperature; when the subsequent probiotics enter the digestive tract after being ingested by the oral cavity of a human body, the subsequent probiotics are further inactivated after the action of gastric juice, digestive enzymes and bile salts, and finally the number of the viable bacteria planted in the intestinal tract is usually lower than the minimum value which can play physiological action theoretically.

The probiotics products produced at home at present have more defects, such as unstable product quality and poor storage stability of probiotics, and the number of live probiotics in the probiotics products is sharply reduced in a short time in the processes of repeated freezing and thawing and storage, so that the effective intake of the viable bacteria is often not achieved. In order to ensure the survival rate of probiotics, maintain the physiological activity of the probiotics and improve the storage stability of the probiotics in the shelf life of products, the microcapsule embedding technology of the probiotics and the derived multilayer embedding technology are researched mostly at home and abroad at present. In the two types of embedding technologies, the survival rate and stability of probiotics mainly depend on the concentration and variety of thalli, the variety of microcapsule wall materials, the outlet temperature of a spray dryer and the like, the influence factors are more, the preparation process is complicated, the death phenomenon of partial thalli cannot be effectively avoided, the degradation time of the capsule in a machine body is long, and the quality standard of a product is difficult to control; meanwhile, when the probiotic microcapsules are applied to food, the probiotic microcapsules can often have adverse effects on the sensory properties and flavor of the food.

Some researchers embed probiotics in an internal aqueous phase gelled multiple emulsion, but the aqueous phase gelled multiple emulsion is mainly designed aiming at the problem of digestion resistance of the probiotics and the problem of intestinal colonization of the probiotics, and the problem of long-term storage of the probiotics is not considered. In addition, when studying and optimizing the preparation and the characteristics of the gel milk by people of Cao morning (Chinese fats 2019, 44(12), 143-148) and Guo Zhanyang and Zheng Zhan (Chinese fats 2019, 44(08), 65-71), the influence of the freeze-thaw process on the gel milk is large, and the freeze-thaw stability of the emulsion is poor, so that when the emulsion is applied to food, the freeze-thaw process influences the stability of the emulsion and simultaneously influences the activity of probiotics.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a W-based optical fiber cable₁/O/W₂Probiotic preparation with double emulsion structure, preparation method and application, which aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The invention develops a novel probiotic-entrapped emulsion system, which can be used for low-temperature product systems such as ice cream, low-temperature yoghourt, cheese, low-temperature beverage, low-temperature animal health-care products and the like, solves the problem that probiotics in the system are easy to inactivate in the processes of freeze thawing and storage, improves the freeze thawing and storage stability of the probiotics, and is beneficial to the probiotics to better exert the physiological activity.

The invention is realized in such a way that₁/O/W₂The preparation method of the probiotic preparation with the double-emulsion structure comprises the following steps:

s1: mixing probiotic bacteria and probiotic bacteria protective agent, and vortexing to uniformly disperse the mixture to form an internal water phase W₁；

S2: dissolving oil soluble emulsifier in edible oil to form oil phase O, and mixing oil phase O with internal water phase W₁Mixing, stirring, emulsifying by low energy emulsification method or high energy emulsification method to obtain water-in-oil type colostrum W₁/O；

S3: using hydrophilic emulsifier as external water phase W₂To the colostrum W₁Adding external water phase W into the solution₂Stirring, emulsifying by low energy emulsification method or high energy emulsification method to obtain W₁/O/W₂And (4) storing the double emulsion at low temperature to obtain the probiotic preparation.

Further, the internal aqueous phase W in step S1₁The loading concentration of the probiotics is more than or equal to 5log/mL；

The probiotic protecting agent includes but is not limited to at least one of saccharides, proteins, alcohols, inorganic salts, antioxidants, polymers and complexes; the mass percentage of the protective agent in the internal water phase is 0.5-25 wt%;

the probiotic protecting agent in step S1 includes, but is not limited to, at least one of glycerol, skim milk powder, ascorbic acid, whey protein isolate, trehalose, sucrose, fructose, maltose, lactose, dextran, gelatin, peptone, methyl cellulose, sodium glutamate, xylitol, and sodium dodecyl sulfate.

Further, the low energy emulsification method in step S2 includes, but is not limited to, phase transition method, spontaneous emulsification method, membrane emulsification method; the high energy emulsification method includes but is not limited to high speed shearing, high pressure homogeneous emulsification, micro jet emulsification.

Further, the oil-soluble emulsifier in step S2 includes, but is not limited to, the oil-soluble emulsifier is at least one of oil-soluble small molecule emulsifier, oil-soluble protein emulsifier, oil-soluble polymer and complex;

the oil-soluble emulsifier in step S2 includes, but is not limited to, at least one of polyglycerol ricinoleate (PGPR), span 20, span 60, span 65, span 80, span 85, ethylene glycol fatty acid ester, propylene glycol monostearate, glyceryl monostearate, and zein.

Furthermore, the addition amount of the oil-soluble emulsifier is 1-15% of the mass of the oil phase O.

Further, the oil phase O and the internal water phase W₁The mass ratio of (A) to (B) is 1-5: 1.

Further, the low energy emulsification method in step S3 includes, but is not limited to, phase transition method, spontaneous emulsification method, membrane emulsification method; the high energy emulsification method includes but is not limited to high speed shearing, high pressure homogeneous emulsification, micro jet emulsification.

Further, the hydrophilic emulsifier includes, but is not limited to, at least one of a small molecule emulsifier, a polysaccharide emulsifier, a polypeptide emulsifier, a protein emulsifier, a polymer, and a complex.

The hydrophilic emulsifier includes but is not limited to at least one of tween 80, pectin, whey protein isolate, whey protein concentrate, casein.

Further, an external aqueous phase W₂In the formula, the mass percent of the emulsifier is 0.1-15 wt%.

Further, an external aqueous phase W₂With colostrum W₁The volume ratio of the oxygen to the oxygen is 1-5: 1.

Based on W₁/O/W₂The probiotic preparation with the double-emulsion structure is prepared by the preparation method.

Based on W as described above₁/O/W₂The application of the probiotic preparation with the double-emulsion structure in probiotic products.

Further, the probiotic product comprises a low temperature product.

Further, the low-temperature product includes any one of ice cream, ice cream bar, low-temperature yogurt, cheese, low-temperature drink, and low-temperature animal health product.

In summary, the advantages and positive effects of the invention are:

the inventor of the application focuses on the processing research of the probiotic preparation for a long time, and obtains the processing technology for preparing the probiotic preparation through a large amount of research and exploration. The probiotic preparation prepared by the process can ensure the survival rate of the probiotics and prolong the storage time of the probiotics, thereby improving the stability of the probiotics and preparing the probiotic preparation which is beneficial to the probiotics to better exert the physiological activity.

In the invention, W is used₁/O/W₂The double emulsion system carries out freeze-proof protection embedding on the probiotics, and the probiotic embedding system can keep the activity of the probiotics in the freezing storage and freezing-thawing processes, reduce the influence of the external environment on the probiotics and improve the freezing-thawing and storage stability of the probiotics in the shelf life of products. Meanwhile, the probiotics are embedded in the water phase in the double emulsion, and the oil film and the interfacial film can prevent the probiotics from being damaged by external environments such as oxygen and the like, so that the survival rate, freeze thawing and storage stability of the probiotic preparation are further improved, and the problem that the quality of probiotic products is unstable is effectively solvedAnd finally, the number of effective live bacteria is insufficient, the survival time of the probiotics in the probiotic product is short, and the like.

The preparation method disclosed by the invention is simple in preparation process, effectively reduces the cost, and has important application values for prolonging the survival rate, freezing and thawing stability and storage stability of the probiotics in the shelf life of the probiotic products and guaranteeing the activity function of the probiotic products. The probiotic preparation of the double emulsion can be used for low-temperature products such as ice cream, low-temperature yoghourt, cheese, low-temperature beverage, low-temperature animal health-care products and the like, and has a wide application prospect.

Drawings

FIG. 1 is a diagram of a probiotic-embedded double emulsion sample;

FIG. 2 is a diagram of probiotic embedded double emulsion CLSM;

FIG. 3 is a graph showing the plate count detection results of example 1;

FIG. 4 is a graph showing the plate count detection results of example 2;

FIG. 5 is a graph showing the plate count detection results of example 3;

FIG. 6 is a graph showing the plate count detection results of example 4.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The invention discloses a W-based₁/O/W₂The probiotic preparation with the double-emulsion structure, the preparation method and the application are shown in the following embodiments. The probiotic bacteria in the present invention may be any probiotic bacteria, including but not limited to lactobacillus plantarum, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus bulgaricus, bifidobacterium bifidum, bifidobacterium longum, lactococcus lactis or streptococcus thermophilus, and since the respective probiotic bacteria do not cause significant differences when adapted to the technical solution of the present invention, the following description will be made in detail by taking only a part of the probiotic bacteria as an example. The edible oil is not particularly limited, and can meet the relevant edible requirements. Unless otherwise specified, the present inventionThe reagents, methods and equipment adopted are conventional in the technical field, and glassware, centrifuge tubes and pipette tips used in each embodiment, and suspensions and solutions used in the method are sterilized at 121 ℃ for 15 min. Example 1 Dual emulsion probiotic formulation preparation and Performance testing

1. The preparation method mainly comprises the following steps:

(1) preparation of bacterial suspension: activating probiotic lactobacillus plantarum by using MRS broth culture medium, culturing for 14h in a constant-temperature incubator at 30 ℃, and centrifuging the bacterial liquid to obtain thalli.

(2) Internal water phase W₁The preparation of (1): preparing 10 wt% of protective agent (trehalose in the embodiment) solution as probiotic protective agent, sterilizing at 121 deg.C for 15min, cooling to room temperature, mixing thallus with protective agent, and vortexing to uniformly disperse thallus to form internal water phase W₁。

The mass percentage of the probiotic protective agent is 0.5-25 wt%, preferably 2-20 wt%. Internal water phase W₁The loading concentration of the probiotics is more than or equal to 5 log/mL.

Probiotic protectant formulations include, but are not limited to, at least one of sugars, proteins, alcohols, inorganic salts, antioxidants, polymers or complexes, and the like; preferably, it includes, but is not limited to, at least one of glycerol, skim milk powder, ascorbic acid, whey protein isolate, trehalose, sucrose, fructose, maltose, lactose, dextran, gelatin, peptone, methyl cellulose, sodium glutamate, xylitol, sodium dodecyl sulfate.

(3) Preparation of oil phase O: adding 6g of an oil-soluble emulsifier (polyglycerol ricinoleate (PGPR) in the embodiment) into 94g of edible oil (soybean oil in the embodiment), and magnetically stirring at 500rpm for 5-45 min to obtain 6 wt% of polyglycerol ricinoleate, namely oil phase O.

The addition amount of the oil-soluble emulsifier is 1-15% of the mass of the oil phase O, and preferably 3-10%. The oil-soluble emulsifier comprises at least one of oil-soluble small molecule emulsifier, oil-soluble protein emulsifier, oil-soluble polymer and compound; preferably, oil-soluble emulsifiers including, but not limited to, polyglycerol ricinoleate (PGPR), span 20, span 60, span 65, span 80, span 85, ethylene glycol fatty acid ester, propylene glycol monostearate, glycerol monostearate, zein and the like. The magnetic stirring conditions were: magnetically stirring at 200-800 rpm for 5-45 min.

(4) Colostrum W₁Preparation of/O: mixing the internal water phase W₁Dropping the mixture into the oil phase O while stirring, wherein the stirring speed is 1000 rpm; the dripping speed is 10-100 mL/min, then stirring is continued for 2-5 min, and a high-energy emulsification method (in the embodiment, the rotating speed of 10000-18000 rpm is used for shearing for 8-15 min) is used for emulsification to obtain primary emulsion W₁/O。

Oil phase O and internal water phase W₁The volume ratio of (A) to (B) is 1 to 5:1, preferably 1 to 3: 1. The stirring speed is 500 to 2000rpm, preferably 500 to 1500 rpm.

(5) Outer water phase W₂The preparation of (1): adding 3g of hydrophilic emulsifier (whey protein isolate in the embodiment) into 97g of deionized water, stirring for 2h to fully dissolve the hydrophilic emulsifier, and putting the mixture into a refrigerator at 4 ℃ overnight to fully hydrate the mixture to obtain a 3 wt% whey protein isolate solution, namely an external water phase W₂。

The hydrophilic emulsifier includes, but is not limited to, at least one of small molecule emulsifier, polysaccharide emulsifier, polypeptide emulsifier, protein emulsifier, polymer and complex, preferably at least one of tween 80, pectin, whey protein isolate, whey protein concentrate, casein. Outer water phase W₂The mass percentage of the emulsifier is 0.1-15 wt%, preferably 1-10 wt%.

(6)W₁/O/W₂Preparation of type double emulsion formulation: mixing the primary emulsion W₁O and external water phase W₂Mixing according to the volume ratio of 1:2, and emulsifying by using a high-energy emulsification method (in the embodiment, shearing at the rotating speed of 10000-18000 rpm for 2-5 min) to obtain double emulsion W₁/O/W₂And storing at a low temperature of-20-4 ℃ to obtain the probiotic preparation.

Outer water phase W₂With colostrum W₁The volume ratio of the oxygen to the oxygen is 1-5: 1, preferably 2-4: 1.

2. And (3) performance detection:

taking original bacterial liquid and lactobacillus plantarum suspended in sterile water as W₁W prepared by low speed stirring and stirring plus shearing respectively₁/O/W₂Double emulsion, Lactobacillus plantarum suspended in 10 wt% trehalose solution, W₁W prepared with stirring only₁/O/W₂Double emulsion as control group, embedded in double emulsion W of example 1₁/O/W₂The lactobacillus plantarum in (III) is an experimental group, and the preparation conditions of the emulsion and the addition of the protective agent to W are examined₁/O/W₂The effect of the viable count of lactobacillus plantarum in the double emulsion. The control group and the experimental group take an equal amount of samples before and after freeze thawing, and the viable count of the samples is determined by adopting a dilution coating flat plate method.

The emulsion prepared by the invention is shown in figure 1, the CLSM graph detection is shown in figure 2 (gray is oil phase, dyed by Nile red dye, excitation wavelength is 483nm, and black part is water phase which is not dyed), the analysis result is shown in figure 3, and the result in the figure shows that the viable count of the emulsion prepared by stirring is reasonably reduced for the lactobacillus plantarum before freeze thawing, and the viable count of the emulsion prepared by shearing is approximately equal to or higher than that of the emulsion prepared by stirring before freeze thawing. Therefore, the shearing is only used for making the emulsion liquid drops smaller, and in the same amount of emulsion, the liquid drops coated on the solid culture medium are more, and the viable bacteria are more, further explaining that the preparation conditions have no influence on the survival rate of the probiotics.

After freeze thawing, the viable count of the lactobacillus plantarum is obviously reduced, namely the viable count of the original bacterial liquid before and after freeze thawing is 4.30 × 10¹²cfu/mL and 4.26 × 10⁹cfu/mL, and the viable count of the sample without the protective agent in the emulsion prepared by shearing before and after freeze thawing is 3.05 × 10¹¹cfu/mL and 6.12 × 10⁹cfu/mL, and the viable count of the sample added with the protectant before and after freeze thawing was 2.91 × 10¹¹cfu/mL and 1.79 × 10¹¹cfu/mL, these data indicate W₁/O/W₂The double emulsion structure and trehalose have a certain protective effect on plant lactobacillus, and particularly, the viable count of the product prepared in the embodiment of the invention is obviously higher than that of other groups after freeze thawing, which shows that the probiotic preparation of the invention can reduce beneficial bacteriaThe inactivation of the bacteria makes the bacteria better play the physiological activity in the subsequent application.