CN112568445A - Fucobiose with prebiotic effect, and preparation method and application thereof - Google Patents

Abstract

The invention discloses fucobiose with prebiotic effect and a preparation method and application thereof, belonging to the technical field of oligosaccharide preparation. The preparation method comprises the following steps: preparing extracellular polysaccharide by using liquid fermentation enterobacter F-CE2, and carrying out acidolysis on the extracellular polysaccharide at high temperature and high pressure to obtain degraded sugar liquid; and (3) removing undegraded macromolecular sugar fragments from the degraded sugar solution by ultrafiltration fractionation, collecting oligosaccharide components with the molecular weight less than or equal to 500Da, and freeze-drying to obtain the fucobiose. The structures of the fucobiose obtained by identification are beta-D-Glcp- (1 → 4) -beta-L-Fucp and alpha-D-Galp- (1 → 3) -beta-L-Fucp. The technology for preparing the fucoidin has the advantages of simple operation, low cost, easy expanded production and high disaccharide yield. The fucobiose obtained by the invention can promote the proliferation of Akkermansia muciniphila and multiple strains of bifidobacteria, and has wide application prospect as a novel prebiotic.

Description

Fucobiose with prebiotic effect, and preparation method and application thereof

Technical Field

The invention relates to the technical field of oligosaccharide preparation, in particular to fucobiose with prebiotic effect and a preparation method and application thereof.

Background

Various oligosaccharides (HMOs) exist in breast milk, wherein 2 '-fucosyllactose (2' -FL) is fucosyloligosaccharide with the highest content, and has the functions of repairing nerves, promoting brain development, improving allergic symptoms, regulating intestinal flora stability and the like for newborns. With the realization of in vitro synthesis of 2 '-FL, clinical studies also show that the addition of 2' -FL in milk powder can promote the brain development of infants and has good tolerance. Due to the complex synthesis and high price of 2 '-FL, many infant formula milk powder is added with galacto-oligosaccharides and fructo-oligosaccharides with functions similar to HMOs, but the health benefits of the oligosaccharides are different due to the different structures of the oligosaccharides and the 2' -FL. At the present stage, the finding of the fucooligosaccharide which has similar structure and function with 2' -FL and can be produced in large batch is of great significance.

Akkermansia muciniphila is a strictly anaerobic intestinal bacterium which is isolated and cultured from human excrement in recent years, and is closely related to the health of organisms. The relative abundance of Akkermansia muciniphila is inversely related to diseases such as inflammatory bowel disease, appendicitis, obesity, type 2 diabetes mellitus and juvenile autism. In 1899, Bifidobacterium was first isolated from the faeces of infants by doctor Tisser, France, and the health of infants was closely related to the presence and quantity of Bifidobacterium in the body. Bifidobacteria are present in the gut of breast-fed infants in much greater amounts than in milk-fed infants, where breast-milk oligosaccharides play an important role. In addition, the short-chain fatty acids, metabolites of Akkermansia muciniphila and bifidobacteria, can also be used as an energy source for intestinal epithelial cells to promote the proliferation of the intestinal epithelial cells, thereby improving intestinal tissues. A plurality of studies show that the fucooligosaccharide can be metabolized and utilized by Akkermansia muciniphila and bifidobacteria, and the fucooligosaccharide is added into food or feed as a prebiotic, which is beneficial to regulating the intestinal flora of human or animals.

Most of the existing fucoidan oligosaccharides are prepared by carrying out acidolysis or enzymolysis on fucoidan sulfate from brown algae or sea cucumbers, the yield is low, the cost is high, and degradation products have large molecular weight and are not easy to be rapidly utilized by probiotics. The preparation of Extracellular Polysaccharide (EPS) has the advantages of small influence of environmental factors, short period, high yield and the like, and has wide research and development values. In recent years, studies have been made on the production of fucose-containing exopolysaccharides by fermentation of specific strains, such as Enterobacter A47, Bacillus licheniformis BioE-BL11, Corynebacterium michiganensis Cm542, and the like. However, the extracellular polysaccharide of bacteria has high molecular weight and high viscosity, and cannot be effectively degraded and utilized by probiotics, which limits the application of the extracellular polysaccharide in activity. Therefore, the method for degrading the macromolecular fucose-rich exopolysaccharide into the micromolecular fucoidin by adopting a proper method can effectively improve the utilization value of the exopolysaccharide and provides a new technical means for realizing the industrial production of the novel fucoidin.

Disclosure of Invention

The invention aims to provide fucobiose with prebiotic effect, a preparation method and application thereof, so as to solve the problems in the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides fucobiose with prebiotics effect, wherein the structure of the fucobiose is beta-D-Glcp- (1 → 4) -beta-L-Fucp and alpha-D-Galp- (1 → 3) -beta-L-Fucp.

The invention provides a method for preparing fucobiose with prebiotic effect, which comprises the following steps:

(1) fermenting Enterobacter (Enterobacter sp.) F-CE2 to extract exopolysaccharide;

(2) preparing a solution of the extracellular polysaccharide obtained in the step (1) and carrying out acidolysis under the conditions of high temperature and high pressure to obtain a degraded sugar solution;

(3) removing undegraded macromolecular sugar from the degraded sugar solution obtained in the step (2) by ultrafiltration, and freeze-drying the obtained filtrate to obtain the fucobiose.

Furthermore, the Enterobacter (Enterobacter sp.) F-CE2 in the step (1) has a preservation number of CGMCC No.20359, and is preserved in China general microbiological culture Collection center of China Committee for culture Collection, No.3 of West Lu No.1 Hospital, Navy, the Beijing area on 7-14 days in 2020, wherein the molar ratio of fucose in the extracellular polysaccharide of the Enterobacter F-CE2 is 30-45%.

Further, the specific preparation method of the exopolysaccharide in the step (1) comprises the following steps: after the enterobacter F-CE2 is activated for three times, the enterobacter F-CE2 is inoculated into a fermentation medium for shaking fermentation, the obtained fermentation liquid is centrifuged at 6000r/min for 15min, the supernatant is collected and concentrated by rotary evaporation, the extracellular polysaccharide is precipitated by adding 95 percent ethanol with three times of volume after concentration, the extracellular polysaccharide is collected and dissolved in ultrapure water, and the extracellular polysaccharide is dialyzed in a dialysis bag for 48 hours and then is dried in vacuum.

Further, the formula of the fermentation medium is as follows: 5-15 g/L beef extract, 2-6 g/L yeast extract, 10-20 g/L peptone and 20-40 g/L, NH glucose₄Cl 0.3g/L、KCl 1g/L、 (NH₄)₂SO₄0.3 g/L、Na₂HPO₄10 g/L、KH₂PO₄3 g/L、K₂SO₄1 g/L、NaCl 1g/L、 MgSO₄·7H₂O 0.2g/L、CaCl₂·6H₂O 0.02g/L、FeSO₄0.001g/L and pH of 7.0-7.2.

Furthermore, the inoculation amount of the fermentation medium is 0.1-1% (V/V), the fermentation temperature is 27-38 ℃, the oscillation speed is 120-180 r/min, and the fermentation time is 24-72 h.

Further, the specific conditions of high pressure and high temperature in the step (2) are as follows: the pressure is 0.1-0.3 Mpa, and the temperature is 130-150 ℃.

Further, the acid treatment conditions of the exopolysaccharide solution in the step (2) are as follows: and adding hydrochloric acid into the extracellular polysaccharide solution, adjusting the pH to 2-4, treating for 2-5 h, and adjusting the concentration of the extracellular polysaccharide solution to 10-50 mg/ml.

Further, the molecular weight of the sugar liquid obtained by ultrafiltration in the step (3) is less than or equal to 500 Da.

The invention also provides application of the fucobiose in promoting proliferation of probiotics.

The invention discloses the following technical effects:

the technology for preparing the fucoidin has the advantages of simple operation, low cost, easy expanded production and high yield of the fucoidin.

The fucoidin provided by the invention has novel structure, high purity and high yield, can be fermented and utilized by intestinal probiotics, reduces the pH value of the intestinal environment, promotes the proliferation of multiple bifidobacteria including Akkermansia muciniphila, bifidobacterium infantis and bifidobacterium, and has wide application prospect as a novel prebiotic.

Drawings

FIG. 1 is a gram-stained microscopic image of Enterobacter according to example 1;

FIG. 2 is the infrared spectrum of exopolysaccharide of Enterobacter of example 3;

FIG. 3 is a GPC chart of extracellular polysaccharide of Enterobacter in example 3;

FIG. 4 is a GPC chart of the monosaccharide composition of fucooligosaccharide of example 5;

FIG. 5 is the mass spectrum of fucooligosaccharide of example 5; FIG. 5-A is ESI-MS in positive ion mode, and FIG. 5-B is ESI-CID-MS/MS in positive ion mode;

FIG. 6 shows the NMR one-dimensional of fucobiose in example 5¹H, spectrogram;

FIG. 7 shows the NMR one-dimensional of fucobiose in example 5¹³C, spectrum;

FIG. 8 is the two-dimensional TOCSY spectrum of the nuclear magnetic resonance of fucobiose in example 5;

FIG. 9 is the two-dimensional HSQC spectrum of the nuclear magnetic resonance of fucobiose in example 5;

FIG. 10 is the two-dimensional HMBC spectrum of the nuclear magnetic resonance of the fucose of example 5;

FIG. 11 is the proliferation promoting effect of fucoidan and 2' -FL prebiotics on 4 probiotics and the acid production capacity of the strains in example 6; FIG. 11-A is Bifidobacterium breve, FIG. 11-B is Bifidobacterium infantis, FIG. 11-C is Bifidobacterium bifidum, and FIG. 11-D is Akkermansia muciniphila;

FIG. 12 is the example 6 fucobiose and 2' -FL prebiotics promote the production of 4 probiotic short chain fatty acids; FIG. 12-A is Bifidobacterium breve, FIG. 12-B is Bifidobacterium infantis, FIG. 12-C is Bifidobacterium bifidum, and FIG. 12-D is Akkermansia muciniphila.

Detailed Description

The following further illustrates embodiments of the invention, taken in conjunction with the accompanying drawings, which are not to be considered limiting of the invention, but are to be understood as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 screening and identification of Enterobacteriaceae

A sewage sample was collected from a sewage treatment plant in the south area of Qingdao City, Shandong province. A30 mL sewage sample is taken, centrifuged at 8000r/min at 4 ℃ for 20min, the precipitate is resuspended by using a proper amount of PBS solution, coated on a nutrient agar plate by a coating method, and cultured overnight at 37 ℃. After the culture is finished, picking 20 single colonies on the plate, numbering 1-20, enriching 20 single colonies by using LB liquid culture medium respectively, and culturing for 24h at 37 ℃.

Extracting DNA of 20 bacteria according to the method for extracting the bacterial genome kit, taking the DNA as a template, and using a 16S rDNA universal primer 27F (shown as SEQ ID NO. 1): 5'-AGAGTTTG ATCCTGGCTCAG-3' and 1492R (shown in SEQ ID NO. 2): 5'-ATTACCGCGG CTGCTGGC-3' colony PCR was performed. The PCR reaction system is as follows: 8 μ L of Taq enzyme, 10 μ L of sterile water, 0.5 μ L of each primer, and 1 μ L of template. The PCR reaction conditions are as follows: (1) pre-denaturation at 95 ℃ for 5 min; (2) denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 45s, and extension at 72 ℃ for 2min for 32 cycles; (3) re-extension at 72 ℃ for 1 min. After the amplification is finished, the PCR amplification product is sent to a biological company for sequencing, the sequencing result is subjected to Blast comparison, the result shows that 1 strain of bacteria is enterobacter, the sequencing result is shown as SEQ ID NO.3, the microscopic examination result also shows that the bacteria is bacillus-shaped gram-negative bacteria as shown in figure 1, namely the enterobacter is successfully separated from the sewage. The enterobacter is named enterobacter F-CE2 and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No.3 of Xilu No.1 of Beijing, Chaoyang, the preservation number is CGMCC No.20359, and the preservation date is 2020, 7 and 14 days.

Example 2 conditions for the optimized preparation of extracellular polysaccharides of Enterobacter

Activating Enterobacter F-CE2 with LB liquid culture medium for three generations, inoculating into liquid fermentation culture medium with inoculation amount of 0.5% (V/V), shake culturing the culture medium in a shaking table at 32 deg.C and rotation speed of 150r/min for 48 h; after fermentation is finished, centrifuging the fermentation liquor at 6000r/min for 15min, removing thalli sediment and collecting supernatant; rotatably evaporating the supernatant in a rotary evaporator at 50 ℃ to 1/5 of the original volume, then adding 95% ethanol with 3 times of volume for precipitation, and standing for 4 hours; centrifuging at 6000r/min for 10min, collecting precipitate, redissolving the precipitate with ultrapure water, dialyzing with 10kDa dialysis bag for 48h, vacuum freeze drying, and calculating extracellular polysaccharide yield. Pretreating exopolysaccharide by using a PMP pre-column derivatization method, performing high-efficiency liquid phase analysis on monosaccharide composition, and calculating fucose content. The result shows that the yield of the enterobacter extracellular polysaccharide is 3-6.5 g/L, and the molar ratio of fucose in the extracellular polysaccharide is 30-45% (see tables 2 and 4).

The specific operation method of the high performance liquid phase analysis comprises the following steps:

5mg of the sample is accurately weighed and placed in an ampoule tube, 1mL of trifluoroacetic acid (TFA) with the concentration of 2mol/L is added into the ampoule tube, the tube is sealed, and then the ampoule tube is placed in an oven to be hydrolyzed for 6 hours under the constant temperature of 110 ℃. The hydrolyzed sample was concentrated by evaporation, and then reconstituted with methanol, and TFA was removed several times. Preparing a standard monosaccharide sample according to an equal molar ratio, and carrying out PMP (1-phenyl-3-methyl-5-pyrazolone) derivatization on a product obtained after hydrolysis of the oligosaccharide sample. The derivation method comprises the following steps: mixing 100 mu L of sample mixed solution to be detected with 210 mu L of NaOH solution with the concentration of 0.3mol/L, adding 200 mu L of 0.5mol/L PMP methanol solution, uniformly mixing, and placing in a 70 ℃ oven for reaction for 60 min; after the reaction, the reaction mixture was cooled at room temperature for 10min, and 210. mu.L of 0.3mol/L HCl was added to neutralize any excess base. Adding 3mL of dichloromethane into the mixed solution, shaking, standing, removing the lower layer after the mixture is layered, and repeating the extraction process for 3 times. The aqueous phase was filtered through a 0.22 μm microfiltration membrane and analyzed by HPLC.

Liquid phase apparatus: agilent 1260 high performance liquid chromatograph; ultraviolet detector (245nm)

Type of liquid phase column: AgilentZORBAX 300XDB-C18

Liquid phase conditions: 0.05mol/LKH₂PO₄Phosphate buffer (pH 6.7)/CH₃CN (83:17, V: V) is a mobile phase; the column temperature was 35 ℃; the flow rate is 1 mL/min; the amount of sample was 20. mu.L.

The liquid fermentation media are shown in table 1 below:

TABLE 1

Inorganic substanceThe formula of the salt is as follows: NH (NH)₄Cl 0.3g/L、KCl 1g/L、(NH₄)₂SO₄0.3 g/L、Na₂HPO₄ 10g/L、KH₂PO₄3 g/L、K₂SO₄1 g/L、NaCl 1g/L、MgSO₄·7H₂O 0.2g/L、 CaCl₂·6H₂O 0.02g/L、FeSO₄0.001 g/L; adjusting the pH value of the culture medium to 7.0-7.2.

Table 2 shows the exopolysaccharide yields and fucose contents of the different fermentation media of Enterobacter in example 2.

TABLE 2

The parameters of the optimized enterobacter fermentation medium are shown in the table 3:

TABLE 3

Table 4 shows the exopolysaccharide yields and fucose contents of the enterobacteria under different fermentation culture conditions of example 2.

TABLE 4

EXAMPLE 3 determination of basic Properties of exopolysaccharides

Optimizing the conditions of the enterobacter fermentation medium according to the example 2, selecting the culture conditions with the highest EPS yield and fucose content, and finally determining the formula of the culture medium as follows: 10g/L beef extract, 6g/L yeast extract, 10g/L peptone and 30g/L, NH glucose₄Cl 0.3g/L、KCl 1g/L、(NH₄)₂SO₄0.3 g/L、 Na₂HPO₄10 g/L、KH₂PO₄3 g/L、K₂SO₄1 g/L、NaCl 1g/L、MgSO₄·7H₂O 0.2 g/L、CaCl₂·6H₂O 0.02g/L、FeSO₄0.001 g/L; adjusting the pH value of the culture medium to 7.0-7.2. Culturing at 32 deg.C and 150r/min for 48 h. As shown in table 5, the monosaccharide composition of exopolysaccharide prepared under optimal conditions comprises fucose, glucose, galactose, glucuronic acid, galacturonic acid, mannose and rhamnose, wherein the molar ratio of fucose is about 45.02%.

Table 5 shows the monosaccharide composition molar ratio of exopolysaccharide in example 3.

TABLE 5

In the experiment, infrared spectroscopy is carried out on the sample by adopting a KBr tablet pressing method. The sample treatment method is as follows: weighing about 2-3mg of sample and 100-600 mg of KBr according to the mass ratio of the sample to the KBr of 1:200, transferring the sample and the KBr into an agate mortar to be uniformly ground into powder, transferring the ground powder into a sheet-making die, keeping the pressure for 2min under the pressure of 10 tons, removing the pressure, taking out a test piece, and visually observing the prepared test piece to be transparent. The test piece is taken out and put into a sample holder for infrared spectrometry, and the model of the instrument is a Nicolet Nexus 470 type infrared spectrometer. As shown in fig. 2, fig. 2 is an infrared spectrum of extracellular polysaccharide of enterobacter; the samples were found to be 3427.69, 2926.56, 1639.69 and 1404.60cm, respectively^-1A characteristic absorption peak of stretching vibration, which is a characteristic absorption peak generated by stretching vibration of polysaccharide O-H, C-H, C ═ O, C-O; 1067.13cm^-1And 1026.77 cm^-1Two absorption peaks indicate the presence of pyranose sugars in the polysaccharide; indicating the presence of carboxyl structures in the polysaccharide, which is consistent with the results of liquid chromatography showing the presence of uronic acid.

The molecular weight of EPS was determined using a high performance liquid chromatography system, a differential detector and a TSK gel G4000 PWXL chromatography column. The system temperature was set at 35 ℃; the mobile phase is composed of 0.2mol/L NaNO₃And 0.01mol/L NaH₂PO₄Composition is carried out; the flow rate was set to 0.5mL/min, and the amount of sample was 20. mu.L. Different molecular weight dextrans (80, 150, 270, 410 and 670kDa) were used to plot standard curves. ResultsAs shown in FIG. 3, FIG. 3 shows that the molecular weight of extracellular polysaccharide of Enterobacter is 5.2 × 10 as calculated by standard curve when the extracellular polysaccharide peaks at 19.674min⁶Da。

Example 4 preparation of fucooligosaccharides by high temperature and high pressure acid hydrolysis

The high pressure acidolysis conditions are shown in table 6. Preparing the enterobacter F-CE2 extracellular polysaccharide into a 1-5% polysaccharide solution by using ultrapure water, adding hydrochloric acid into the polysaccharide solution, adjusting the pH to 2-4, the temperature to 130-150 ℃, the pressure to 0.1-0.3 Mpa, treating for 2-5 h, and adjusting the pH to 7.0 by using 2mol/L NaOH. Centrifuging the obtained oligose liquid at 5000r/min for 10min, drying and weighing the precipitate, and collecting the supernatant. Intercepting the supernatant by using a dialysis bag with molecular weight cutoff of 500Da to obtain oligosaccharide sugar solution with molecular weight less than 500Da, freeze-drying the sugar solution into powder, and weighing. The yield of the exopolysaccharide oligosaccharide is shown in table 7, and the result shows that after the enterobacter exopolysaccharide is subjected to acid treatment under the high-temperature and high-pressure environment, the yield of the oligosaccharide with the molecular weight of less than 500Da is 50-70%, and the optimal reaction conditions are as follows: treating for 4-5 h with polysaccharide concentration of 30mg/ml, pH of 3 and vacuum degree of 0.2 Mpa.

Table 6 shows the exopolysaccharide conditions of high-pressure acidolysis of Enterobacter in example 4.

TABLE 6

Table 7 shows the yield of exopolysaccharide-oligosaccharides from Enterobacter hyperbaric acid hydrolysis in example 4.

TABLE 7

Example 5 structural analysis of fucooligosaccharides

(1) Analysis of the monosaccharide composition of fucooligosaccharides

The monosaccharide composition of fucooligosaccharide was measured according to the method of high performance liquid chromatography for monosaccharide composition in example 2, and the results are shown in fig. 4, and fig. 4 is a GPC diagram of the monosaccharide composition of fucooligosaccharide, indicating that fucooligosaccharide is composed of glucose, galactose and fucose, and the ratio is 1:1: 2.

(2) Mass spectrometric analysis of fucooligosaccharides

The molecular weight of the fucooligosaccharide is identified by positive and negative ion electrospray ionization mass spectrometry (ESI-MS) and electrospray collision induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) by using an Agilent 1290Infinity II system provided with an Agilent 6460 triple quadrupole mass spectrometer. The mobile phase was acetonitrile/water (1:1, V/V), the flow rate was 0.4mL/min, the atomizer pressure was 40psi, the capillary voltage was 3500V, argon was used as the collision gas, and the collision energy was 10-55 eV. FIG. 5 is a graph showing the results of mass spectrometry of oligosaccharide samples, as shown in FIG. 5; FIG. 5-A is ESI-MS in positive ion mode, and FIG. 5-B is ESI-CID-MS/MS in positive ion mode; it is known that the fucooligosaccharide is fucobiose having a molecular weight of 326Da, and as a result of the combination of monosaccharides, the fucobiose may be a disaccharide composed of glucose and fucose, or a disaccharide composed of galactose and fucose.

(3) Nuclear magnetic analysis of fucooligosaccharides

10mg of a fucobiose sample was dissolved in 0.5mL of D₂O and freeze drying. The sample was then re-dissolved in 0.5mL of D₂In O, chemical shifts were calibrated using acetone as an internal standard. All one dimensions (¹H，¹³C, DEPT 90 °, 135 °) and two dimensions (¹H-¹H COSY，¹H-¹H TOCSY，¹H-¹³C HSQC，¹H-¹³C HMBC) NMR spectra were recorded using an Agilent DD2-500 spectrometer with a frequency of 500MHz and a temperature of 25 ℃. As shown in FIGS. 6 to 10, FIGS. 6 to 10 are nuclear magnetic resonance results of fucobiose, and FIG. 6 is a nuclear magnetic resonance one-dimensional graph¹H spectrum, FIG. 7 is one dimension of nuclear magnetic resonance¹³A spectrum C, a two-dimensional TOCSY spectrum of Nuclear Magnetic Resonance (NMR), a two-dimensional HSQC spectrum of nuclear magnetic resonance (HSQC) spectrum of nuclear magnetic resonance (HSBC) spectrum of nuclear magnetic resonance (HMBC) spectrum of; table 8 shows each monosaccharide residue of fucobiose¹H and¹³chemical shift of C. The results showed that the fucobiose structure was β -D-Glcp- (1 → 4) - β -L-Fucp and α -D-Galp- (1 → 3) - β -L-Fucp.

Table 8 shows each monosaccharide residue of fucobiose in example 5¹H and¹³chemical position of CAnd (6) moving.

TABLE 8

Example 6 prebiotic Effect of fucobiose

In order to explore the probiotic effect of the fucobiose on the intestinal flora, the experimental process simulates the intestinal anaerobic fermentation environment, and the proliferation effect and the substrate utilization efficiency of the fucobiose on the bifidobacterium breve, the bifidobacterium infantis, the bifidobacterium bifidum and the Akkermansia muciniphila are explored. Using an oxygen-removed YCFA fermentation culture medium, wherein the single strain inoculation amount is 0.5%, fermenting for 48h at 37 ℃, and respectively taking fermentation products of 0 h, 12h, 24h, 36 h and 48h for subsequent analysis. The results are shown in fig. 11, fig. 11 shows the proliferation promoting effect of the fucobiose and 2' -FL prebiotics on 4 probiotics and the acid production capacity of the strains; FIG. 11-A is Bifidobacterium breve, FIG. 11-B is Bifidobacterium infantis, FIG. 11-C is Bifidobacterium bifidum, and FIG. 11-D is Akkermansia muciniphila; compared with 2' -FL, the fucobiose can remarkably promote the proliferation of bifidobacterium breve, Akkermansia muciniphila, bifidobacterium infantis and bifidobacterium bifidum (p is less than 0.05), and the pH value can be reduced (by 1-2) within 12 h. After 24h fermentation, fucobiose promoted production of more formate, acetate and lactate by bifidobacteria compared to 2 '-FL, which promoted production of a small amount of butyrate by Akkermansia muciniphila, whereas 2' -FL failed to promote production of butyrate by Akkermansia muciniphila; as shown in fig. 12, fig. 12 is the results of the fucobiose and 2' -FL prebiotics promoting the production of 4 probiotic short chain fatty acids; FIG. 12-A is Bifidobacterium breve, FIG. 12-B is Bifidobacterium infantis, FIG. 12-C is Bifidobacterium bifidum, and FIG. 12-D is Akkermansia muciniphila.

It will be understood that modifications and variations can be made by persons skilled in the art in light of the above teachings and all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.

Sequence listing

<110> Nanjing Yixue Biotech Co., Ltd; china ocean university

<120> fucobiose with prebiotics effect, and preparation method and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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agagtttgat cctggctcag 20

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

attaccgcgg ctgctggc 18

<210> 3

<211> 1443

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgcggggcgg cacggtacca tgcagtcgag cggtaacaca gggagcttgc tcctgggtga 60

cgagcggcgg acgggtgagt aatgtctggg aaactgcctg atggaggggg ataactactg 120

gaaacggtag ctaataccgc ataacgtcgc aagaccaaag agggggacct tcgggcctct 180

tgccatcaga tgtgcccaga tgggattagc tagtaggtgg ggtaatggct cacctaggcg 240

acgatcccta gctggtctga gaggatgacc agccacactg gaactgagac acggtccaga 300

ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgcaagcctg atgcagccat 360

gccgcgtgta tgaagatccc cttcgggttg taaagtactt tcagcgggga ggaaggtgtt 420

gaggttaata acctcagcaa ttgacgttac ccgcagaaga agcaccggct aactccgtgc 480

cagcagccgc ggtaatacgg agggtgcaag cgttaatcgg aattactggg cgtaaagcgc 540

acgcaggcgg tctgtcaagt cggatgtgaa atccccgggc tcaacctggg aactgcattc 600

gaaactggca ggctagagtc ttgtagaggg gggtagaatt ccaggtgtag cggtgttttg 660

cgtagagatc tggaggaata ccggtggcga aggcggcccc ctggacaaag actgacgctc 720

aggtgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg 780

atgtcgactt ggaggttgtg cccttgaggc gtggcttccg gagctaacgc gttaagtcga 840

ccgcctgggg agtacggccg caaggttaaa actcaaatga attgacgggg gcccgcacaa 900

gcggtggagc atgtggttta attcgatgca acgcgaagaa ccttacctac tcttgacatc 960

cagagaactt agcagagatg ctttggtgcc ttcgggaact ctgagacagg tgctgcatcc 1020

gtgtcgtcag ctcgtgttgt gaaatgttgg gttaagtccc gcaacgagcg caacccttat 1080

cctttgttgc cagcggttcg gccgggaact caaaggagac tgccagtgat aaactggagg 1140

aaggtgggga tgacgtcaac agatcatggc ccttacgagt agggctacac acgtgctaca 1200

atggcgcata caaagagaag cgacctcgcg agagcaagcg gacctcataa agtgcgtcgt 1260

agtccggatt ggagtctgca actcgactcc atgaagtcgg aatcgctagt aatcgtagat 1320

cagaatgcta cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggga 1380

gtgggttgca aaagaagtag gtagcttaac cttcgggagg gcgctaccac tttgtattag 1440

ttg 1443

Claims (10)

1. Fucobiose having prebiotic effect, wherein the structure of fucobiose is β -D-Glcp- (1 → 4) - β -L-Fucp and α -D-Galp- (1 → 3) - β -L-Fucp.

2. A method for preparing the fucobiose with prebiotic effect as defined in claim 1, which comprises the following steps:

(1) fermenting enterobacter F-CE2 and extracting extracellular polysaccharide;

(2) preparing a solution of the extracellular polysaccharide obtained in the step (1) and carrying out acidolysis under the conditions of high temperature and high pressure to obtain a degraded sugar solution;

(3) removing undegraded macromolecular sugar from the degraded sugar solution obtained in the step (2) by ultrafiltration, and freeze-drying the obtained filtrate to obtain the fucobiose.

3. The method according to claim 2, wherein the Enterobacter F-CE2 with a collection number of CGMCC No.20359 in step (1) is collected at the China general microbiological culture Collection center of China Committee for culture Collection, China school No.3 of Xilu 1 on North Chen of the Yang facing district, Beijing, 7 and 14 days in 2020, and the molar ratio of fucose in the extracellular polysaccharide of the Enterobacter F-CE2 is 30 to 45 percent.

4. The method according to claim 2, wherein the specific preparation method of exopolysaccharide in step (1) comprises: after the enterobacter F-CE2 is activated for three times, the enterobacter F-CE2 is inoculated into a fermentation medium for shaking fermentation, the obtained fermentation liquid is centrifuged at 6000r/min for 15min, the supernatant is collected and concentrated by rotary evaporation, the extracellular polysaccharide is precipitated by adding 95 percent ethanol with three times of volume after concentration, the extracellular polysaccharide is collected and dissolved in ultrapure water, and the extracellular polysaccharide is dialyzed in a dialysis bag for 48 hours and then is dried in vacuum.

5. The method of claim 4, wherein the fermentation medium is formulated as: 5-15 g/L beef extract, 2-6 g/L yeast extract, 10-20 g/L peptone and 20-40 g/L, NH glucose₄Cl 0.3g/L、KCl 1g/L、(NH₄)₂SO₄ 0.3g/L、Na₂HPO₄ 10g/L、KH₂PO₄ 3g/L、K₂SO₄ 1g/L、NaCl 1g/L、MgSO₄·7H₂O 0.2g/L、CaCl₂·6H₂O 0.02g/L、FeSO₄0.001g/L and a pH of 7.0 to 7.2.

6. The method as claimed in claim 4 or 5, wherein the strain inoculation amount of the fermentation medium is 0.1-1% (V/V), the fermentation temperature is 27-38 ℃, the oscillation speed is 120-180 r/min, and the fermentation time is 24-72 h.

7. The method according to claim 2, wherein the specific conditions of high pressure and high temperature in step (2) are as follows: the pressure is 0.1-0.3 Mpa, and the temperature is 130-150 ℃.

8. The method as claimed in claim 2, wherein the acid hydrolysis of the exopolysaccharide solution of step (2) is carried out under the following conditions: and adding hydrochloric acid into the extracellular polysaccharide solution, adjusting the pH to 2-4, treating for 2-5 h, and adjusting the concentration of the extracellular polysaccharide solution to 10-50 mg/ml.

9. The process of claim 2, wherein the degraded saccharide solution obtained by ultrafiltration in step (3) has a molecular weight of ≦ 500 Da.

10. Use of the fucobiose of claim 1 for promoting proliferation of a probiotic.

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