A method for improving the yield of exopolysaccharide of probiotics, exopolysaccharide and application thereof

Abstract

The present application relates to the field of microbial polysaccharide, and particularly relates to a method for preparing exopolysaccharide of probiotics for improving polysaccharide yield and application. The present application provides a method for preparing exopolysaccharide of probiotics for improving polysaccharide yield, which comprises fermenting and culturing probiotics by using the aforementioned fermentation medium, collecting fermentation liquor; obtaining fermentation supernatant from the fermentation liquor, precipitating the fermentation supernatant, and collecting the precipitate; dissolving the precipitate and mixing it with trichloroacetic acid, collecting supernatant, and freeze-drying to obtain exopolysaccharide of probiotics; the fermentation medium comprises 80-160 g / L sweet whey powder, 1-5% v / v hydrochloric acid, and the rest is water, and the pH of the fermentation medium is 6.0-7.0. The fermentation medium of the present application can improve the yield of exopolysaccharide of probiotics.

Description

Technical Field

[0001] This invention relates to the field of microbial polysaccharide technology, specifically to a method for increasing the yield of extracellular polysaccharides in probiotics, extracellular polysaccharides, and their applications. Background Technology

[0002] Polysaccharides are high-molecular-weight carbohydrates composed of ten or more monosaccharides linked by glycosidic bonds, and are widely found in plants, animals, microorganisms, and algae in nature. Since the 1960s, polysaccharides have been considered a broad-spectrum, non-specific immune booster that can enhance the cellular and humoral immune functions of host cells, such as activating macrophages, T cells, B cells, and NK cells, activating complement, and inducing interferon production. Their role is to activate the body's non-specific defense mechanisms, showing good therapeutic effects in antiviral, antitumor, and anti-radiation applications.

[0003] Depending on their origin, polysaccharides can be classified into animal polysaccharides, plant polysaccharides, and microbial polysaccharides. Among them, microbial polysaccharides are produced by microorganisms metabolizing carbohydrates, but are less studied due to their low yield. Currently, most probiotics, even in preferred culture media (such as MRS, TPY, etc.), produce less than 200 mg / L. In addition, the aforementioned preferred culture media are all artificially synthesized and non-natural, which greatly limits the prospects for structural characterization, subsequent functional exploration, and industrial application of such extracellular polysaccharides.

[0004] Therefore, finding a natural and safe culture medium to increase the yield of probiotic extracellular polysaccharides is an important scientific problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for increasing the yield of probiotic extracellular polysaccharides, the extracellular polysaccharides, and their applications.

[0006] To achieve the above and other related objectives, the first aspect of this application provides a method for preparing probiotic extracellular polysaccharides to increase polysaccharide production, comprising the following steps:

[0007] 1) Ferment probiotics using a fermentation medium and collect the fermentation broth;

[0008] 2) Obtain the fermentation supernatant from the fermentation broth, precipitate the fermentation supernatant, and collect the precipitate;

[0009] 3) Dissolve the precipitate and mix it with trichloroacetic acid. Collect the supernatant and freeze-dry it to obtain probiotic extracellular polysaccharide.

[0010] The fermentation medium comprises: 80-160 g / L sweet whey powder, 1-5% w / v hydrochloric acid, and the remainder is water, with a pH of 6.0-7.0.

[0011] The second aspect of this application provides probiotic extracellular polysaccharides prepared by the above-described method.

[0012] In summary, this invention provides a method for increasing the yield of extracellular polysaccharides in probiotics, the extracellular polysaccharides themselves, and their applications, and achieves the following beneficial effects:

[0013] Compared with the prior art, the present invention uses sweet whey hydrolysate as a fermentation medium to cultivate probiotics, and found that using this medium to cultivate probiotics can increase the yield of extracellular polysaccharides, and the yield of extracellular polysaccharides can reach ≥450 mg / mL compared with existing fermentation media. Detailed Implementation

[0014] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0016] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0017] This application first provides a method for preparing probiotic extracellular polysaccharides to increase polysaccharide production, comprising the following steps:

[0018] 1) Ferment probiotics using a fermentation medium and collect the fermentation broth;

[0019] 2) Obtain the fermentation supernatant from the fermentation broth, precipitate the fermentation supernatant, and collect the precipitate;

[0020] 3) Dissolve the precipitate and mix it with trichloroacetic acid. Collect the supernatant and freeze-dry it to obtain probiotic extracellular polysaccharide.

[0021] The fermentation medium comprises: 80-160 g / L sweet whey powder, 0.5-0.8% v / v hydrochloric acid, and the remainder is water, with a pH of 6.0-7.0.

[0022] In this invention, increasing polysaccharide yield means that the extracellular polysaccharide yield in the fermentation broth is at least 450 mg / mL.

[0023] In some embodiments, the concentration of sweet whey powder in the fermentation medium is 100-140 g / L; more preferably, the concentration of sweet whey powder is 120 g / L.

[0024] Sweet Whey powder refers to the whey produced during the production of hard cheese, which is pasteurized, concentrated, and spray-dried into a powder. Sweet Whey powder can be a ready-made product or prepared in-house.

[0025] In some embodiments, the hydrochloric acid in the fermentation medium is 0.7-0.8% w / v; more preferably, it is 7.4% v / v.

[0026] In some embodiments, the pH of the fermentation medium is 6.0 to 7.0; preferably, it is 6.3 to 6.8; more preferably, it is 6.5.

[0027] In some embodiments of the present invention, the preparation method of the fermentation culture medium includes the following steps: dissolving sweet whey powder in hydrochloric acid solution, reacting in a constant temperature water bath, adjusting the pH, and sterilizing to obtain the culture medium.

[0028] Preferably, the temperature of the constant temperature water bath reaction is 40~100℃, more preferably, 50~80℃; more preferably, 60℃.

[0029] In some embodiments, the probiotic is an extracellular polysaccharide-producing probiotic. Preferably, the probiotic is one or both of *Lactobacillus fermentum* and *Lactobacillus rhamnosus*.

[0030] In some embodiments of the present invention, the fermenting *Lactobacillus* is *Lactobacillus* B44; and the *Lactobacillus rhamnosus* is *Lactobacillus rhamnosus* BD4047.

[0031] Preferably, the preservation number of Lactobacillus rhamnosus BD4047 is CGMCC No. 13310. This strain was deposited on November 15, 2016, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. Its Latin name is Lactobacillus rhamnosus. It is also known as Lactobacillus rhamnosus B6. See reference 1 (Han Jin. Screening, identification and optimization of sugar-producing lactic acid bacteria. Journal of Microbiology [J]. 2021, 5: 43-51) and reference 2 (Li Ruiying et al. Study on antioxidant activity and cell protection of Lactobacillus rhamnosus B6. Food and Fermentation Industries [J]. 2022, 17: 57-63).

[0032] Preferably, the preservation number of Lactobacillus fermentum B44 is CGMCC No.17321. This strain was deposited on March 8, 2019 at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No.3, Beichen West Road, Chaoyang District, Beijing. Its Latin name is Lactobacillus fermentum.

[0033] In a specific embodiment of this application, in step 1), the inoculation amount of the probiotics can be 1×10⁻⁶. 7 ~5×10 7 CFU / mL, for example, can be 1×10⁻⁶. 7 1.5×10 7 2.0×10 7 2.5×10 7 3×10 7 3.5×10 7 4.0×10 7 4.5×10 7 5.0×10 7 .

[0034] In a specific embodiment of this application, in step 1), the fermentation time can be 12 to 36 hours, for example, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, or 36 hours; in a preferred embodiment, the fermentation time is 18°C ​​to 30°C. More preferably, the fermentation time is 24 hours.

[0035] In a specific embodiment of this application, in step 1), the fermentation temperature can be 25–42°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C; in a preferred embodiment, the fermentation temperature is 30°C–44°C. More preferably, the fermentation temperature is 37°C. Fermenting probiotics at the above inoculum size, fermentation time, and fermentation temperature can increase the yield of extracellular polysaccharides produced by probiotics.

[0036] In a specific embodiment of this application, step 2) further includes inactivating the fermentation broth, specifically by inactivating probiotics at high temperature, in order to reduce the risk of live microorganisms in the product, ensure the safety of the product, maintain the stability of EPS (exoploysaccharides), and improve its purity.

[0037] In a specific embodiment of this application, in step 2), the inactivation temperature can be 80-100℃, for example, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, or 100℃. In one embodiment of this application, boiling water can be used.

[0038] In a specific embodiment of this application, in step 2), the inactivation time can be 15 to 35 minutes; for example, it can be 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, or 35 minutes.

[0039] Obtaining fermentation supernatant refers to separating the liquid portion (supernatant) from the solid portion of a mixture during solid-liquid separation. Methods such as sedimentation, centrifugation, filtration, vacuum filtration, ultrafiltration, dialysis, evaporation, reverse osmosis, electrophoresis, and chromatography can be used. In the specific embodiment of this application, in step 2), the method for obtaining fermentation supernatant is centrifugation.

[0040] In a specific embodiment of this application, in step 2), the centrifugation speed can be 8000 to 10000 rpm, for example, 8100 rpm, 8200 rpm, 8300 rpm, 8400 rpm, 8500 rpm, 8600 rpm, 8700 rpm, 8800 rpm, 8900 rpm, 9000 rpm, 9100 rpm, 9200 rpm, 9300 rpm, 9400 rpm, 9500 rpm, 9600 rpm, 9700 rpm, 9800 rpm, 9900 rpm, or 10000 rpm.

[0041] In a specific embodiment of this application, in step 2), the centrifugation time can be 10 to 25 minutes, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, or 25 minutes.

[0042] Precipitation of fermentation supernatant refers to the separation of target substances, such as proteins, nucleic acids, extracellular polysaccharides (EPS), enzymes, and other biomolecules, from the liquid (supernatant) obtained after the fermentation process using specific methods. Precipitation of fermentation supernatant can be carried out by methods such as alcohol precipitation, salting out precipitation, organic solvent precipitation, temperature change, acid-base adjustment, precipitation with precipitating agents, affinity precipitation, metal ion precipitation, and nonionic surfactants. In the specific embodiment of this application, in step 2), the method for precipitating fermentation supernatant is alcohol precipitation.

[0043] Alcohol precipitation can be performed using anhydrous ethanol, aqueous solutions of ethanol at different concentrations, isopropanol, or polyethylene glycol. In a specific embodiment of this application, in step 2), alcohol precipitation involves adding anhydrous ethanol.

[0044] In a specific embodiment of this application, in step 2), the volume ratio of the added anhydrous ethanol to the supernatant can be 2.5 to 4:1, for example, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, or 4.0:1. Under this ratio, the extracellular polysaccharides in the supernatant can be precipitated efficiently.

[0045] The precipitate can be collected by methods such as centrifugation, filtration, vacuum filtration, sedimentation, decantation, pipetting, ultrafiltration, freeze drying, electrophoresis, chromatography, evaporation to dryness, and back extraction. In the specific embodiment of this application, in step 3), the method for collecting the precipitate is centrifugation.

[0046] In a specific embodiment of this application, the centrifugal speed can be 8000 to 10000 rpm, for example, 8100 rpm, 8200 rpm, 8300 rpm, 8400 rpm, 8500 rpm, 8600 rpm, 8700 rpm, 8800 rpm, 8900 rpm, 9000 rpm, 9100 rpm, 9200 rpm, 9300 rpm, 9400 rpm, 9500 rpm, 9600 rpm, 9700 rpm, 9800 rpm, 9900 rpm, or 10000 rpm.

[0047] In a specific embodiment of this application, the centrifugation time can be 10 to 25 minutes, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, or 25 minutes.

[0048] In a specific embodiment of this application, in step 3), dissolving the precipitate is done by dissolving the precipitate in water.

[0049] In a specific embodiment of this application, in step 3), the final concentration of trichloroacetic acid in the mixed system can be 4-9% by mass, for example, 4%, 5%, 6%, 7%, 8%, or 9%. The mixed system refers to the mixture of the solution after the precipitate is dissolved in water and trichloroacetic acid. The final concentration refers to the final concentration of a specific solute or component in the solution or mixture. In this application, it refers to the final concentration of trichloroacetic acid in the mixture after the trichloroacetic acid and the solution after the precipitate is dissolved in water are mixed. At this concentration, it is beneficial for the removal of excess protein from the precipitate.

[0050] Collecting supernatant refers to obtaining the liquid portion during solid-liquid separation. Supernatant can be collected by centrifugation, filtration, vacuum filtration, sedimentation, decantation, pipetting, ultrafiltration, gel filtration, or back extraction. In the specific embodiment of this application, in step 3), the method for collecting supernatant is centrifugation.

[0051] In a specific embodiment of this application, in step 3), the centrifugation speed can be 8000 to 10000 rpm, for example, 8100 rpm, 8200 rpm, 8300 rpm, 8400 rpm, 8500 rpm, 8600 rpm, 8700 rpm, 8800 rpm, 8900 rpm, 9000 rpm, 9100 rpm, 9200 rpm, 9300 rpm, 9400 rpm, 9500 rpm, 9600 rpm, 9700 rpm, 9800 rpm, 9900 rpm, or 10000 rpm.

[0052] In a specific embodiment of this application, in step 3), the centrifugation time can be 10 to 25 minutes, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, or 25 minutes.

[0053] In a specific embodiment of this application, step 3) may further include dialysis after collecting the supernatant to remove small molecule impurities from the EPS sample, such as salts, monosaccharides, unreacted monomers, buffers, etc., which helps to improve the purity of EPS. The dialysis involves placing the supernatant into a dialysis bag with a molecular weight cutoff of 14,000 Daltons and dialyzing for 72 hours, changing the water every 8 hours. Finally, the dialyzed solution is freeze-dried to obtain the extracellular polysaccharide.

[0054] This application also provides extracellular polysaccharides prepared by the above-described preparation method.

[0055] The present invention also provides the application of the above-mentioned extracellular polysaccharides in the preparation of food or cosmetics.

[0056] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0057] Example 1: Preparation of probiotic extracellular polysaccharides

[0058] Preparation of seed culture (fermentation strain): Lyophilized powders of *Lactobacillus fermentatus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 were dissolved separately in a small amount of sterile distilled water. A loopful was used to streak a single colony onto MRS solid medium (purchased from Merck Co., Germany, catalog number 1.10660.0500). After anaerobic incubation at 37°C for 48 h, a single colony was picked up and placed into 10 mL of MRS liquid medium (purchased from Merck Co., Germany, catalog number 1.10661.0500). The colony was evenly dispersed in the liquid medium using a vortex mixer and anaerobic incubated at 37°C for 48 h. The culture was then inoculated into MRS liquid medium at a 2% (v / v) inoculum. After anaerobic incubation at 37°C for 24 h, the culture was cultured to 15,000 cells / mL. Centrifuge at rpm for 10 minutes, discard the supernatant, wash the bacterial cells twice with sterile distilled water, and then suspend them in the original culture volume of sterile distilled water to obtain the seed culture for fermentation. The concentration of the seed culture for fermenting *Lactobacillus myxitis* CGMCC No. 17321 was 1.5 × 10⁻⁶. 9 The CFU / mL concentration of *Lactobacillus rhamnosus* seed culture (CGMCC No. 13310) was 3.7 × 10⁻⁶. 9 CFU / mL. The cost of the relevant culture medium can be found online on the official website.

[0059] Preparation of probiotic extracellular polysaccharides: 12 g of sweet whey powder (purchased from Shanghai Golden Palm Food Co., Ltd., China, item number ZL-ARG01) was completely dissolved in 100 mL of hydrochloric acid solution with a volume ratio of 2 mL:98 mL. The hydrochloric acid solution contained concentrated hydrochloric acid (Shanghai Lingfeng Chemical Reagent Co., Ltd., China, item number 7647-01-0) and water in a volume ratio of 2 mL:98 mL. The solution was placed in a 60℃ constant temperature water bath for continuous hydrolysis for 12 h. After cooling to room temperature, the pH was adjusted to 6.5 with 5 M NaOH solution, sterilized at 118℃ for 15 min, and cooled to room temperature to obtain sweet whey hydrolysate.

[0060] The seed culture of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 was prepared at a final concentration of 3 x 10⁻⁶. 7CFU / mL was aseptically inoculated into the above-mentioned sweet whey hydrolysate and anaerobically cultured at 37℃ for 24 h to obtain the fermentation broth. The fermentation broth was heated to boiling for 15 min to inactivate the bacteria, centrifuged (4℃, 10000 rpm, 10 min), and the supernatant was collected. Three volumes of anhydrous ethanol were added to precipitate the bacteria, and centrifuged (4℃, 10000 rpm, 10 min) to dissolve the precipitate in water. Trichloroacetic acid solution was added to a final concentration of 4%, and the mixture was refrigerated at 4℃ overnight. The supernatant was then centrifuged (4℃, 10000 rpm, 10 min) and dialyzed against a dialysis bag with a molecular weight cutoff of 14000 Daltons for 72 h (with water changed every 8 h). The mixture was then freeze-dried to obtain crude polysaccharides. After weighing and conversion, the crude polysaccharide yields of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 were 550 mg / L and 500 mg / L, respectively.

[0061] Example 2 - Preparation of probiotic extracellular polysaccharides

[0062] Preparation of seeds (fermentation strains): Same as in Example 1.

[0063] Preparation of probiotic extracellular polysaccharides: 16 g of sweet whey powder was completely dissolved in 100 mL of hydrochloric acid solution with a volume ratio of 2 mL: 98 mL. The solution was placed in a constant temperature water bath at 100 °C for 3 h for continuous hydrolysis. After cooling to room temperature, the pH was adjusted to 6.8 with 5 M NaOH solution, sterilized at 118 °C for 15 min, and cooled to room temperature to obtain sweet whey hydrolysate.

[0064] The seed culture of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 was fermented at a final concentration of 5 x 10⁻⁶. 7 CFU / mL was aseptically inoculated into the above-mentioned sweet whey hydrolysate, and anaerobic cultured at 34℃ for 12 h to obtain fermentation broth. The fermentation broth was heated to boiling for 15 min to inactivate the bacteria, centrifuged (4℃, 10000 rpm, 10 min), and the supernatant was collected. Three volumes of anhydrous ethanol were added for precipitation, centrifuged (4℃, 10000 rpm, 10 min), and the precipitate was dissolved in water. Trichloroacetic acid solution was added to a final concentration of 4%, and the mixture was refrigerated at 4℃ overnight. The supernatant was centrifuged (4℃, 10000 rpm, 10 min), dialyzed using a dialysis bag with a molecular weight cutoff of 14000 Daltons for 72 h (with water changed every 8 h), and then freeze-dried to obtain crude polysaccharides. After weighing and conversion, the crude polysaccharide yields of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 were 530 mg / L and 463 mg / L, respectively.

[0065] Example 3 - Preparation of probiotic extracellular polysaccharides

[0066] Preparation of seeds (fermentation starter): Same as in Example 1.

[0067] Preparation of probiotic extracellular polysaccharides: 8 g of sweet whey powder was completely dissolved in 100 mL of hydrochloric acid solution with a volume ratio of 5 mL: 95 mL. The solution was placed in a constant temperature water bath at 40 °C and hydrolyzed for 18 h. After cooling to room temperature, the pH was adjusted to 6.3 with 5 M NaOH solution, sterilized at 118 °C for 15 min, and cooled to room temperature to obtain sweet whey hydrolysate.

[0068] The seed culture of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 was fermented at a final concentration of 1 x 10⁻⁶. 7 CFU / mL was aseptically inoculated into the above-mentioned sweet whey hydrolysate, and anaerobic cultured at 30℃ for 36 h to obtain fermentation broth. The fermentation broth was heated to boiling for 15 min to inactivate the bacteria, centrifuged (4℃, 10000 rpm, 10 min), and the supernatant was collected. Three volumes of anhydrous ethanol were added to precipitate the bacteria, and centrifuged (4℃, 10000 rpm, 10 min) to dissolve the precipitate in water. Trichloroacetic acid solution was added to a final concentration of 4%, and the mixture was refrigerated at 4℃ overnight. The supernatant was collected by centrifugation (4℃, 10000 rpm, 10 min), dialyzed using a dialysis bag with a molecular weight cutoff of 14000 Daltons for 72 h (with water changed every 8 h), and then freeze-dried to obtain crude polysaccharides. After weighing and conversion, the crude polysaccharide yields of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 were 522 mg / L and 487 mg / L, respectively.

[0069] Example 4 - Preparation of probiotic extracellular polysaccharides

[0070] Preparation of seeds (fermentation strains): Same as in Example 1.

[0071] Preparation of probiotic extracellular polysaccharides: 14 g of sweet whey powder was completely dissolved in 100 mL of hydrochloric acid solution with a volume ratio of 1 mL: 99 mL. The solution was placed in an 80℃ constant temperature water bath for continuous hydrolysis for 6 h. After cooling to room temperature, the pH was adjusted to 7.0 with 5 M NaOH solution, sterilized at 118℃ for 15 min, and cooled to room temperature to obtain sweet whey hydrolysate.

[0072] The seed culture of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 was fermented at a final concentration of 4 x 10⁻⁶. 7CFU / mL was aseptically inoculated into the above-mentioned sweet whey hydrolysate, and anaerobic cultured at 44℃ for 30 h to obtain fermentation broth. The fermentation broth was heated to boiling for 15 min to inactivate the bacteria, centrifuged (4℃, 10000 rpm, 10 min), and the supernatant was collected. Three volumes of anhydrous ethanol were added to precipitate the bacteria, and centrifuged (4℃, 10000 rpm, 10 min) to dissolve the precipitate in water. Trichloroacetic acid solution was added to a final concentration of 4%, and the mixture was refrigerated at 4℃ overnight. The supernatant was collected by centrifugation (4℃, 10000 rpm, 10 min), dialyzed using a dialysis bag with a molecular weight cutoff of 14000 Daltons for 72 h (with water changed every 8 h), and then freeze-dried to obtain crude polysaccharides. After weighing and conversion, the crude polysaccharide yields of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 were 537 mg / L and 466 mg / L, respectively.

[0073] Example 5 - Preparation of probiotic extracellular polysaccharides

[0074] Preparation of seeds (fermentation strains): Same as in Example 1.

[0075] Preparation of probiotic extracellular polysaccharides: 10 g of sweet whey powder was completely dissolved in 100 mL of hydrochloric acid solution with a volume ratio of 3 mL: 97 mL. The solution was placed in a constant temperature water bath at 50 °C and hydrolyzed for 15 h. After cooling to room temperature, the pH was adjusted to 6.0 with 5 M NaOH solution, sterilized at 118 °C for 15 min, and cooled to room temperature to obtain sweet whey hydrolysate.

[0076] The seed culture of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 was fermented at a final concentration of 2 x 10⁻⁶. 7 CFU / mL was aseptically inoculated into the above-mentioned sweet whey hydrolysate and anaerobically cultured at 41℃ for 18 h to obtain the fermentation broth. The fermentation broth was heated to boiling for 15 min to inactivate the bacteria, centrifuged (4℃, 10000 rpm, 10 min), and the supernatant was collected. Three volumes of anhydrous ethanol were added to precipitate the bacteria, and centrifuged (4℃, 10000 rpm, 10 min) to dissolve the precipitate in water. Trichloroacetic acid solution was added to a final concentration of 4%, and the mixture was refrigerated at 4℃ overnight. The supernatant was then centrifuged (4℃, 10000 rpm, 10 min) and dialyzed against a dialysis bag with a molecular weight cutoff of 14000 Daltons for 72 h (with water changed every 8 h). The mixture was then freeze-dried to obtain crude polysaccharides. After weighing and conversion, the crude polysaccharide yields of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 were 509 mg / L and 450 mg / L, respectively.

[0077] Comparative Example 1

[0078] Referring to Example 1, the extracellular polysaccharide yields of *Lactobacillus mucinus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 fermented in SKM fermentation medium and sweet whey hydrolysate fermentation medium were compared.

[0079] Preparation of seeds (fermentation strains): Same as in Example 1.

[0080] Preparation of probiotic extracellular polysaccharides:

[0081] Preparation of fermentation medium for sweet whey hydrolysate: 12 g of sweet whey powder was completely dissolved in 100 mL of hydrochloric acid solution with a volume ratio of 2 mL: 98 mL. The solution was placed in a constant temperature water bath at 60 °C and hydrolyzed for 12 h. After cooling to room temperature, the pH was adjusted to 6.5 with 5 M NaOH solution, sterilized at 118 °C for 15 min, and cooled to room temperature to obtain sweet whey hydrolysate.

[0082] Dissolve 12g of skim milk powder (purchased from Foterra, New Zealand, product number 6395) completely in 100mL of distilled water, sterilize at 118℃ for 15 min, and cool to room temperature to obtain the skim milk solution.

[0083] The seed culture of *Lactobacillus mucilaginosus* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 was prepared at a final concentration of 3 x 10⁻⁶. 7 CFU / mL was aseptically inoculated into the above-mentioned sweet whey hydrolysate or skim milk solution, and anaerobically cultured at 37℃ for 24 h to obtain fermentation broth. The fermentation broth was heated to boiling for 15 min to inactivate the bacteria, centrifuged (4℃, 10000 rpm, 10 min), and the supernatant was collected. Three volumes of anhydrous ethanol were added for precipitation, centrifuged (4℃, 10000 rpm, 10 min), and the precipitate was dissolved in water. Trichloroacetic acid solution was added to a final concentration of 4%, refrigerated at 4℃ overnight, centrifuged (4℃, 10000 rpm, 10 min), and the supernatant was collected. Dialyzed using a dialysis bag with a molecular weight cutoff of 14000 Daltons for 72 h (with water changed every 8 h), and then freeze-dried to obtain crude polysaccharides. The crude polysaccharide yields of *Lactobacillus myxitis* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 in sweet whey hydrolysate and skim milk solution after weighing and conversion are shown in Table 1.

[0084] Table 1. Crude polysaccharide yields of *Lactobacillus fermentum* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 in sweet whey hydrolysate and skim milk solution.

[0085]

[0086] As shown in Table 1, the crude polysaccharide yield of *Lactobacillus myxoides* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310 in sweet whey hydrolysate was significantly higher than that in skim milk solution, indicating that sweet whey hydrolysate is more suitable for fermentation and sugar production by *Lactobacillus myxoides* CGMCC No. 17321 and *Lactobacillus rhamnosus* CGMCC No. 13310.

[0087] Comparative Example 2

[0088] The concentration of hydrochloric acid solution, the hydrolysis temperature of sweet whey powder, the hydrolysis time of sweet whey powder, the amount of probiotics inoculated, and the fermentation time in Example 1 were adjusted one by one to obtain the following group of fermented milk prepared by different methods. The crude polysaccharide yield of each group is shown in Table 2.

[0089] Table 2. Yields of crude polysaccharides prepared by different methods

[0090]

[0091] The results shown in Table 2 indicate that when the concentration of hydrochloric acid solution, the hydrolysis temperature of sweet whey powder, the hydrolysis time of sweet whey powder, the inoculum amount of probiotics, and the fermentation time are adjusted to outside the preferred range in the method for increasing the yield of probiotic extracellular polysaccharides, the yield of probiotic extracellular polysaccharides prepared decreases significantly. This shows that fermented Lactobacillus mucilaginosus CGMCC No. 17321 and Lactobacillus rhamnosus CGMCC No. 13310 cannot synthesize a large amount of extracellular polysaccharides under fermentation conditions other than the preferred ones.

[0092] In summary, the above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing probiotic extracellular polysaccharides to increase polysaccharide production, the method comprising the following steps: 1) Ferment probiotics using a fermentation medium and collect the fermentation broth; 2) Obtain the fermentation supernatant from the fermentation broth, precipitate the fermentation supernatant, and collect the precipitate; 3) Dissolve the precipitate and mix it with trichloroacetic acid, collect the supernatant, and freeze-dry it to obtain probiotic extracellular polysaccharide; The fermentation medium comprises: 80-160 g / L sweet whey powder, 0.5-0.8% w / v hydrochloric acid, and the remainder is water. The pH of the medium is 6.0-7.

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2. The preparation method according to claim 1, characterized in that, In the fermentation medium, the mass concentration of sweet whey powder is 100~140 g / L; and / or, the mass percentage of hydrochloric acid is 0.7~0.8% w / v; and / or, the pH of the fermentation medium is 6.0~7.

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3. The preparation method according to claim 1, characterized in that, The preparation method of the fermentation medium includes the following steps: dissolving sweet whey powder in hydrochloric acid solution, reacting in a constant temperature water bath, adjusting the pH, and sterilizing to obtain the culture medium.

4. The preparation method according to claim 3, characterized in that, The temperature of the constant temperature water bath reaction is 40~100℃.

5. The preparation method according to claim 4, characterized in that, The probiotics are one or both of Lactobacillus fermentum and Lactobacillus rhamnosus.

6. The preparation method according to claim 5, characterized in that, The fermenting *Lactobacillus* is *Lactobacillus fermentatus* B44, with accession number CGMCC No. 17321; and / or, the *Lactobacillus rhamnosus* is *Lactobacillus rhamnosus* BD4047, with accession number CGMCC No. 13310.

7. The preparation method according to claim 1, characterized in that, Step 1) also includes one or more of the following features: 11) The inoculation amount of the probiotics is 1×10⁻⁶. 7 ~5×10 7 CFU / mL; 12) The fermentation time is 12–36 hours; 13) The fermentation temperature is 25-42℃.

8. The preparation method according to claim 1, characterized in that, Step 2) also includes one or more of the following features: 21) It also includes inactivating the fermentation broth; preferably, the inactivation temperature is 80-100°C and the inactivation time is 15-35 min; 22) The method for obtaining the fermentation supernatant is centrifugation; preferably, the centrifugation speed is 8000-10000 rpm and the centrifugation time is 10-25 min; 23) The method for precipitating the fermentation supernatant is alcohol precipitation; preferably, the alcohol precipitation is the addition of anhydrous ethanol; more preferably, the volume ratio of anhydrous ethanol to supernatant is 2.5~4:1; 24) The method for collecting the precipitate is centrifugation; preferably, the centrifugation speed is 8000-10000 rpm and the centrifugation time is 10-25 min.

9. The preparation method according to claim 1, characterized in that, Step 3) also includes one or more of the following features: 31) The final concentration of the trichloroacetic acid in the mixed system is 4–9% w / v; 32) The method for collecting the supernatant is centrifugation; preferably, the centrifugation speed is 8000-10000 rpm and the centrifugation time is 10-25 min.

10. A probiotic extracellular polysaccharide, wherein the probiotic extracellular polysaccharide is prepared by the preparation method according to any one of claims 1 to 9.

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