A kind of probiotic RG-I pectin microcapsule and preparation method thereof

Abstract

The present invention discloses a probiotic RG-I pectin microcapsule and a preparation method thereof, and relates to the field of biotechnology. The preparation method comprises the following steps: (1) adding a probiotic suspension and a calcium carbonate solution to an RG-I pectin aqueous solution, mixing uniformly, and obtaining a mixed solution; (2) adding an oil phase containing an emulsifier to the mixed solution, performing an emulsification reaction, then adding an oil phase containing glacial acetic acid, continuing the reaction, and finally adding a sodium acetate solution, standing, separating to obtain an aqueous phase, then sieving, washing, and obtaining the RG-I pectin microcapsule. The probiotic RG-I pectin microcapsule provided by the present invention can enhance the tolerance of probiotics to harsh environments, reduce the loss of probiotics during gastrointestinal digestion, and extend the shelf life of probiotics.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a probiotic RG-I pectin microcapsule and a preparation method thereof. Background Art

[0002] As an important food functional factor, probiotics play an important role in physiological processes such as digestion, intestinal immune development, and pathogen colonization in the intestine, and have been widely studied. However, probiotics are affected by many factors during processing and storage (such as temperature, humidity, nutrients, acidity, etc.), which leads to a decrease in the activity of probiotics and the inability to exert their physiological functions. Moreover, when probiotics are ingested by the human body, the special environment of the human digestive tract (gastric acid, bile salts, digestive tract peristalsis, etc.) will also affect the activity of probiotics. Therefore, it is of great significance to implement effective encapsulation technology for probiotics to enhance their survival ability in harsh environments for their commercialization. Microcapsules are capsules that encapsulate a small amount of active ingredients into micron-sized capsules. By establishing a functional barrier between the active ingredients and the environment, potential harmful chemical and physical reactions are avoided and the active ingredients are protected from the surrounding environment. Due to its advantages such as good protection, size controllability, and simple operation, it has become a research hotspot for improving the survival of probiotics.

[0003] Although microcapsules can reach specific target areas for release without being adversely affected by environmental factors. However, this also limits the choice of microcapsule wall materials. Rhamnogalacturonan-I (RG-I) pectin is one of the three domains that constitute pectin. Due to the steric hindrance between its main chain and the neutral side chain rhamnose carboxyl group and the cross-linking agent, the cross-linking between the two is limited, resulting in its inability to form a gel. Therefore, RG-I pectin is considered unsuitable as a wall material for microcapsules. Although studies have found that when the degree of esterification of RG-I pectin is low, it can combine with divalent metal ions to form a gel, whether it can form microcapsules to protect probiotics still needs further study. Summary of the invention

[0004] The purpose of the present invention is to provide a probiotic RG-I pectin microcapsule and a preparation method thereof to solve the problems existing in the above-mentioned prior art. The probiotic RG-I pectin microcapsule provided by the present invention can enhance the tolerance of probiotics to harsh environments, reduce the loss of probiotics during gastrointestinal digestion, and extend the shelf life of probiotics.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for preparing probiotic RG-I pectin microcapsules, comprising the following steps:

[0007] (1) adding a probiotic suspension and a calcium carbonate solution to the RG-I pectin aqueous solution and mixing them evenly to obtain a mixed solution;

[0008] (2) adding an oil phase containing an emulsifier to the mixed solution to carry out an emulsification reaction, then adding an oil phase containing glacial acetic acid, continuing the reaction, and then adding a sodium acetate solution. The mixture is allowed to stand and separated to obtain an aqueous phase, which is then sieved and washed to obtain the RG-I pectin microcapsules.

[0009] Furthermore, in step (1), the concentration of RG-I pectin in the RG-I pectin aqueous solution is 0.8-1.8 wt %.

[0010] Furthermore, in step (1), the mass ratio of RG-I pectin in the RG-I pectin aqueous solution to calcium carbonate in the calcium carbonate solution is 1:1-5.

[0011] Furthermore, in step (2), the emulsifier includes Tween 80.

[0012] Furthermore, the volume fraction of the emulsifier in the oil phase containing the emulsifier is 0.5-2.5%.

[0013] Furthermore, in step (2), after adding the oil phase containing glacial acetic acid, the water-oil volume ratio in the reaction system is 1:1-5.

[0014] The present invention also provides probiotic RG-I pectin microcapsules prepared according to the above preparation method.

[0015] The present invention also provides a method for preparing probiotic RG-I pectin microcapsules containing biofilms, comprising the steps of stimulating and culturing the above-mentioned probiotic RG-I pectin microcapsules to allow the probiotics embedded in the probiotic RG-I pectin microcapsules to form biofilms, thereby obtaining probiotic RG-I pectin microcapsules containing biofilms.

[0016] Furthermore, calcium carbonate is added to the culture medium for the stimulation culture in an amount of 10-100 mM.

[0017] The present invention also provides probiotic RG-I pectin microcapsules containing biofilm prepared according to the above preparation method.

[0018] The present invention discloses the following technical effects:

[0019] The present invention uses an endogenous emulsification method to prepare RG-I pectin microcapsules, and by embedding probiotics, the tolerance of the probiotics to a harsh environment is enhanced, the loss of the probiotics during gastrointestinal digestion is reduced, and the shelf life of the probiotics is extended.

[0020] The present invention uses Ca2+ As a cross-linking agent of RG-I pectin, it successfully formed microcapsules of RG-I pectin, broadening the application of RG-I pectin.

[0021] Although microcapsules can provide a physical barrier for probiotics, the protection of microcapsules to probiotics is still insufficient. The use of multi-layer embedding will increase the volume of microcapsules, thereby affecting the taste of ingestion and increasing costs. The present invention considers that the growth of probiotics themselves can generate biofilms to improve the tolerance of probiotics to the environment. Therefore, after using the endogenous emulsification method to form RG-I pectin microcapsules to embed probiotics, the probiotics are stimulated to form biofilms in the microcapsules through culture. The method of the present invention not only significantly improves the protective effect of microcapsules on probiotics, but also avoids the problem of increased microcapsule particle size caused by multi-layer embedding. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Lactobacillus plantarum and Lactobacillus rhamnosus used in the following examples were purchased from the Institute of Microbiology, Guangdong Academy of Sciences.

[0028] RG-I pectin is extracted from acidic and alkaline wastewater discharged from citrus canning factories. The specific method is as follows: first filter the acidic wastewater through a 400-mesh filter bag. Then, adjust the pH to 3 (up to 4) with NaOH, and precipitate for 2 hours with 95% ethanol in a volume ratio of 1:1 to obtain PA pectin. The alkaline wastewater is first adjusted to 6 (up to 7) with 2M HCl, and after natural precipitation, the precipitate is washed with 95% ethanol by volume, and placed in an oven at 55°C for 24 hours to obtain PB pectin. Finally, PA pectin and PB pectin are mixed in a mass ratio of 1:1 to obtain RG-I pectin.

[0029] Commercial pectin was purchased from Shanghai Yantian Biotechnology Co., Ltd.

[0030] Example 1

[0031] (1) Activation of strains and preparation of bacterial suspension: The glycerol culture of Lactobacillus plantarum and Lactobacillus rhamnosus stored at -80°C was inoculated into sterile MRS liquid medium, cultured at 37°C for 24 hours, and the bacteria were activated twice under the same conditions. The activated strains were inoculated into MRS liquid medium at an inoculum rate of 2% (v / v), cultured at 37°C, and the bacterial suspension was collected at the late logarithmic growth stage, centrifuged at 8000 r / min for 10 minutes, washed twice with physiological saline (9 g / L NaCl), and resuspended with physiological saline to obtain about 10 9 CFU / mL of bacterial suspension of two probiotics.

[0032] (2) Preparation of Lactobacillus plantarum / Lactobacillus rhamnosus microcapsules: RG-I pectin was dissolved in 20 mL of sterile water to a final concentration of 1.5 wt% (0.8-1.8 wt% can achieve the same effect), and then mixed with 10 mL of Lactobacillus plantarum / Lactobacillus rhamnosus bacterial suspension, and then 0.5 g of CaCO3 (the mass ratio of CaCO3 to RG-I pectin was controlled at 1:1-5) was added and stirred evenly to obtain a mixed solution.

[0033] Then, 40 mL of soybean oil containing Tween 80 (the volume fraction of Tween 80 is 0.1%, and the volume fraction of Tween 80 is required to be controlled at 0.5-2.5%) is added to the mixed solution, and emulsified at a speed of 300 r / min (200-800 r / min can achieve the same effect) for 15 minutes. Subsequently, 20 mL of soybean oil containing glacial acetic acid (the volume fraction of glacial acetic acid is 0.5%) is added and stirred for 30 minutes. After adding the soybean oil containing glacial acetic acid, the water-oil volume ratio in the reaction system is controlled at 1:1-5.

[0034] Finally, 60 mL of sodium acetate solution (the volume fraction of sodium acetate is 0.5%) is added and allowed to stand for 60 min. After all the microcapsules to be formed have settled to the bottom of the sodium acetate solution, the aqueous phase is separated and passed through a 200 mesh sieve (up to 400 mesh), and the microcapsules are collected and washed twice to remove the remaining oil phase and surface bacteria to obtain plant lactobacillus / rhamnosus lactobacillus microcapsules, which are then stored in a refrigerator at 4°C.

[0035] (3) 1 mL of Lactobacillus plantarum / Lactobacillus rhamnosus microcapsules were placed in a MRS liquid culture medium containing 50 mM CaCO3, and cultured at 37°C for 12 h to obtain Lactobacillus plantarum / Lactobacillus rhamnosus microcapsules containing biofilms. The amount of CaCO3 added to the MRS liquid culture medium was controlled to be 10-100 mM.

[0036] (4) Determination of microcapsule embedding efficiency: Take 1 mL of the plant lactobacillus / rhamnosus microcapsules prepared in step (2) and the plant lactobacillus / rhamnosus microcapsules containing biofilm prepared in step (3), add them to 9 mL of sterile PBS (pH 7.5) solution, homogenize at a speed of 10000 r / min for 30 seconds, and then gently shake in a rotary stirrer for 10 minutes to release the bacteria in the microcapsules. The total colony count was determined by the plate coating method. The formula of the encapsulation efficiency (EE%) is EE (%) = (the total colony count released from the microcapsule (Log CFU / mL) / the total colony count of the bacterial suspension of the two probiotics used for microcapsule preparation (Log CFU / mL)) × 100%.

[0037] The results of the embedding rate showed that the embedding rate of Lactobacillus plantarum in the microcapsules was 65.08%, and the embedding rate of Lactobacillus rhamnosus was 65.95%.

[0038] Example 2

[0039] The Lactobacillus plantarum / Lactobacillus rhamnosus microcapsules containing biofilm prepared in Example 1 were subjected to a gastrointestinal digestion tolerance test, and the specific steps were as follows:

[0040] In the test tube containing 8mL simulated gastric juice, add respectively the plant lactobacillus suspension, rhamnosus lactobacillus suspension, plant lactobacillus microcapsule, rhamnosus lactobacillus microcapsule, plant lactobacillus microcapsule containing biofilm and rhamnosus lactobacillus microcapsule containing biofilm prepared by 2mL embodiment 1, vibrate at a speed of 150rpm in the vibration incubator for gastric digestion, sample at 0, 60 and 120min respectively.Gastric juice is composed of 0.02M PBS and 10mg / mL pepsin, adjusts pH to 2.0 with 1MHCl, and digestion temperature is 37 ℃.

[0041] During small intestinal digestion, in the test tube containing 8mL simulated intestinal fluid, add the plant lactobacillus suspension, rhamnosus lactobacillus suspension, plant lactobacillus microcapsule, rhamnosus lactobacillus microcapsule, plant lactobacillus microcapsule containing biofilm and rhamnosus lactobacillus microcapsule containing biofilm prepared by 2mL embodiment 1, shake with the speed of 150rpm in the vibration incubator for small intestinal digestion, sample at 0,120 and 240min respectively.Small intestinal digestion fluid is 0.02M PBS, 10mg / mL trypsin, 0.3wt% bile salt, 1M NaOH regulates pH6.8, and digestion temperature is 37 ℃.Digestion sample is centrifuged for 5min under 6000r / min, to remove digestion fluid.Resuspend sample in PBS buffer, determine total colony count by plate count method.

[0042] The shelf life survival rate test method is: place the Lactobacillus plantarum / Lactobacillus rhamnosus microcapsules containing biofilm in a storage bottle, seal it, and store it at room temperature (25°C). After 45 days, the number of live probiotics is determined by the plate colony counting method.

[0043] The survival rates of the two probiotics in gastric digestion are shown in Table 1, where the probiotic survival rate at 0 min is used as the benchmark.

[0044] Table 1

[0045]

[0046] As shown in Table 1, the survival rates of free probiotics, probiotic microcapsules and probiotic microcapsules containing biofilms showed different downward trends after simulated gastric juice digestion. At 120 minutes, the survival rate of free Lactobacillus plantarum decreased to 33%, and the survival rate of free Lactobacillus rhamnosus decreased to 46%. Compared with the free Lactobacillus plantarum, the downward trend of Lactobacillus plantarum encapsulated by microcapsules was greatly slowed down. At 120 minutes, its survival rate reached 51%. In addition, Lactobacillus rhamnosus also showed a slowing trend in the decline of survival rate. Among the three samples, microcapsules containing biofilms showed the best effect. After 120 minutes of simulated digestion, Lactobacillus plantarum encapsulated in microcapsules containing biofilms still showed a survival rate of 65%. Similarly, Lactobacillus rhamnosus also showed the same trend. Therefore, according to the results, RG-I microcapsules can enhance the tolerance of probiotics in gastric digestion, and microcapsules with biofilms further improve the protection of microcapsules to probiotics.

[0047] The survival rates of the two probiotics in small intestinal digestion are shown in Table 2, where the probiotic survival rate at 0 min is used as the benchmark.

[0048] Table 2

[0049]

[0050] As shown in Table 2, microcapsule-embedded probiotics significantly improve the survival rate of probiotics in the small intestine, and both probiotics show good effects. At the same time, the formation of biofilm can also improve the survival rate of microcapsules in the small intestine. This shows that the present invention has a good improvement effect on the survival rate of probiotics in the digestive process of the gastrointestinal tract.

[0051] After storage at 25°C for 45 days, the survival rates of free Lactobacillus plantarum and Lactobacillus rhamnosus were 45% and 59% respectively, while the survival rates of Lactobacillus plantarum and Lactobacillus rhamnosus encapsulated in microcapsules were 66% and 77% respectively, indicating that the microcapsules significantly improved their survival rates. The survival rates of the microcapsules of the two bacteria with biofilm were 74% and 83% respectively, which more significantly improved the effect of the microcapsules, indicating that the present invention can significantly improve the storage stability of probiotics.

[0052] Comparative Example 1

[0053] The same method as in Example 1 was used to prepare a Lactobacillus rhamnosus suspension, Lactobacillus rhamnosus microcapsules and Lactobacillus rhamnosus microcapsules containing biofilms, the only difference being that commercial pectin was used instead of RG-I pectin.

[0054] The embedding rate results showed that the embedding rate of the Lactobacillus rhamnosus microcapsules prepared in Comparative Example 1 was 45%.

[0055] The same method as in Example 2 was used to detect the survival rates of the Lactobacillus rhamnosus suspension, Lactobacillus rhamnosus microcapsules and Lactobacillus rhamnosus microcapsules containing biofilm prepared in Comparative Example 1 in simulated gastric juice. The results are shown in Table 3, where the probiotic survival rate at 0 min was used as the benchmark.

[0056] Table 3

[0057]

[0058] As shown in Table 3, the survival rate of free Lactobacillus rhamnosus is greatly affected by gastric acid during simulated gastric digestion. After 60 minutes of simulated digestion, its survival rate is 63%. After 120 minutes of simulated digestion, its survival rate is less than half, 46%; after microcapsule embedding, its survival rate is slightly improved, after 120 minutes of simulated digestion, its survival rate is 53%; and the probiotics embedded in microcapsules with biofilm show better effects during simulated digestion, after 120 minutes of simulated digestion, its survival rate can still reach 72%. The results show that after the probiotics are embedded according to the method of comparative example 1, the survival of the probiotics is slightly improved, but the tolerance of the microcapsules to gastric digestion caused by the materials and the production method is relatively poor. The microcapsules are easily eroded by gastric acid, causing the structure of the microcapsules to be destroyed, thereby causing the survival rate of the probiotics to decrease, and its protection effect is significantly lower than that of the microcapsules prepared in Example 1 of the present invention.

[0059] Comparative Example 2

[0060] The same method as in Example 1 was used to prepare a Lactobacillus rhamnosus suspension, Lactobacillus rhamnosus microcapsules and Lactobacillus rhamnosus microcapsules containing biofilms, the only difference being that sodium alginate was used instead of RG-I pectin.

[0061] The embedding rate results showed that the embedding rate of Lactobacillus rhamnosus microcapsules prepared in Comparative Example 2 was 63%.

[0062] The survival rates of the Lactobacillus rhamnosus suspension, Lactobacillus rhamnosus microcapsules and Lactobacillus rhamnosus microcapsules containing biofilm prepared in Comparative Example 2 were tested in simulated gastric fluid. The results are shown in Table 4, where the probiotic survival rate at 0 min was used as the benchmark.

[0063] Table 4

[0064]

[0065] As shown in Table 4, when free Lactobacillus rhamnosus is in simulated gastric digestion, its survival rate will be greatly affected because the probiotics have poor tolerance to acidity. After 120 minutes of simulated gastric digestion, its survival rate is less than half, which is 46%; microcapsules can provide a shell for probiotics, which can reduce the penetration of gastric acid, thereby improving the survival rate of probiotics. Therefore, after microcapsule embedding, its survival rate is slightly improved, and after 120 minutes of simulated gastric digestion, its survival rate is 54%; and the probiotics embedded in microcapsules with biofilms have a significantly increased survival rate during simulated digestion due to the formation of biofilms. After 120 minutes of simulated digestion, its survival rate can still reach 72%. The results show that after embedding the probiotics according to the method of Comparative Example 2, although the survival of the probiotics is slightly improved, the microcapsules made of sodium alginate have poor tolerance to gastric acid, which leads to a decrease in survival rate, and its protective effect is significantly lower than that of the microcapsules prepared in Example 1 of the present invention.

[0066] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing probiotic RG-I pectin microcapsules, characterized in that: The following steps are involved: (1) Adding the probiotic suspension and the calcium carbonate solution to the RG-I pectin aqueous solution, mixing evenly to obtain a mixed solution; (2) adding an oil phase containing an emulsifier to the mixed solution to carry out an emulsification reaction, then adding an oil phase containing glacial acetic acid to continue the reaction, and finally adding a sodium acetate solution, standing, separating to obtain an aqueous phase, and then sieving and washing to obtain the RG-I pectin microcapsules; In step (1), the concentration of RG-I pectin in the RG-I pectin aqueous solution is 0.8-1.8 wt %; In step (1), the mass ratio of RG-I pectin in the RG-I pectin aqueous solution to calcium carbonate in the calcium carbonate solution is 1:1-5; In step (2), the emulsifier is Tween 80; The volume fraction of the emulsifier in the oil phase containing the emulsifier is 0.5-2.5%; In step (2), after adding the oil phase containing glacial acetic acid, the water-oil volume ratio in the reaction system is 1:1-5; The probiotics are Lactobacillus plantarum ( Lactobacillus plantarum ) or Lactobacillus rhamnosus ( Lactobacillus rhamnosus ).

2. A probiotic RG-I pectin microcapsule prepared according to the preparation method according to claim 1.

3. A method for preparing probiotic RG-I pectin microcapsules containing biofilm, characterized in that: The method comprises the steps of stimulating and culturing the probiotic RG-I pectin microcapsules described in claim 2 to allow the probiotics embedded in the probiotic RG-I pectin microcapsules to form biofilms, thereby obtaining probiotic RG-I pectin microcapsules containing biofilms.

4. The preparation method according to claim 3, characterized in that: Calcium carbonate is added to the culture medium for the stimulation culture in an amount of 10-100 mM; the stimulation culture time is 12 hours.

5. A probiotic RG-I pectin microcapsule containing biofilm prepared according to the preparation method of claim 3 or 4.

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