Method for improving gastrointestinal tolerance of probiotics and probiotic liquid core gel ball

Abstract

The invention discloses a method for improving gastrointestinal tolerance of probiotics and a probiotic liquid core gel ball, the probiotic liquid core gel ball is prepared by adopting a calcium alginate cross-linked reverse spheroidizing method, and various indexes influencing the characteristics of the probiotic liquid core ball after re-culture are optimized, the process is simple in preparation, raw materials are natural and safe, the cost is low, and the method is suitable for industrial production. The tolerance and the survival rate of the probiotics in gastrointestinal digestion can be well improved, and the probiotic liquid core gel ball prepared by the method disclosed by the invention has good mechanical property, swelling rate and storage survival rate; by optimizing various parameters, such as re-culture time, core material liquid viscosity, polysaccharide molecular weight and a storage solution, which influence the characteristics of the probiotic liquid core ball after re-culture, the re-cultured liquid core ball has better mechanical properties, swelling rate and survival rate, and by combining with a gastric acid-cholate resistance reaction result, the survival rate of the liquid core ball is improved. The invention provides the preparation process of the re-culture solution core ball capable of effectively improving the gastrointestinal tolerance of the probiotics whether the core ball is broken or not.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic processing, and particularly relates to a method for improving the gastrointestinal tolerance of probiotics and a probiotic liquid-core gel ball. Background Art

[0002] Probiotics have multiple physiological functions, including regulating the balance of the human intestinal flora, enhancing immunity, and alleviating allergies and inflammation. Their market size is growing steadily. However, current problems with oral probiotic preparations primarily stem from their vulnerability to inactivation during the production process and their destruction after oral administration. For example, exposure of anaerobic probiotics to air during production can affect the activity of intracellular enzymes and nucleic acids. Disinfection can easily cause cellular stress, altering cell structure, and even inactivation. Gastric acid and bile acids can significantly reduce the survival rate of ingested probiotics. For probiotics to be effective, they must be able to reach and colonize in areas like the colon at higher concentrations. Therefore, how to better protect probiotics and minimize their loss during gastrointestinal digestion is a key issue.

[0003] Existing research and production have found that traditional preparations (such as powders and tablets) are easily eroded by gastric acid, resulting in low survival rates of live bacteria. Products stored at room temperature are easily inactivated and rely on cold chain transportation. Although multi-layer encapsulation technology can improve the protection effect, the process is complex and the steps are cumbersome. There are problems such as large fluctuations in survival rates between batches and high difficulty in industrial production. In addition, existing materials (such as gelatin and whey protein) lack a dynamic response mechanism and are prone to swelling and disintegration in gastric juice, resulting in premature release or retention failure of probiotics. At the same time, there are significant individual differences. Some strains are difficult to colonize due to rejection of the intestinal environment. There is an urgent need to develop innovative technologies that take into account efficient protection, process stability and targeted release.

[0004] Due to its pH-responsive nature, calcium alginate has been proven to be an ideal wall material for protecting probiotics in the gastrointestinal tract. Microencapsulation of probiotics also offers sustained-release benefits, allowing them to reach the intestines in greater numbers before being released to exert their desired effects. However, directly encapsulating probiotics with calcium alginate provides only a single layer of physical protection. Once ruptured, the probiotics are directly released, significantly reducing the protective effect and making it unsuitable for practical applications. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for improving the gastrointestinal tolerance of probiotics.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions: comprising:

[0009] Probiotics were inoculated into MRS liquid medium for anaerobically culture, and diluted after culture to obtain a probiotic suspension;

[0010] adding sodium hyaluronate to the calcium chloride solution to obtain a calcium chloride-sodium hyaluronate solution;

[0011] The probiotic suspension is mixed with the calcium chloride-sodium hyaluronate solution to obtain a core liquid;

[0012] The sodium alginate solution is allowed to stand to remove internal bubbles, thereby obtaining a coagulation bath;

[0013] The core material droplets are added into the coagulation bath, taken out after the reaction, and quickly filtered and cleaned to obtain the liquid core ball;

[0014] The liquid core ball is hardened in a calcium chloride solution, filtered and cleaned to obtain a hardened liquid core ball;

[0015] The hardened liquid core balls are transferred to MRS culture medium for anaerobically cultured again. The obtained re-cultured probiotic liquid core gel balls are filtered and cleaned and then transferred to a phosphate-free modified MRS culture medium for storage, thereby improving the gastrointestinal tolerance of the probiotics.

[0016] As a preferred embodiment of the method for improving gastrointestinal tolerance of probiotics according to the present invention, the probiotics are inoculated into MRS liquid culture medium and cultured anaerobic for 12 to 48 hours.

[0017] As a preferred embodiment of the method for improving gastrointestinal tolerance of probiotics according to the present invention, the bacterial concentration of the probiotic suspension is 10 6～7 CFU / mL.

[0018] As a preferred embodiment of the method for improving the gastrointestinal tolerance of probiotics according to the present invention, the concentration of the calcium chloride solution is 0.5-5% (w / v).

[0019] As a preferred embodiment of the method for improving the gastrointestinal tolerance of probiotics according to the present invention, the concentration of sodium hyaluronate in the calcium chloride-sodium hyaluronate solution is 0.5-1.0 (w / v).

[0020] As a preferred embodiment of the method for improving gastrointestinal tolerance of probiotics according to the present invention, the mixing volume ratio of the probiotic suspension to the calcium chloride-sodium hyaluronate solution is 1:10 to 1:30.

[0021] As a preferred embodiment of the method for improving gastrointestinal tolerance of probiotics according to the present invention, the concentration of the sodium alginate solution is 0.5-0.8% (w / v), wherein the molecular weight of the sodium alginate is 300,000-450,000 Da.

[0022] As a preferred embodiment of the method for improving the gastrointestinal tolerance of probiotics according to the present invention, the core material liquid is uniformly added dropwise at a rate of 3.0 to 5.0 ml / min into a coagulation bath stirred at 600 to 1200 r / min using a peristaltic pump, and is removed after reacting for 1 to 3 minutes.

[0023] As a preferred embodiment of the method for improving gastrointestinal tolerance of probiotics according to the present invention, the time for the second anaerobic culture is 12 to 48 hours.

[0024] As a preferred embodiment of the method for improving the gastrointestinal tolerance of probiotics according to the present invention, the formula of the modified MRS culture medium includes:

[0025] 8.0-12.0 g / L peptone, 3.0-6.0 g / L yeast extract, 5.0-20.0 g / L glucose, 3.0-5.0 g / L sodium acetate, 0.5-1.0 g / L Tween 80.

[0026] Another object of the present invention is to provide a re-cultured probiotic liquid core gel ball, wherein the re-cultured probiotic liquid core ball has a gastric acid resistance survival rate increased by 50-55% and a bile salt resistance survival rate increased by 40-45% compared with naked bacteria.

[0027] Beneficial effects of the present invention:

[0028] The present invention provides a method for improving the gastrointestinal tolerance of probiotics based on recultivation. The method adopts a reverse spheroidization method of calcium alginate cross-linking to prepare probiotic liquid-core gel spheres, and optimizes various indicators affecting the characteristics of the probiotic liquid-core spheres after recultivation. The process is simple to prepare, the raw materials are natural and safe, and the tolerance and survival rate of probiotics in gastrointestinal digestion can be greatly improved. The probiotic liquid-core gel spheres prepared by the method of the present invention have good mechanical properties, swelling rate and storage survival rate.

[0029] The present invention uses a reverse spheroidization method to drip a core liquid containing probiotics, a calcium ion solution, and sodium hyaluronate into a stirring sodium alginate coagulation bath at a fixed rate to form liquid-core gel spheres that encapsulate the probiotics. The encapsulated probiotic microcapsules are then recultured, and the probiotics within the calcium alginate shell spontaneously form a biofilm on the bacterial surface. This biofilm is composed of microbial cells and their secreted extracellular polymers (EPS). After the physical shell ruptures, the probiotics can be further protected from adverse environmental influences such as gastric acid, bile salts, and antibiotics, thereby enhancing the colonization and survival ability of the probiotics in the gastrointestinal tract. The probiotics in the biofilm can also stably release beneficial metabolites (such as short-chain fatty acids and antimicrobial peptides), thereby enhancing the health-promoting effect on the host. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0031] Figure 1 The gastric acid and bile salt resistance of the Lactobacillus rhamnosus LRa05 re-cultured probiotic liquid core gel spheres prepared in Example 1 of the present invention is shown in FIG.

[0032] Figure 2 The gastric acid and bile salt resistance of the animal Bifidobacterium BLa80 re-cultured probiotic liquid core gel spheres prepared in Example 1 of the present invention is shown in FIG.

[0033] Figure 3 This is a comparison of the probiotic liquid core balls (Bifidobacterium animalis BLa80) prepared in Example 1 and Comparative Example 1 after re-cultivation.

[0034] Figure 4 The figures are a comparison of the blasting force and mass changes of the spheres in Example 1, Comparative Examples 2, and Comparative Examples 3 after storage for 28 days in different storage solutions and then incubation with the liquid core.

[0035] Figure 5 This is a comparison chart of the core material liquid viscosity corresponding to different sodium hyaluronate addition amounts in Example 2 of the present invention.

[0036] Figure 6 This is a relationship diagram established between the molecular weights of the three sodium alginates in Example 1 and Example 3 determined by high performance gel exclusion chromatography and the viscosities determined by a rapid viscometer.

[0037] Figure 7 The figure shows a comparison of the blasting force and mass change of the re-culture liquid core balls made from three kinds of sodium alginate in Examples 1 and 3 of the present invention.

[0038] Figure 8 The figure shows a comparison of the bile salt tolerance of the re-culture medium core balls prepared from three sodium alginate compositions in Examples 1 and 3 of the present invention.

[0039] Figure 9 This is a comparison chart of the number of gelatinous cocci before and after re-cultivation in Example 4 of the present invention.

[0040] Figure 10 The bacterial counts of the two bacteria corresponding to different re-cultivation times in Example 4 of the present invention are shown. DETAILED DESCRIPTION

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0042] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0043] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0044] Unless otherwise specified, the raw materials used in the present invention are commonly available in the market.

[0045] Specific embodiment of the present invention Before the experiment, MRS broth, 0.9% physiological saline and other experimental tools were sterilized by high pressure at 115° C. for 20 minutes.

[0046] The gastric acid resistance test method of the present invention is:

[0047] 0.1 ml of naked bacteria were added to 4.9 ml of simulated gastric acid solution and reacted at 37°C and 150 r / min for 2 h. 0.5 g of intact and broken re-cultured probiotic liquid core balls were added to 4.5 ml of simulated gastric acid solution and reacted at 37°C and 150 r / min for 2 h. The bacterial count was determined by taking the bacterial suspension at time points of 0, 20, 40, 60, 80, 100, and 120 min.

[0048] Bile salt tolerance test method:

[0049] Take 0.1 ml of naked bacteria and add it to 4.9 ml of bile salt solution, and react at 37°C and 150 r / min for 1 hour. Take 0.5 g of intact and broken re-cultured probiotic liquid core balls and add them to 4.5 ml of bile salt solution, and react at 37°C and 150 r / min for 1 hour. Take the bacterial suspensions before and after the reaction in the bile salt solution for 1 hour to determine the internal bacterial count.

[0050] Example 1

[0051] This embodiment provides a method for improving the gastrointestinal tolerance of probiotics (Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis subsp. lactis BLa80), specifically:

[0052] 1) Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis subsp. lactis BLa80 were inoculated into MRS liquid medium and placed in an anaerobic workstation for 24 h (0.5 g / L L-cysteine hydrochloride was added when inoculating BLa80), then washed with physiological saline and centrifuged twice (4 ° C, 5000 g, 10 min), and the probiotic suspension was prepared after dilution (10 7 CFU / mL);

[0053] 2) Calcium chloride was added to deionized water and stirred until completely dissolved to obtain a calcium chloride solution with a mass concentration of 1% (w / v);

[0054] Sodium hyaluronate (molecular weight of sodium hyaluronate is 1.8 million Da) is added to the calcium chloride solution and stirred until completely dissolved to obtain a calcium chloride-sodium hyaluronate solution with a mass concentration of sodium hyaluronate of 0.8% (w / v);

[0055] 3) Add the probiotic suspension to the calcium chloride-sodium hyaluronate solution at a volume ratio of 1:20 and stir and disperse for 30 minutes to obtain a core liquid;

[0056] 4) Sodium alginate was added to deionized water and stirred until completely dissolved to obtain a sodium alginate solution with a mass concentration of 0.6% (w / v) and a molecular weight of approximately 360,000 Da. The solution was allowed to stand to remove internal bubbles to obtain a coagulation bath;

[0057] 5) The core material liquid was added dropwise at a rate of 5.0 ml / min into a coagulation bath stirred at a speed of 1200 r / min by a peristaltic pump. After reacting for 2 minutes, the liquid was taken out and quickly filtered and washed to obtain a liquid core ball;

[0058] 6) The liquid core ball is transferred to a newly prepared calcium chloride solution with a mass concentration of 1% (w / v), hardened for 10 minutes, and then filtered and washed to obtain a hardened liquid core ball;

[0059] 7) The hardened liquid core balls were transferred to MRS broth and anaerobically cultured in an anaerobic workstation for 24 hours to obtain recultured probiotic liquid core gel balls. After filtration and cleaning, the balls were transferred to modified MRS broth GM (formula see Table 1) and stored at 4°C to improve the gastrointestinal tolerance of the probiotics (Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis subsp. lactis BLa80).

[0060] Table 1 Modified MRS broth formula

[0061]

[0062] The gastric acid and bile salt resistance of the two re-cultured probiotic liquid core gel balls (Lactobacillus rhamnosus LRa05 and Bifidobacterium animalis BLa80) prepared in this example were tested in the presence and absence of rupture. Figure 1 、 Figure 2 As shown, Figure 1 The gastric acid and bile salt resistance of Lactobacillus rhamnosus LRa05 re-cultivated probiotic liquid core gel spheres in the ruptured and unruptured conditions. Figure 2 The gastric acid and bile salt resistance of the re-cultured probiotic liquid-core gel balls of animal Bifidobacterium BLa80 in the ruptured and unruptured conditions was studied. It can be seen that the liquid-core balls after rupture and unruptured re-culture have stronger gastric acid and bile salt resistance than naked bacteria, which can protect the probiotics well and still have a good protective effect after rupture. This may be related to the fact that the probiotics secrete more extracellular metabolites on the surface of the bacteria with the help of the outer shell of the liquid-core balls during the re-culture process, which helps to resist the damage of gastrointestinal digestion to the bacteria, so that it still has a high tolerance after rupture.

[0063] Comparative Example 1

[0064] The difference between this comparative example and Example 1 is that the calcium chloride solution in step 2) and step 6) is replaced with a calcium lactate solution of the same concentration, and the remaining steps and processes are the same as those in Example 1 to obtain the re-cultured probiotic liquid core gel spheres of this comparative example.

[0065] Figure 3 This is a comparison of the probiotic liquid core balls (Bifidobacterium animalis BLa80) prepared in Example 1 and Comparative Example 1 after re-cultivation. It can be seen that the solution of the liquid core balls prepared with calcium lactate in the system of the present invention becomes more turbid after re-cultivation, that is, the effect of embedding the probiotics is not good, there will be more leakage, and it is not conducive to the protection of the internal probiotics. Therefore, calcium chloride is a more suitable calcium ion choice.

[0066] Comparative Example 2

[0067] The difference between this comparative example and Example 1 is that in step 7), the probiotic liquid core gel balls are re-cultured, filtered and washed, and then transferred to physiological saline (NaCl) and stored at 4° C. The remaining steps and processes are the same as those in Example 1.

[0068] Comparative Example 3

[0069] The difference between this comparative example and Example 1 is that in step 7), the probiotic liquid core gel balls are re-cultured, filtered, cleaned, and then transferred to MRS broth and stored at 4° C. The remaining steps and processes are the same as those in Example 1.

[0070] Figure 4 The explosive force and mass changes of the liquid core balls after storage for 28 days in different storage solutions of Example 1, Comparative Example 2 and Comparative Example 3 are compared. Among them, Ori is the liquid core ball in the initial state, MRS is MRS broth, GM is modified MRS solution, and NaCl is physiological saline. Figure 4 It can be seen that compared with the use of MRS to store liquid core balls, the modified MRS with components such as phosphate removed can make the liquid core balls after re-culture have higher explosive force and lower swelling phenomenon, and compared with normal saline, it has the basic nutrients to maintain the growth of probiotics. Therefore, the probiotic liquid core gel balls after modified MRS storage and re-culture are preferred.

[0071] Comparative Example 4

[0072] The difference between this comparative example and Example 1 is that the concentration of the probiotic suspension in step 1) is adjusted to 10 9 CFU / mL, and the remaining steps and processes were referred to Example 1 to obtain the re-cultured probiotic liquid core gel ball of this comparative example.

[0073] The results showed that the use of a higher concentration of probiotic suspension in this comparative example did not significantly increase the number of bacteria in the later culture, and the internal bacteria were more likely to die.

[0074] Example 2

[0075] This example differs from Example 1 in that the mass concentration of sodium hyaluronate in the calcium chloride-sodium hyaluronate solution in step 2) is adjusted to 0, 0.5, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0% (w / v), respectively. The remaining steps are the same as in Example 1, to obtain re-cultured probiotic liquid-core gel spheres prepared with different sodium hyaluronate concentrations in this example.

[0076] Figure 5 The following is a comparison chart of the core liquid viscosity corresponding to different sodium hyaluronate addition amounts when the encapsulated bacteria is Lactobacillus rhamnosus LRa05. It can be seen that when the sodium hyaluronate addition amount is 0.7-0.9% (w / v), the viscosity range is 300-500 cP (rapid viscometer No. 63 rotor, speed 100 rpm). This viscosity range has a suitable core liquid viscosity and a better probiotic suspension state. When the encapsulated bacteria is Bifidobacterium animalis subspecies lactis BLa80, the results are consistent.

[0077] Example 3

[0078] The difference between this embodiment and embodiment 1 is that the molecular weight of the sodium alginate solution in step 4) is adjusted to 360,000 Da and 450,000 Da respectively. The remaining steps are referred to embodiment 1, and the re-cultivated probiotic liquid core gel balls prepared at different re-cultivation times in this embodiment are obtained. Among them, the re-cultivated probiotic liquid core gel balls with a molecular weight of 300,000 Da are recorded as SA-1, the re-cultivated probiotic liquid core gel balls with a molecular weight of 360,000 Da are recorded as SA-2, and the re-cultivated probiotic liquid core gel balls with a molecular weight of 450,000 Da are recorded as SA-3.

[0079] The molecular weights of the three sodium alginate samples in Example 1 and Example 3 were determined by high performance gel exclusion chromatography and correlated with the viscosity measured by a rapid viscometer (rapid viscometer rotor No. 63, speed 100 rpm). The results are shown in the figure. Figure 6 As shown, Figure 7 、 Figure 8 The effects of three types of sodium alginate on the characteristics of re-cultured liquid core balls and their bile salt tolerance can be seen. It can be seen that the liquid core balls prepared by sodium alginate (SA-2) with medium molecular weight and viscosity have the highest explosive force and the lowest quality among the two bacteria after re-culture, indicating that it absorbs less water and swells, which is conducive to the maintenance of the liquid core ball shape. In addition, the number of the two probiotics surviving after 1 hour of bile salt treatment is also greater than that of the other two types. Therefore, sodium alginate with a molecular weight of 360,000 Da is preferred for the preparation of re-cultured liquid core balls.

[0080] Example 4

[0081] The difference between this example and Example 3 is that the molecular weight of the sodium alginate solution was adjusted to 360,000 Da and 450,000 Da, respectively, and the anaerobic incubation time of the hardened liquid core balls in step 7) was adjusted to 12, 36, and 48 hours, respectively. The remaining steps were the same as in Example 1, and the re-cultivated probiotic liquid core gel balls prepared in this example at different re-cultivation times were obtained.

[0082] Figure 9 This is a comparison chart of the number of gel cocci before and after re-cultivation. Compared with before re-cultivation, the number of bacteria inside the liquid core ball can be greatly increased after re-cultivation (about 4logCFU), and the re-cultivation process can make the probiotics secrete more extracellular metabolites and spontaneously form biofilms, which helps the probiotics better protect themselves in the gastrointestinal tract.

[0083] Figure 10 The bacterial counts of the two bacteria corresponding to different re-cultivation times are shown. It can be seen that the probiotic liquid core balls prepared after re-cultivation for 24 hours have the highest bacterial count, and the pattern is consistent in different sodium alginate. Therefore, it is preferred to take out the probiotic liquid core balls after re-cultivation for 24 hours.

[0084] In summary, the present invention provides a method for improving the gastrointestinal tolerance of probiotics based on recultivation, wherein probiotic liquid-core gel spheres are prepared by a reverse spheroidization method of calcium alginate cross-linking, and various indicators affecting the characteristics of the probiotic liquid-core spheres after recultivation are optimized. The process is simple to prepare, the raw materials are natural and safe, and the tolerance and survival rate of probiotics in gastrointestinal digestion can be greatly improved. The probiotic liquid-core gel spheres prepared by the method of the present invention have good mechanical properties, swelling rate and storage survival rate.

[0085] The present invention uses a reverse spheroidization method to drip a core liquid containing probiotics, a calcium ion solution, and sodium hyaluronate into a stirring sodium alginate coagulation bath at a fixed rate to form liquid-core gel spheres that encapsulate the probiotics. The encapsulated probiotic microcapsules are then recultured, and the probiotics within the calcium alginate shell spontaneously form a biofilm on the bacterial surface. This biofilm is composed of microbial cells and their secreted extracellular polymers (EPS). After the physical shell ruptures, the probiotics can be further protected from adverse environmental influences such as gastric acid, bile salts, and antibiotics, thereby enhancing the colonization and survival ability of the probiotics in the gastrointestinal tract. The probiotics in the biofilm can also stably release beneficial metabolites (such as short-chain fatty acids and antimicrobial peptides), thereby enhancing the health-promoting effect on the host.

[0086] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for improving gastrointestinal tolerance of probiotics, characterized by: include, Probiotics were inoculated into MRS liquid medium for anaerobically culture, and diluted after culture to obtain a probiotic suspension; adding sodium hyaluronate to the calcium chloride solution to obtain a calcium chloride-sodium hyaluronate solution; The probiotic suspension is mixed with the calcium chloride-sodium hyaluronate solution to obtain a core liquid; The sodium alginate solution is allowed to stand to remove internal bubbles, thereby obtaining a coagulation bath; The core material droplets are added into the coagulation bath, taken out after the reaction, and quickly filtered and cleaned to obtain the liquid core ball; The liquid core ball is hardened in a calcium chloride solution, filtered and cleaned to obtain a hardened liquid core ball; The hardened liquid core balls are transferred to MRS culture medium for anaerobically cultured again. The obtained re-cultured probiotic liquid core gel balls are filtered and cleaned and then transferred to a phosphate-free modified MRS culture medium for storage, thereby improving the gastrointestinal tolerance of the probiotics.

2. The method for improving gastrointestinal tolerance of probiotics according to claim 1, wherein: The probiotics are inoculated into the MRS liquid culture medium and the anaerobic culture time is 12 to 48 hours.

3. The method for improving gastrointestinal tolerance of probiotics according to claim 2, wherein: The bacterial concentration of the probiotic suspension is 10 6～7 CFU / mL.

4. The method for improving gastrointestinal tolerance of probiotics according to claim 3, wherein: The concentration of the calcium chloride solution is 0.5-5% (w / v), and the concentration of sodium hyaluronate in the calcium chloride-sodium hyaluronate solution is 0.5-1.0 (w / v).

5. The method for improving gastrointestinal tolerance of probiotics according to claim 4, characterized in that: The mixing volume ratio of the probiotic suspension to the calcium chloride-sodium hyaluronate solution is 1:10 to 1:

30.

6. The method for improving gastrointestinal tolerance of probiotics according to claim 1, wherein: The concentration of the sodium alginate solution is 0.5-0.8% (w / v), wherein the molecular weight of the sodium alginate is 300,000-450,000 Da.

7. The method for improving gastrointestinal tolerance of probiotics according to claim 6, characterized in that: The core material liquid is uniformly added dropwise into a coagulation bath stirred at 600-1200 r / min using a peristaltic pump at a rate of 3.0-5.0 ml / min, and is taken out after reacting for 1-3 minutes.

8. The method for improving gastrointestinal tolerance of probiotics according to claim 6, wherein: The anaerobic culture is carried out for a period of 12 to 48 hours.

9. The method for improving gastrointestinal tolerance of probiotics according to claim 8, characterized in that: The formula of the improved MRS culture medium includes, 8.0-12.0 g / L peptone, 3.0-6.0 g / L yeast extract, 5.0-20.0 g / L glucose, 3.0-5.0 g / L sodium acetate, 0.5-1.0 g / L Tween 80.

10. The re-cultured probiotic liquid core gel sphere prepared by the method for improving gastrointestinal tolerance of probiotics according to any one of claims 1 to 9, characterized in that: Compared with naked bacteria, the gastric acid resistance survival rate of the re-cultured probiotic liquid core ball is increased by 50-55%, and the bile salt resistance survival rate is increased by 40-45%.

Images