CN117964787A - Extracellular polysaccharide derived from lactobacillus rhamnosus strain BD 4047 and preparation method and application thereof - Google Patents

Abstract

The invention provides extracellular polysaccharide derived from lactobacillus rhamnosus strain BD 4047, and a preparation method and application thereof, and belongs to the technical field of microbial polysaccharide. The invention separates an extracellular polysaccharide from lactobacillus rhamnosus strain BD 4047 from supernatant obtained by fermenting lactobacillus rhamnosus strain BD 4047 in skim milk, wherein the extracellular polysaccharide comprises three monosaccharides of rhamnose, glucose and galactose; the average molecular weight is (1.419-1.735) x 10 ⁶ daltons. The extracellular polysaccharide has the effects of improving phagocytic activity of macrophages, improving secretion of TNF-alpha and NO release amount in the macrophages, and activating NF- κB signal paths in the macrophages, thereby achieving the effect of regulating immunity. And the extracellular polysaccharide is safe and nontoxic, and provides a new means for preparing an immunoregulation medicament and treating immune-related diseases.

Description

Extracellular polysaccharide derived from lactobacillus rhamnosus strain BD 4047 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of microbial polysaccharide, and particularly relates to extracellular polysaccharide derived from lactobacillus rhamnosus strain BD 4047, and a preparation method and application thereof.

Background

The polysaccharide is considered as a broad-spectrum nonspecific immunity promoter, can enhance cellular immunity and humoral immunity functions of host cells, such as activating macrophages, T cells, B cells, NK cells and the like, and also can activate complement and induce the production of interferon and the like, has the effect of activating nonspecific defensive functions of human bodies, and has good curative effects on antiviral, antitumor, anti-radiation and the like.

Polysaccharides can be classified into animal polysaccharides, plant polysaccharides and microbial polysaccharides according to their sources, wherein microbial polysaccharides are produced by metabolizing carbohydrates by microorganisms, and are less studied due to their lower yields. In the case of lactobacillus rhamnosus, at present, most of extracellular polysaccharide of the strain is extracted from a synthetic culture medium, and few reports from edible culture media exist. Therefore, the structural characterization and functional judgment of the extracellular polysaccharide of lactobacillus rhamnosus are an important scientific problem to be solved in the field, and are also important indexes for evaluating the application potential of the strain, so that the development of novel microbial polysaccharide has important significance for enriching the variety of immune promoters.

Lactobacillus rhamnosus is a known beneficial bacterium, and the patent of publication No. CN115216422A discloses that the lactobacillus rhamnosus has the characteristics of high extracellular polysaccharide yield, acid resistance, choline resistance and gastrointestinal digestive enzyme resistance, and has the effects of improving glucose tolerance, controlling liver index, controlling blood fat and reducing insulin. The patent of publication No. CN111154676A discloses that the extracellular of lactobacillus rhamnosus source has stronger antioxidant capacity, lipase inhibition capacity and blood fat reducing effect. Patent publication No. CN106635924A discloses that exopolysaccharides derived from Lactobacillus rhamnosus have the effects of promoting macrophage proliferation and treating colitis.

Disclosure of Invention

Accordingly, the present invention is directed to an extracellular polysaccharide derived from lactobacillus rhamnosus strain BD 4047, which has the effects of improving phagocytic activity of macrophages and promoting secretion or release of TNF- α and NO, and achieving the purpose of immunomodulation by activating NF- κb signaling pathway.

The invention provides an extracellular polysaccharide derived from lactobacillus rhamnosus strain BD 4047, wherein the repeating unit of the extracellular polysaccharide comprises the following monosaccharide types: rhamnose, glucose and galactose; the molar ratio of the rhamnose, the glucose and the galactose is (3.67-3.72): (1.47-1.52): 1, a step of; the average molecular weight is (1.419-1.735) x 10 ⁶ daltons.

Preferably, the extracellular polysaccharide is a backbone formed by alternating 1, 3-linked rhamnose residues, 1, 2-linked glucose residues and 1, 3-linked glucose residues, forming branched side chains at the O2 position of the 1,2, 3-linked rhamnose residues, said branched side chains being formed by galactose residues terminally modified with a pyruvic acid substituent.

Preferably, the repeating units of the extracellular polysaccharide are represented by formula I:

The invention provides a preparation method of the extracellular polysaccharide, which comprises the following steps of;

performing alcohol precipitation on fermentation supernatant of lactobacillus rhamnosus strain BD 4047, and collecting precipitate;

dissolving the precipitate in water, mixing with trichloroacetic acid, and collecting supernatant to obtain crude polysaccharide;

and (3) performing gel column chromatography separation on the crude polysaccharide, and collecting the earliest component peak fraction to obtain extracellular polysaccharide.

Preferably, the preparation method of the fermentation supernatant of the lactobacillus rhamnosus strain BD 4047 comprises inoculating the lactobacillus rhamnosus strain BD 4047 into skimmed milk, standing and fermenting to obtain fermented milk, inactivating, and performing solid-liquid separation, wherein the collected liquid phase is the fermentation supernatant;

preferably, the skim milk comprises skim milk powder and water;

the mass of the skim milk powder accounts for 6-12% of the mass of the skim milk;

the inoculation amount of the lactobacillus rhamnosus is 1.25 multiplied by 10 ⁷～1×10⁸ CFU/mL;

The temperature of the standing fermentation is 25-45 ℃; the standing fermentation time is 12-36 h.

Preferably, the volume ratio of the fermentation supernatant of lactobacillus rhamnosus strain BD 4047 to the alcohol is 1: (3-4);

the final mass concentration of the trichloroacetic acid is 5% -9%.

Preferably, when the gel column chromatography is used for separation,

The packing of the gel column is Sepharose 6FastFlow; the specification of the gel column is 2.6cm multiplied by 30cm;

The eluent is 0.2MNaCl water solution;

the flow rate of the eluent is 0.2-0.3 mL/min.

The invention provides application of the extracellular polysaccharide or the extracellular polysaccharide prepared by the preparation method in preparation of immunoregulation products.

Preferably, the immunomodulation comprises at least one of:

Improving phagocytic activity of macrophages;

Promoting secretion levels of TNF-a from macrophages;

promoting the release of NO from macrophages;

activating the NF- κB signaling pathway.

The invention provides application of the extracellular polysaccharide or the extracellular polysaccharide prepared by the preparation method in preparing macrophage function promoter.

The invention provides an extracellular polysaccharide from lactobacillus rhamnosus strain BD 4047, which is shown by structural characterization of extracellular polysaccharide produced by lactobacillus rhamnosus fermented skim milk, wherein the extracellular polysaccharide comprises the following types of monosaccharides: rhamnose, glucose and galactose; the molar ratio of the rhamnose, the glucose and the galactose is (3.67-3.72): (1.47-1.52): 1, a step of; the average molecular weight is (1.419-1.735) x 10 ⁶ daltons. The extracellular polysaccharide has the effects of improving phagocytic activity of macrophages, improving secretion of TNF-alpha and NO release amount in the macrophages, and activating NF- κB signal paths in the macrophages, thereby achieving the effect of regulating immunity. And the extracellular polysaccharide is safe and nontoxic, and provides a new means for preparing an immunoregulation medicament and treating immune-related diseases.

Drawings

FIG. 1 is a gel column chromatography of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 2 is a 1H-NMR spectrum of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 3 is a 13C-NMR spectrum of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 4 is a HSQCAD map of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 5 is a gHMBCAD map of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 6 is a NOESY profile of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 7 is a gCOSY map of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 8 is a TOCSY profile of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 9 is a cytotoxicity experiment of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047;

FIG. 10 is the effect of Lactobacillus rhamnosus strain BD 4047-derived extracellular polysaccharide on phagocytic activity of cells;

FIG. 11 is the effect of Lactobacillus rhamnosus strain BD 4047-derived extracellular polysaccharide on NO release;

FIG. 12 is the effect of Lactobacillus rhamnosus strain BD 4047-derived extracellular polysaccharide on TNF- α production;

FIG. 13 is the effect of extracellular polysaccharide derived from Lactobacillus rhamnosus strain BD 4047 on NF- κB activation.

Biological preservation information

The invention provides a lactobacillus rhamnosus (Lactobacillus rhamnoides) strain BD 4047 which is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) at 11 and 15 of 2016: the preservation number of the Qingyang area North Star Xili No.1 and 3 of Beijing is CGMCC No.13310.

Detailed Description

The invention provides a preparation method of extracellular polysaccharide from lactobacillus rhamnosus strain BD 4047, which comprises the following steps of;

performing alcohol precipitation on fermentation supernatant of lactobacillus rhamnosus strain BD 4047, and collecting precipitate;

dissolving the precipitate in water, mixing with trichloroacetic acid, and collecting supernatant to obtain crude polysaccharide;

And (3) performing gel column chromatography separation on the crude polysaccharide, collecting the earliest component peak fractions, and combining to obtain extracellular polysaccharide.

The invention mixes the fermentation supernatant of lactobacillus rhamnosus strain BD 4047 with absolute ethyl alcohol, and collects the precipitate.

In the invention, the lactobacillus rhamnosus strain BD 4047 has a preservation number of CGMCC No.13310 and is disclosed in a patent with a publication number of CN 112322554A.

In the invention, the preparation method of the fermentation supernatant of the lactobacillus rhamnosus strain BD 4047 preferably comprises inoculating the lactobacillus rhamnosus strain BD 4047 into skimmed milk, standing and fermenting, inactivating the obtained fermented milk, and carrying out solid-liquid separation, wherein the collected liquid phase is the fermentation supernatant.

In the present invention, the skim milk preferably includes fresh skim milk or artificially formulated skim milk. The raw materials of the skim milk prepared artificially comprise skim milk powder and water. The skim milk powder accounts for 6-12% of the mass of the skim milk, and more preferably 8%. The preparation method of the manually formulated skim milk can comprise the following steps: mixing the skim milk powder with water, sterilizing at 95-125 deg.c for 5-20 min, and cooling. The inoculation amount of the lactobacillus rhamnosus strain BD 4047 is preferably 1.25 multiplied by 10 ⁷～1×10⁸ CFU/mL; more preferably 2.5X10 ⁷～7.5×10⁷ CFU/mL, most preferably 5X 10 ⁷ CFU/mL. The stationary fermentation is preferably anaerobic fermentation. Lactobacillus rhamnosus is anaerobic bacteria, and can grow and reproduce rapidly under the condition of static culture. The temperature of the stationary fermentation is preferably 25℃to 45℃and more preferably 30℃to 40℃and most preferably 37 ℃. The time for the stationary fermentation is preferably 12 to 36 hours, more preferably 18 to 30 hours, and most preferably 24 hours. The method for inactivating the fermented milk is preferably heat inactivation, and the temperature of the heat inactivation is preferably 90-100 ℃. The solid-liquid separation method is preferably centrifugation. The rotational speed of the centrifugation is preferably 8,000 to 12,000g, more preferably 9,000 to 11,000g, most preferably 10,000g. The time of the centrifugation is preferably 8 to 12 minutes, more preferably 9 to 11 minutes, and most preferably 10 minutes.

In the present invention, the volume ratio of the fermentation supernatant of lactobacillus rhamnosus strain BD 4047 to the alcohol is preferably 1: (3-4), more preferably 1:3. The alcohol is preferably absolute ethanol. The absolute ethanol is beneficial to precipitation of polysaccharide components in the fermentation supernatant.

After the sediment is obtained, the sediment is dissolved in water and then is mixed with trichloroacetic acid, and the supernatant is collected to obtain crude polysaccharide.

In the present invention, the final mass concentration of the trichloroacetic acid is preferably 5% to 9%, more preferably 7%. The trichloroacetic acid is advantageous for removing residual proteins. After the supernatant is obtained, the trichloroacetic acid remaining in the supernatant is preferably removed. The method for removing trichloroacetic acid in the supernatant is preferably dialysis. The dialysis bag preferably has a molecular weight cut-off of 14000 daltons during dialysis. The dialysis time is preferably 72 hours. The frequency of changing water during dialysis is preferably one time per 8 hours of dialysis.

After crude polysaccharide is obtained, the crude polysaccharide is subjected to gel column chromatography separation, and the earliest component peak fraction is collected to obtain extracellular polysaccharide.

In the invention, the packing material of the gel column is preferably Sepharose 6Fast Flow during the chromatographic separation of the gel column; the gel column is preferably 2.6cm by 30cm in size. The eluent is preferably 0.2M NaCl aqueous solution. The flow rate of the eluent is preferably 0.2-0.3 mL/min. After elution, the fraction of the earliest component peak is collected, preferably at a frequency of 1 mL/tube. The combined extracellular polysaccharide is preferably dialyzed again to remove residual small molecules, and then freeze-dried to obtain the extracellular polysaccharide.

In the present invention, the inoculum size, the culture temperature and the culture time are all influencing factors influencing the yield of extracellular polysaccharide. Experiments show that both a lower inoculum size (0.1%), a lower fermentation temperature (below 10 ℃) and a shorter fermentation time (6 hours) are detrimental to extracellular polysaccharide production.

In the invention, the prepared extracellular polysaccharide is subjected to detection of molecular weight, monosaccharide composition and connection relation, and the repeating unit of the extracellular polysaccharide from the lactobacillus rhamnosus strain BD 4047 comprises the following types of monosaccharides: rhamnose, glucose and galactose; the molar ratio of the rhamnose, the glucose and the galactose is (3.67-3.72): (1.47-1.52): 1, a step of; the average molecular weight is (1.419-1.735) x 10 ⁶ daltons. The extracellular polysaccharide is preferably a backbone formed by alternating 1, 3-linked rhamnose residues, 1, 2-linked glucose residues and 1, 3-linked glucose residues, forming branched side chains at the O2 position of the 1,2, 3-linked rhamnose residues, said branched side chains being formed by galactose residues terminally modified with a pyruvic acid substituent.

In the present invention, the repeating unit of the extracellular polysaccharide is preferably represented by formula I:

In the invention, toxicity measurement is carried out on the extracellular polysaccharide by adopting an MTT method, and the result shows that the extracellular polysaccharide can promote proliferation of macrophages at low concentration (6.25-25 mug/mL) or high concentration (50-200 mug/mL) and is dose-dependent, so that the extracellular polysaccharide of the lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is safe and nontoxic within the range of 6.25-200 mug/mL.

The invention provides application of the extracellular polysaccharide or the extracellular polysaccharide prepared by the preparation method in preparation of immunoregulation products.

In the present invention, the immunomodulation preferably comprises at least one of the following:

Improving phagocytic activity of macrophages;

Promoting secretion levels of TNF-a from macrophages;

promoting the release of NO from macrophages;

activating the NF- κB signaling pathway.

The invention provides application of the extracellular polysaccharide or the extracellular polysaccharide prepared by the preparation method in preparing macrophage function promoter.

In the embodiment of the invention, the influence of the extracellular polysaccharide on the phagocytic activity of macrophages is detected by using a neutral red method, and the result shows that the phagocytic rate of the extracellular polysaccharide A treated phagocytes of 25-200 mug/mL is obviously enhanced in a dose-dependent manner, and the maximum phagocytic capacity reaches 110% of that of a positive control group at 200 mug/mL. It can be seen that the extracellular polysaccharide derived from lactobacillus rhamnosus strain BD 4047 can stimulate macrophages and enhance the phagocytic capacity thereof.

In another embodiment of the invention, the Griess method is adopted to detect the amount of the extracellular polysaccharide released by macrophages, and the high concentration (200 mug/mL) of the extracellular polysaccharide of the lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) can obviously promote the macrophages to release the NO.

In another embodiment of the invention, the levels of TNF- α in macrophage culture supernatants were measured using ELISA kits, and the results indicate that medium concentrations (50 μg/mL) of extracellular polysaccharide significantly promote secretion of TNF- α.

In another embodiment of the invention, the influence of extracellular polysaccharide on NF- κB activation is studied, and the result shows that the extracellular polysaccharide can improve the overall expression level of NF- κB by activating the NF- κB signal channel, thereby achieving the effect of regulating immunity.

The following examples are provided to illustrate in detail the extracellular polysaccharide derived from lactobacillus rhamnosus strain BD 4047, and the preparation method and application thereof, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Materials and methods

(A) Preparation of seeds (fermentation strains): the method comprises the steps of dissolving freeze-dried powder of lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) with a small amount of sterile distilled water, taking a ring of streaks on an MRS solid medium (purchased from Merck Co., germany) by an inoculating loop, taking out the strain after anaerobic culture at 37 ℃ for 48 hours, taking single colony out by the inoculating loop, putting the single colony into a 10mLMRS liquid medium (purchased from Merck Co., germany), uniformly dispersing the colony into the liquid medium by a vortex oscillator, taking out the colony after anaerobic culture at 37 ℃ for 48 hours, inoculating the colony into the MRS liquid medium with an inoculum size of 2% (v/v), centrifuging the culture for 10 minutes at 15,000rpm after anaerobic culture at 37 ℃ for 24 hours, discarding the supernatant, washing the thallus with sterile distilled water for 2 times, and suspending the thallus with sterile distilled water of an original culture volume to obtain seeds for fermentation, wherein the bacterial concentration of the seed solution is 2.5x ⁹ CFU/mL.

(B) Preparation of skim milk: mixing 10% skimmed milk powder with distilled water, dissolving, sterilizing at 125deg.C for 5min, and cooling to room temperature to obtain skimmed milk with desired concentration.

2. Preparation of lactobacillus rhamnosus extracellular polysaccharide

Seed of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is aseptically inoculated into skimmed milk (10% by mass) with an inoculum size of 2% (v/v, the seed solution accounts for the volume percentage of the fermentation broth, and the following is the same), and anaerobic culture is carried out at 37 ℃ for 24 hours to obtain the fermented milk. Heating and inactivating fermented milk, centrifuging at 10,000g for 10min, collecting supernatant, adding three times volume of absolute ethanol for precipitation, centrifuging at 10,000g for 10min again, dissolving the precipitate in water, adding trichloroacetic acid to reach final concentration of 7%, refrigerating and standing overnight, centrifuging at 10,000g for 10min again, collecting supernatant, dialyzing in dialysis bag with molecular weight cutoff of 14000 dalton for 72h,8h, changing water once, and lyophilizing the solution in the bag to obtain crude polysaccharide A (yield is 340 mg/L).

100Mg of crude polysaccharide A was dissolved in 5mLNaCl (0.2M) solution, loaded onto a Sepharose6Fast Flow (available from GE company, USA) column (D2.6cm. Times.30 cm), eluted with 0.2M NaCl solution at a Flow rate of 0.25mL/min, and 1mL per tube was collected. Detecting polysaccharide content in each collecting tube by sulfuric acid-phenol method, mixing the eluted products of the collected component peaks F1 (figure 1), dialyzing in dialysis bag with molecular weight cut-off of 14000 dalton for 72h, changing water once for 8h, and freeze drying the solution in the bag to obtain extracellular polysaccharide A.

Example 2

1. Materials and methods

(A) Preparation of seeds (fermentation strains): as in example 1.

(B) Preparation of skim milk: mixing skimmed milk powder with weight percentage of 12% with distilled water, dissolving thoroughly, sterilizing at 95deg.C for 20min, and cooling to room temperature to obtain skimmed milk with desired concentration.

2. Preparation of lactobacillus rhamnosus microbial inoculum

Seed of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is aseptically inoculated into skimmed milk (12% by mass) with an inoculum size of 0.5% (v/v), and anaerobically cultured at 25deg.C for 36 hr to obtain fermented milk. Heating and inactivating fermented milk, centrifuging at 12,000g for 8min, collecting supernatant, adding three times volume of absolute ethanol for precipitation, centrifuging at 12,000g for 8min again, dissolving the precipitate in water, adding trichloroacetic acid to reach final concentration of 9%, refrigerating and standing overnight, centrifuging at 12,000g for 8min again, collecting supernatant, dialyzing in dialysis bag with molecular weight cutoff of 14000 dalton for 72h,8h, changing water once, and lyophilizing the solution in the bag to obtain crude polysaccharide B (yield is 316 mg/L).

And (3) performing chromatographic column separation and purification on the crude polysaccharide B in reference example 1 to obtain extracellular polysaccharide B.

Example 3

1. Materials and methods

(A) Preparation of seeds (fermentation strains): as in example 1.

(B) Preparation of skim milk: mixing skimmed milk powder with mass percentage of 6% with distilled water, dissolving thoroughly, sterilizing at 100deg.C for 15min, and cooling to room temperature to obtain skimmed milk with desired concentration.

2. Preparation of lactobacillus rhamnosus microbial inoculum

Seed of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is aseptically inoculated into skimmed milk (6% by mass) with an inoculum size of 4% (v/v), and anaerobically cultured at 45deg.C for 12 hr to obtain fermented milk. Heating and inactivating fermented milk, centrifuging at 8,000g for 12min, collecting supernatant, adding three times volume of absolute ethanol for precipitation, centrifuging at 8,000g for 12min again, dissolving the precipitate in water, adding trichloroacetic acid to reach final concentration of 5%, refrigerating and standing overnight, centrifuging at 8,000g for 12min again, collecting supernatant, dialyzing in dialysis bag with molecular weight cutoff of 14000 dalton for 72h, changing water for 8h once, and freeze drying the solution in the bag to obtain crude polysaccharide C (yield is 324 mg/L).

And (3) performing chromatographic column separation and purification on the crude polysaccharide C in reference example 1 to obtain extracellular polysaccharide C.

Example 4

1. Materials and methods

(A) Preparation of seeds (fermentation strains): as in example 1.

(B) Preparation of skim milk: mixing skimmed milk powder with mass percentage of 8% with distilled water, dissolving thoroughly, sterilizing at 120deg.C for 10min, and cooling to room temperature to obtain skimmed milk with desired concentration.

2. Preparation of lactobacillus rhamnosus microbial inoculum

Seed of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is aseptically inoculated into skimmed milk (mass percentage is 8%) with an inoculum size of 3% (v/v), and anaerobically cultured at 30deg.C for 18h to obtain fermented milk. Heating and inactivating fermented milk, centrifuging at 11,000g for 9min, collecting supernatant, adding three times volume of absolute ethanol for precipitation, centrifuging at 11,000g for 9min again, dissolving the precipitate in water, adding trichloroacetic acid to reach final concentration of 5%, refrigerating and standing overnight, centrifuging at 11,000g for 9min again, collecting supernatant, dialyzing in dialysis bag with molecular weight cut-off of 14000 dalton for 72h,8h, changing water once, and lyophilizing the solution in the bag to obtain crude polysaccharide D (yield 309 mg/L).

The crude polysaccharide D was purified by column chromatography in reference example 1 to obtain extracellular polysaccharide D.

Example 5

1. Materials and methods

(A) Preparation of seeds (fermentation strains): as in example 1.

(B) Preparation of skim milk: uniformly mixing the skim milk powder with the mass percentage of 9% with distilled water, fully dissolving, sterilizing at 115 ℃ for 12min, and cooling to room temperature to obtain the skim milk with the required concentration.

2. Preparation of lactobacillus rhamnosus microbial inoculum

Seed of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is aseptically inoculated into skimmed milk (9% by mass) with an inoculum size of 1% (v/v), and anaerobically cultured at 40deg.C for 30 hr to obtain fermented milk. Heating and inactivating fermented milk, centrifuging at 9,000g for 11min, collecting supernatant, adding three times volume of absolute ethanol for precipitation, centrifuging at 9,000g for 11min again, dissolving the precipitate in water, adding trichloroacetic acid to reach final concentration of 5%, refrigerating and standing overnight, centrifuging at 9,000g for 11min again, collecting supernatant, dialyzing in dialysis bag with molecular weight cut-off of 14000 dalton for 72h,8h, changing water once, and lyophilizing the solution in the bag to obtain crude polysaccharide E (yield is 330 mg/L).

The crude polysaccharide E was purified by column chromatography in reference example 1 to obtain extracellular polysaccharide E.

Comparative example 1

The inoculum size, culture temperature and culture time in example 1 were adjusted one by one, and the following group of crude polysaccharide of lactobacillus rhamnosus prepared by different methods was obtained, and the yields of the respective groups of crude polysaccharide of extracellular polysaccharide are shown in table 3.

TABLE 3 production of crude polysaccharide from Lactobacillus rhamnosus extracellular polysaccharide prepared by different methods

Example 6

Molecular weight distribution of lactobacillus rhamnosus extracellular polysaccharide

Dextran with different molecular weights is used as a standard substance: STD-1 (mw=5,000), STD-2 (mw=12,000), STD-3 (mw=50,000), STD-4 (mw=270,000), STD-5 (mw=670,000). The above series of standard polysaccharides and extracellular polysaccharides A, B, C, D and E were dissolved in mobile phase (0.1 mol/LNaNO ₃ solution) to obtain 1mg/mL solutions, which were filtered through 0.45 μm filters, respectively, and analyzed by Agilent 1100 high performance liquid chromatograph. And drawing a standard curve by taking the logarithm LgMw of the molecular weight of the standard polysaccharide as an abscissa and the retention time tR as an ordinate to obtain a linear regression equation of the logarithm of the molecular weight and the retention time. The molecular weight of the samples can be calculated according to the regression equation and the results are shown in table 2 below.

TABLE 2 determination of molecular weight of Lactobacillus rhamnosus extracellular polysaccharide

Conclusion: the average weight molecular weight of the exopolysaccharide of lactobacillus rhamnosus strain BD 4047 (cgmccno. 13310) is 1.419×10 ⁶～1.735×10⁶ daltons.

Example 7

Determination of the monosaccharide composition of the extracellular polysaccharide of Lactobacillus rhamnosus

(1) Hydrolysis of polysaccharide samples

2.0Mg of extracellular polysaccharide A, B, C, D and E samples were taken and placed in respective ampoules, 3mL of 2mol/L trifluoroacetic acid (TFA) was added, and the mixture was sealed and hydrolyzed at 110℃for 5 hours. Cooling the hydrolysate, steaming at 45 deg.C under reduced pressure until it is dry, adding methanol, steaming, and repeating for several times to remove excessive TFA to obtain extracellular polysaccharide hydrolysate.

(2) Derivatization of hydrolyzed and mixed monosaccharide standards

The extracellular polysaccharide hydrolysate was dissolved in 1mL of water to obtain a sample solution to be derivatized. 1mL of the sample solution or 9 monosaccharide mixed standard solutions (0.5 mg/mL, rhamnose, fucose, glucuronic acid, galactose, glucose, mannose, galacturonic acid, arabinose and xylose) are taken, 1mL of 0.6mol/LNaOH solution and 1mL of 0.5mol/L PMP methanol solution are added, and the mixture is uniformly mixed to completely dissolve the solid product, and the solid product is placed in a 70 ℃ oven for reaction for 100min. After cooling to room temperature, 0.3mol/L HCl is added dropwise to adjust to neutrality, water phase is collected after 3 times of chloroform extraction, and the water phase is filtered by a 0.45 mu m filter membrane and is subjected to HPLC sample injection analysis.

(3) Chromatographic conditions

A Agilent 1260 high performance liquid chromatograph (available from Agilent, USA) was used, equipped with a DAD detector, and the column was AGILENT ECLIPSE XDB-C18 column (available from Agilent, USA). The column temperature was set at 30℃and the sample injection amount was 20. Mu.L, the mobile phase acetonitrile: 0.1mol/L phosphate buffer (pH 6.8) =16:84 (V/V), and the detection wavelength was 250nm.

(4) Data analysis

The type of monosaccharide composition of the polysaccharide samples was determined with reference to retention times based on different monosaccharide standards (purchased from sigma corporation, usa). And determining the molar ratio of the monosaccharides in the polysaccharide sample according to the peak area ratio of the monosaccharide composition. The results are shown in Table 3.

TABLE 3 monosaccharide composition determination of Lactobacillus rhamnosus extracellular polysaccharide

Conclusion: the extracellular polysaccharide of lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is heteropolysaccharide consisting of rhamnose, glucose and galactose in a molar ratio of 3.67-3.72:1.47-1.52:1.

Example 8

Determination of the way in which lactobacillus rhamnosus extracellular polysaccharide is linked

(1) Methylation analysis

Uronic acid reduction: the uronic acid was reduced by carbodiimide-sodium borohydride (EDC-NaBH ₄) method, the reaction was divided into two stages, the first: 50mg of extracellular polysaccharide A was dissolved in 6mL of ultrapure water, and stirred until completely dissolved. 500mg of EDC was added to the solution in two portions with a 30min interval and the pH was controlled to be always between 4.5 and 4.8 with 0.1mol/L HCl, taking 3h for the whole reaction. And a second stage: 8mL of 2mol/LNaBH ₄ are added dropwise into the system within 40min, the pH of the system is controlled to be about 7.0, the reaction is continued for 1h after the dropwise addition is finished, and the product is placed in a dialysis bag (the molecular weight cut-off is 3500 Da) for dialysis for 24h in running water. The above procedure was repeated 4 times and the completion of the reduction of uronic acid was checked by PMP-HPLC.

Methylation: taking 20mg of the dried polysaccharide sample obtained by completely reducing uronic acid, placing the dried polysaccharide sample into a 10mL reaction bottle, rapidly adding 3mL of anhydrous dimethyl sulfoxide at room temperature, sealing, magnetically stirring for 30min, using ultrasound to assist in dissolving the sample, rapidly adding 50mg of dried NaOH powder, sealing and stirring until most of NaOH is dissolved, then carrying out ice bath for 5min, slowly dropwise adding 1mL of methyl iodide within 30min, stirring at room temperature in a dark place for continuous reaction for 30min, and finally adding 1mL of ultrapure water for reaction termination. And (5) placing the product in a dialysis bag, dialyzing with running water for 24 hours, steaming until the product is dry, and repeating the steps. After methylation is carried out for many times, a small amount of samples are taken for infrared spectrum detection, and if the absorption peak of the polysaccharide sample in O-H stretching vibration of 3400-3000cm ^-1 disappears, the polysaccharide sample is completely methylated; if the sample is not yet fully methylated, the reaction is continued until the sample is fully methylated.

Hydrolysis and acetylation: a sample of the fully methylated extracellular polysaccharide A (2 mg) was placed in an ampoule, 3mL of 2mol/L TFA was added and sealed, and after sealed hydrolysis at 110℃for 4 hours, methanol was added and distilled under reduced pressure several times to completely remove TFA. After spin-drying, 3mL of ultrapure water was added for dissolution, 50mg of NaBH ₄ was added, and the reaction was magnetically stirred at room temperature for 3 hours. After the reaction, acetic acid was added until the solution became slightly acidic (ph=5), methanol was added and the mixture was dried by rotary evaporation, and the reaction was repeated several times to sufficiently remove boric acid. The resulting solid was dried in an oven at 100deg.C for 10min, 3mL of acetic anhydride was added, and reacted at 100deg.C for 100min, after which toluene (3 mL) was added and co-distilled multiple times to remove excess acetic anhydride. Dissolving the product in chloroform (5 mL), extracting with ultrapure water (5 mL×3) for 3 times, recovering chloroform layer, adding anhydrous sodium sulfate powder for dewatering, standing for 30min, evaporating under reduced pressure to dryness, adding 0.5mL of chloroform for dissolving, filtering with 0.22 μm organic filter membrane, and performing GC-MS analysis.

GC-MS conditions: instrument model: agilent 7820A/5977GC-MS (available from Agilent corporation, USA); chromatographic column model: HP-5 capillary column; programming temperature: the initial temperature is 120 ℃, the temperature is raised to 250 ℃ after being kept for 2min, the temperature raising rate is 5 ℃/min, and the temperature is kept for 10min; the sample inlet adopts a split-flow mode, and the split-flow ratio is 3:1; the sample loading was 1. Mu.L. The mass spectrum ion source is an EI source, the voltage of the ion source is 70eV, and the temperature is 180 ℃.

Data analysis: the EI-MS spectrum obtained by GC-MS is compared with a standard PMAA spectrum, and the result of monosaccharide composition is combined, so that the connection mode of each sugar residue of the exopolysaccharide A after reduction can be determined to be 1, 3-connected rhamnose residue, 1, 2-connected glucose residue and 1, 3-connected glucose residue, 1,4, 6-connected galactose residue.

(2) Nuclear magnetic resonance spectroscopy

20Mg of exopolysaccharide A was taken and dissolved with 0.5mL of D ₂ O and transferred to a clean nuclear magnetic resonance tube, after chromatography on a 600MHz nuclear magnetic resonance apparatus (from Bruker, switzerland) 1H-NMR (FIG. 2), 13C-NMR (FIG. 3), HSQCAD (FIG. 4), gHMBCAD (FIG. 5), NOESY (FIG. 6), gCOSY (FIG. 7) and TOCSY (FIG. 8) were obtained, and after comprehensive analysis, it was found that the exopolysaccharide backbone of Lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) consisted of 1, 3-linked rhamnose residues, 1, 2-linked glucose residues and 1, 3-linked glucose residues, the branching point was located at the O2 position of the 1,2, 3-linked rhamnose residues, and the branching consisted of terminal linked galactose residues with pyruvate substituents, the repeating unit composition of which was represented by formula I.

Example 9

Toxicity test of Lactobacillus rhamnosus extracellular polysaccharide (MTT method)

RAW264.7 cells were plated in 96-well plates at a concentration of 1×10 ⁴ cells/well and incubated overnight at 37 ℃ under 5% co ₂ saturated humidity. After pipetting the culture medium, 100. Mu.L of fresh DMEM cell culture medium containing different concentrations (0, 6.25, 12.5, 25, 50, 100, 200. Mu.g/mL) of exopolysaccharide A or LPS (1. Mu.g/mL) was added to the wells and incubated for 24h at 37℃under 5% CO ₂ saturation humidity, then the culture medium was pipetted, 30. Mu. LMTT (5 mg/mL) was added to each well, incubated for 4h at 37℃under 5% CO ₂ saturation humidity, the liquid in the wells was pipetted, 200. Mu.L of DMSO was added to dissolve formazan crystals, and absorbance at 490nm was measured for each well using a microplate counter (Multiskan FC, china) and this value indirectly reflects the number of living cells, as shown in FIG. 9.

Conclusion: compared with the control group, the low concentration of the extracellular polysaccharide A (6.25-25 mu g/mL) has no obvious effect on the cell viability of RAW264.7 macrophages, while the high concentration of the extracellular polysaccharide A (50-200 mu g/mL) can promote the proliferation of the macrophages and is dose-dependent. Therefore, the extracellular polysaccharide of the lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) is safe and nontoxic within the range of 6.25-200 mug/mL.

Example 10

Immunomodulation of lactobacillus rhamnosus exopolysaccharides

(1) Effect of Lactobacillus rhamnosus extracellular polysaccharide on phagocytic Activity (neutral Red method)

RAW264.7 cells were plated in 96-well plates at a concentration of 1×10 ⁴ cells/well and incubated overnight at 37 ℃ under 5% co ₂ saturated humidity. After pipetting the culture medium, 100. Mu.L of fresh DMEM cell culture medium containing different concentrations (6.25, 12.5, 25, 50, 100, 200. Mu.g/mL) of exopolysaccharide A or LPS (1. Mu.g/mL) was added to the wells and incubated for 24h at 37℃under 5% CO ₂ saturated humidity, 100. Mu.L of neutral red solution (8%) was added to each well, incubated for 1h at 37℃under 5% CO ₂ saturated humidity, the wells were pipetted, washed three times with PBS, and the cells were lysed by adding lysate (glacial acetic acid: ethanol=1:1), and absorbance at 540nm was measured for each well by a microplate counter, as shown in FIG. 10.

Conclusion: after RAW264.7 cells are treated by 6.25-12.5 mug/mL exopolysaccharide A, the phagocytosis rate is basically the same as that of a control group. However, with increasing extracellular polysaccharide A concentration (25-200. Mu.g/mL), the phagocytosis rate is remarkably enhanced in a dose-dependent manner, and the maximum phagocytosis capacity reaches 110% of that of the positive control group at 200. Mu.g/mL. The results show that the extracellular polysaccharide of lactobacillus rhamnosus strain BD 4047 (cgmccno. 13310) can stimulate RAW264.7 cells and enhance phagocytic capacity thereof under the experimental concentration.

(2) Effect of Lactobacillus rhamnosus extracellular polysaccharide on NO Release (Griess method)

RAW264.7 cells were plated in 96-well plates at a concentration of 5×10 ⁵ cells/well and incubated overnight at 37 ℃ under 5% co ₂ saturated humidity. After pipetting away the culture medium, 100. Mu.L of fresh DMEM cell culture medium containing different concentrations (6.25, 12.5, 25, 50, 100, 200. Mu.g/mL) of exopolysaccharide A or LPS (1. Mu.g/mL) was added to the wells and incubated at 37℃under 5% CO ₂ saturation humidity for 24h, the culture supernatant was collected, mixed with an equal volume of Griess reagent and incubated at room temperature for 10min. The absorbance at 540nm of each well was measured by a microplate counter, and the amount of NO secretion was calculated by drawing a standard curve using NaNO ₂ as a standard, and the results are shown in FIG. 11.

Conclusion: when the concentration of the extracellular polysaccharide of the lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) reaches 200 mug/mL, the release of NO by macrophages can be obviously promoted.

(3) Effect of Lactobacillus rhamnosus extracellular polysaccharide on TNF-alpha production

RAW264.7 cells were plated in 96-well plates at a concentration of 5×10 ⁵ cells/well and incubated overnight at 37 ℃ under 5% co ₂ saturated humidity. After the culture broth was aspirated, 100. Mu.L of fresh DMEM cell culture broth containing different concentrations (6.25, 12.5, 25, 50, 100, 200. Mu.g/mL) of extracellular polysaccharide A or LPS (1. Mu.g/mL) was added to the well and incubated at 37℃under a saturated humidity condition of 5% CO ₂ for 24 hours, the culture supernatant was collected, the levels of TNF-. Alpha.in the supernatant were measured with ELISA kit, and the TNF-. Alpha.concentration was calculated using a standard curve, and the results are shown in FIG. 12.

Conclusion: when the concentration of the extracellular polysaccharide of the lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) reaches 50 mug/mL, the expression of TNF-alpha can be obviously promoted.

(4) Effect of Lactobacillus rhamnosus extracellular polysaccharide on NF- κB activation

RAW264.7 cells were plated in 96-well plates at a concentration of 5×10 ⁵ cells/well and incubated overnight at 37 ℃ under 5% co ₂ saturated humidity. After the medium was aspirated, 100. Mu.L of fresh DMEM cell culture medium containing extracellular polysaccharide A (200. Mu.g/mL) or LPS (1. Mu.g/mL) was added to the well and incubated at 37℃under saturated humidity with 5% CO ₂ for 4 hours, 4% paraformaldehyde was added for 20 minutes to fix the cells, 5% BSA was added and allowed to stand at room temperature for 1 hour to terminate the reaction, and the mixture was stored at 4℃overnight after the addition of NF-. Kappa. B p65 antibody. After cell removal, CY 3-labeled secondary antibody and DAPI were added and allowed to stand at room temperature for 5min, and each of the above steps was washed three times with a washing buffer for 5 min. Finally, an image was captured using a confocal microscope (A1R, nikon, japan), and the fluorescence intensity was measured, and the result is shown in fig. 13.

Conclusion: the extracellular polysaccharide of lactobacillus rhamnosus strain BD 4047 (CGMCC No. 13310) can improve the overall expression level of NF- κB by activating the NF- κB signal path, thereby achieving the effect of regulating immunity.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. An extracellular polysaccharide derived from lactobacillus rhamnosus (Lactobacillus rhamnosus) strain BD 4047, characterized in that the repeating unit of the extracellular polysaccharide comprises monosaccharides of the following kind: rhamnose, glucose and galactose;

The molar ratio of the rhamnose, the glucose and the galactose is (3.67-3.72): (1.47-1.52): 1, a step of;

The average molecular weight is (1.419-1.735) x 10 ⁶ daltons.

2. The exopolysaccharide of claim 1, wherein the repeating units of the exopolysaccharide are formed by alternating 1, 3-linked rhamnose residues, 1, 2-linked glucose residues and 1, 3-linked glucose residues to form a backbone, and branching side chains are formed at the O2 position of the 1,2, 3-linked rhamnose residues, said branching side chains being formed by galactose residues terminally modified with a pyruvic acid substituent.

3. The exopolysaccharide of claim 2, wherein the repeating units of the exopolysaccharide are represented by formula I:

4. A process for the preparation of an extracellular polysaccharide according to any one of claims 1 to 3, comprising the steps of;

performing alcohol precipitation on fermentation supernatant of lactobacillus rhamnosus strain BD 4047, and collecting precipitate;

dissolving the precipitate in water, mixing with trichloroacetic acid, and collecting supernatant to obtain crude polysaccharide;

and (3) performing gel column chromatography separation on the crude polysaccharide, and collecting the earliest component peak fraction to obtain extracellular polysaccharide.

5. The method according to claim 4, wherein the method for preparing the fermentation supernatant of lactobacillus rhamnosus strain BD 4047 comprises inoculating lactobacillus rhamnosus strain BD 4047 into skimmed milk, standing for fermentation, inactivating the obtained fermented milk, and performing solid-liquid separation, wherein the collected liquid phase is the fermentation supernatant;

preferably, the skim milk comprises skim milk powder and water;

the mass of the skim milk powder accounts for 6-12% of the mass of the skim milk;

the inoculation amount of the lactobacillus rhamnosus is 1.25 multiplied by 10 ⁷～1×10⁸ CFU/mL;

The temperature of the standing fermentation is 25-45 ℃; the standing fermentation time is 12-36 h.

6. The method according to claim 4, wherein the volume ratio of the fermentation supernatant of lactobacillus rhamnosus strain BD 4047 to the alcohol is 1: (3-4);

the final mass concentration of the trichloroacetic acid is 5% -9%.

7. The preparation method according to claim 4, wherein the packing material of the gel column is Sepharose 6FastFlow during the chromatographic separation of the gel column; the specification of the gel column is 2.6cm multiplied by 30cm;

the eluent is 0.2M NaCl water solution;

the flow rate of the eluent is 0.2-0.3 mL/min.

8. Use of an extracellular polysaccharide according to any one of claims 1 to 3 or prepared by a method according to any one of claims 4 to 7 for the preparation of an immunomodulatory product.

9. The use of claim 8, wherein the immunomodulation comprises at least one of:

Improving phagocytic activity of macrophages;

Promoting secretion levels of TNF-a from macrophages;

promoting the release of NO from macrophages;

activating the NF- κB signaling pathway.

10. Use of an extracellular polysaccharide according to any one of claims 1 to 3 or prepared by a method according to any one of claims 4 to 7 for the preparation of a macrophage function promoter.