LU500037B1 - Fucosyl-disaccharide with Prebiotic Effect, Method for Preparing the Same and Application Thereof - Google Patents

Abstract

The present invention discloses fucosyl-disaccharide with prebiotic effect, a method for preparing the same and application thereof, and belongs to the field of oligosaccharide preparation technology. The method has the following specific steps: using liquid fermentation Enterobacter F-CE2 to prepare extracellular polysaccharide (EPS), performing acidolysis on the EPS at high temperature and at high pressure to obtain a degraded sugar solution; performing ultrafiltration on the degraded sugar solution to fractionate and remove undegraded macromolecular sugar fragments, collecting oligosaccharide components with a molecular weight ? 500 Da, lyophilizing the oligosaccharide components to obtain fucosyl-disaccharide. The fucosyl-disaccharide has the following identified structure: ?-D-Glcp-(1?4)-?-L-Fucp and ?-D-Galp-(1?3)-?-L-Fucp. Preparation technology of fucosyl-disaccharide of the present invention has the advantages of simple operation, low cost, easy expansion of production and high yield of disaccharide. The fucosyl-disaccharide obtained in the present invention can promote the proliferation of Akkermansia muciniphila and a plurality of strains of bifidobacteria.

Description

DESCRIPTION Fucosyl-disaccharide with Prebiotic Effect, Method for Preparing the Same and Application Thereof

TECHNICAL FIELD The present invention relates to the field of oligosaccharide preparation technology, and particularly, to a fucosyl-disaccharide with prebiotic effect, a method for preparing the same and application thereof.

BACKGROUND There is a plurality of oligosaccharides (HMOs) in breast milk, among which, 2'-fucosyllactose (2'-FL) is a kind of fucosyl oligosaccharide with the highest content, a variety of functions for newborn infants includes repairing nerves, promoting brain development, improving allergy symptoms, regulating intestinal flora stability, and other functions. With the realization of in vitro synthesis of 2'-FL, clinical studies have also shown that adding 2"-FL into the milk powder can promote the development of brain in infants and young children with good tolerance. Because of the complex synthesis and high price of 2'-FL, many infant formula milk powders have been added with galacto-oligosaccharide and fructo- oligosaccharide with functions similar to HMOs. However, the structures of these oligosaccharides are different from 2'-FL and have different health benefits. Currently, it is of great significance to find the fucose-containing oligosaccharides

(FCOs) that has the similar structure and functions with 2’-FL and can be produced in large quantities.

Akkermansia muciniphila is a strictly anaerobic intestinal bacterium isolated from human feces in recent years, which is closely related to the health of the human body. The relative abundance of the Akkermansia muciniphila is negatively correlated with a plurality of diseases including inflammatory bowel disease, appendicitis, obesity, type 2 diabetes, and adolescent autism. In 1899, bifidobacteria was firstly isolated from infant stool by French scientist Dr. Tisser. The health of infant is closely related to the presence and quantity of bifidobacteria in the body. The number of bifidobacteria in intestines of breast-fed infants is much greater than that of infants fed with a milk powder, and breast milk oligosaccharide plays an important role in it. Furthermore, short-chain fatty acids, the metabolites of the Akkermansia muciniphila and bifidobacteria, can also be used as energy sources for intestinal epithelial cells to promote the proliferation of intestinal epitheliacells, and thereby improve intestinal tissues. Various studies have shown that FCOs can be metabolized by the Akkermansia muciniphila and the bifidobacteria. The supplement of FCOs in food or fodders can work as prebiotics to help regulate intestinal microbiota of humans or animals.

Most of the existing FCOs are prepared from fucoidan derived from brown algae or sea cucumbers using acidolysis or enzymatic hydrolysis, however, the preparation method possesses low yield rates and high costs, further, the degradation product has a large molecular weight and is difficult to be quickly utilized by probiotics. Preparation of bacterial extracellular polysaccharide (EPS) has extensive research and development value for its advantages of being weak- influenced by the surroundings, a short cycle, high yield, etc. In recent years, it has been reported that fucose-containing EPS can be prepared by the fermentation of specific strains, such as Enterobacter A47, Bacillus licheniformis BioE-BL11, and Michigan corynebacteria Cm542 and so on. However, bacterial exopolysaccharide has a high molecular weight and high viscosity, and hence cannot be effectively degraded and utilized by probiotics which limit its application of functional activities. Therefore, using a suitable method to degrade fucose-rich EPS with high molecular weight into FCOs with lower molecular weight can effectively increase the utilization value of EPS while providing new technical means for the industrial production of new FCOs.

SUMMARY The objective of the present invention is to provide fucosyl-disaccharide with prebiotic effect, a method for preparing the same and application thereof, so as to solve the forgoing problems in the prior art.

The present invention provides fucosyl-disaccharide with prebiotic effect, wherein the fucosyl-disaccharide has structure of B-D-Glcp-(1—4)-B-L-Fucp and a-D-Galp- (1—3)-B-L-Fucp.

The present invention provides a method for preparing fucosyl-disaccharide with prebiotic effect, specifically including the following steps: (1) fermenting Enterobacter F-CE2 to extract EPS; (2) preparing a solution with the EPS obtained in step (1) and performing acidolysis for the EPS at high temperature and at high pressure to obtain a degraded sugar solution; (3) performing ultrafiltration for the degraded sugar solution obtained in step (2) to remove undegraded macromolecular sugars, and then lyophilizing the obtained filtrate solution to obtain the fucosyl-disaccharide.

Further, the Enterobacter F-CE2 of step (1) has a preservation number of CGMCC No. 20359, was preserved on July 14, 2020 in China General Microbiological Culture Collection Center, No. 3, 1% Yard, Beichen West Road, Chaoyang District, Beijing. The EPS of Enterobacter F-CE2 presents a mole ratio of 30%-45% of fucose.

Further, a specific preparation method of the EPS in step (1) includes: after the third activation of Enterobacter F-CE2 , the Enterobacter F-CE2 is inoculated into a fermentation medium; after shaking and fermentation, a resulting fermentation broth is centrifuged at 6000 r/min for 15 min to collect a supernatant; the resulting supernatant is rotated, evaporated and concentrated; after the concentration, the supernatant is precipitated with three times volume of 95% ethanol to precipitate the EPS, and the EPS is collected, then dissolved in ultrapure water, dialyzed in a dialysis bag for 48 h and then vacuum-dried.

Further, the fermentation medium has the following formula: beef extract of 5-15 g/L, yeast extract of 2~6 g/L, peptone of 10-20 g/L, glucose of 20-40 g/L, NH4Cl of 0.3 g/L, KCl of 1 g/L, (NHa)2SO4 of 0.3 g/L, NazHPO4 of 10 g/L, KH2PO4 of 3 g/L, KSO4 of 1 g/L, NaCl of 1 g/L, MgSO4 °7H20 of 0.2 g/L, CaCl2*6H20 of

0.02 g/L, and FeSO4 of 0.001 g/L, pH is 7.0~7.2.

Further, the fermentation medium has inoculum volume of 0.1% to 1% (V/V), fermentation temperature of 27 °C to 38 °C, shaking speed of 120 r/min~180 r/min, and fermentation time of 24 h~72 h.

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

Further, an EPS solution in step (2) has the following conditions for acid treatment: adding hydrochloric acid to the EPS solution, and adjusting pH to 2~4, with treating time of 2 to 5 h, and concentration of 10 to 50 mg/ml EPS.

Further, the degraded sugar solution obtained by ultrafiltration in step (3) has a molecular weight of less than or equal to 500 Da.

The present invention further provides an application of fucosyl-disaccharide in promoting the proliferation of probiotics.

The present invention discloses the following technical effects: Fucosyl-disaccharide preparation technology of the present invention has the advantages of simple operation, low cost, easy expansion of production and high yield of the fucosyl-disaccharide.

The fucosyl-disaccharide provided by the present invention has a novel structure, high purity and high yield, can be fermented and utilized by intestinal probiotics, can reduce the pH value of intestinal environment, can promote proliferation of Akkermansia muciniphila and a plurality of bifidobacteria strains including Bifidobacterium infantis, and has broad application prospects as new prebiotics.

DESCRIPTION OF THE FIGURES FIG.1 shows a Gram Staining microscopic view of Enterobacter in Embodiment 1; FIG.2 shows an infrared spectrum of Enterobacter EPS in Embodiment 3; FIG.3 shows a GPC view of the molecular weight of Enterobacter EPS in Embodiment 3; FIG.4 shows a high performance liquid chromatography (HPLC) chromatogram of monosaccharide compositions of FCOs in Embodiment 5;

FIG.5 shows a mass spectrum of FCOs in Embodiment 5; FIG.5-A shows an electrospray lonization-mass spectrometry (ESI-MS) spectra in positive ion mode, and FIG.5-B shows an ESI-CID (collision-induced dissociation) -MS/MS spectra in positive ion mode;

FIG.6 shows a 1D 'H nuclear magnetic resonance (NMR) spectrum of fucosyl- disaccharide in Embodiment 5;

FIG.7 shows a 1D '3C NMR spectrum of fucosyl-disaccharide in Embodiment 5;

FIG.8 shows a 2D NMR 'H -'H TOCSY spectrum of fucosyl-disaccharide in Embodiment 5;

FIG. 9 shows a 2D NMR 'H -3C HSQC spectrum fucosyl-disaccharide in Embodiment 5;

FIG.10 shows 2D NMR 'H -3C HMBC spectrum of fucosyl-disaccharide in Embodiment 5;

FI1G.11 shows the promoting effect of fucosyl-disaccharide and 2'-FL prebiotic on the proliferation ability- and acid-producing ability of four probiotics strains of Embodiment 6, FIG.11-A shows Bifidobacterium breve, FIG.11-B shows Bifidobacterium infantis, FIG.11-C shows Bifidobacterium bifidum, and FIG.11-D shows Akkermansia muciniphila;

FIG. 12 shows promotion of fucosyl-disaccharide and 2'-FL prebiotics on short-chain fatty acids production of four probiotics strains of Embodiment 6, F1G.12-A shows Bifidobacterium breve, F1G.12-B shows Bifidobacterium infantis, FIG.12-C shows Bifidobacterium bifidum, and FIG.12-D shows Akkermansia muciniphila.

DESCRIPTION OF THE INVENTION The embodiments of the present invention are further described below in conjunction with the figures. The detailed description should not be considered as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.

It should be understood that the terms described in the present invention are only used to describe specific embodiments and are not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between an upper limit and a lower limit of the range is also specifically disclosed. Each smaller range between an intermediate value within any stated value or a stated range and an intermediate value within any other stated value or the stated range is also included in the present invention. Upper and lower limits of these smaller ranges can be independently included or excluded from the range.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by the person skilled in the art in the field of the present invention. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated document, the contents of this manual shall prevail.

Without departing from the scope or spirit of the present invention, it is obvious to the person skilled in the art that various improvements and changes can be made to the specific embodiments of the present specification. Other embodiments derived from the specification of the present invention will be obvious to the person skilled in the art. The specification and embodiments of this application are only exemplary.

As used herein, "comprise", "include", "have", "contain", etc., are all open terms, which means including but not limited thereto.

Embodiment 1: Screening and identification of Enterobacter Sewage samples were collected from a sewage treatment plant in Shinan District, Qingdao City, Shandong Province. Sewage sample (30mL) was collected and centrifuged at 8000 r/min for 20 min at 4 °C. The deposit was re-suspended by using an appropriate amount of PBS solution, and coated on a nutrient agar plate by a coating method, and incubated at 37 °C overnight. After cultivation, 20 single bacterial colonies were picked from the plate, and numbered from 1$-to 20". The 20 single bacterial colonies were enriched using LB liquid medium, respectively, and cultivated at 37 °C for 24 h.

The DNA samples of 20 bacteria was extracted according to the method of a bacterial genome extraction kit. The DNA was used as a template. 16S rDNA universal primer 27F (shown in SEQ ID NO. 1) of 5" AGAGTTTGATCCTGGCTCAG-3' and 1492R (shown in SEQ ID No. 2) of 5" ATTACCGCGGCTGCTGGC-3' were used for colony PCR. The PCR reaction system are as follows: 8 uL Tag enzyme, 10 pL sterile water, 0.5 uL each primer, and 1 pL template. PCR reaction conditions are as follows: (1) pre-denaturation at 95 °C for 5 min; (2) denaturation at 95 °C for 30 s, annealing at 52 °C for 45 s, extension at 72 °C for 2 min, a total of 32 cycles; and (3) re-extension at 72 °C for 1 min. After amplification was ended, PCR amplified products were sent to a biological company for sequencing. Sequencing results were compared by Blast. The results showed that one strain was Enterobacter. The sequencing results were shown as SEQ ID NO. 3, and the microscopic examination results also showed that these bacteria were Gram-negative bacteria in shape of bacilli, as shown in

FIG.1, that is, enterobacteria that were successfully isolated from the sewage. This Enterobacter was named Enterobacter F-CE2 and preserved in China General Microbiological Culture Collection Center, No. 3, 1% Yard, Beichen West Road, Chaoyang District, Beijing, and had a preservation number of CGMCC No. 20359 and a preservation date of July 14, 2020.

Embodiment 2: The optimization of preparation conditions of Enterobacter exopolysaccharide After Enterobacter F-CE2 was activated with a LB liquid medium for three generations, the Enterobacter F-CE2 was inoculated into a liquid fermentation medium with inoculation amount of 0.5% (V/V). The medium was placed in a shaker for shaking and culture at 32° C with rotation speed of 150 r/min for 48 h. After fermentation, the fermentation broth was centrifuged at 6000 r/min for 15 min to remove bacterial precipitates and collect the supernatant. The supernatant was rotated and evaporated to the volume of 1/5 of the initial volume in a rotary evaporator at 50 °C. Then supernatant was precipitated with 3 times volume of 95% ethanol, and let it stand for 4 h. Precipitates were collected after centrifugation at 6000 r/min for 10 min. The precipitates were redissolved with ultrapure water, dialyzed in a 10 kDa dialysis bag for 48 h, and lyophilized in vacuum. Yield of EPS was calculated. The EPS were pre-treated by a PMP (1-phenyl-3-methyl-5- pyrazolone) pre-column derivatization method, and then performed with HPLC of monosaccharide compositions. Fucose content was calculated. Results showed that Enterobacter EPS had a yield of 3~6.5 g/L, and fucose in the EPS had a molar ratio of 30%-45% (see Tables 2 and 4).

The specific operation method of HPLC was as follows: Sample of 5 mg was accurately weighed and placed in an ampoule tube, and 1 mL of trifluoroacetic acid (TFA) with concentration of 2 mol/L was added. The tube was sealed and placed in an oven for hydrolysis at temperature of 110 °C for 6 h. The hydrolyzed sample was evaporated and concentrated, and was redissolved using methanol for several times to remove TFA. A mixed standard monosaccharides sample was prepared in an equimolar ratio and was derived with PMP along with the acidolysis products of oligosaccharide sample. A derivation method was as follows: taking 100 pL of a sample mixture solution to be tested and mixing with 210 pL of 0.3 mol/L NaOH solution, adding 200 pL of 0.5 mol/L PMP solution (dissolved in methanol), mixing the solution evenly, placing the mixture in 70 °C oven and reacting for 60 min; after the reaction, cooling at room temperature for 10 min, and adding 210 pL of 0.3 mol/L HCI to neutralize the excessive alkali. The mixed solution was added with 3 mL of dichloromethane, and was shaken and let it stand. The lower layer was discarded after the mixed solution was layered. The extraction process was repeated for 3 times. The aqueous phase was filtered with a 0.22 um microporous membrane and then analyzed by HPLC method.

HPLC instrument: Agilent 1260 high performance liquid chromatograph; UV detector (245 nm) HPLC column model: Agilent ZORBAX 300 XDB-C18 HPLC conditions: 0.05 mol/L KH2PO4 phosphate buffer (pH 6.7) / CH3CN (83:17, V:V) was used as a mobile phase; the column temperature was 35°C; the flow rate was 1 mL/min; and the sample volume was 20 pL.

The liquid fermentation medium was shown in Table 1 below: Table 1 Beef extract Yeast extract Inorganic (g/L) (g/L) Peptone (g/L) Glucose (g/L) salt © 5 6 10 30 V © 10 6 10 30 V ©) 15 6 10 30 V © 10 2 10 30 V © 10 4 10 30 V ® 10 6 20 30 V D 10 6 15 30 V 6 10 20 V © 10 6 10 40 V Inorganic salt formula were as follows: NH4CI of 0.3 g/L, KCI of 1 g/L, (NH4)2S04 of 0.3 g/L, NazHPO4 of 10g/L, KH2PO4 of 3 g/L, KoSO4 of 1 g/L, NaCl of 1 g/L, MgSO4* 7H20 of 0.2 g/L, CaCl2+6H20 of 0.02 g/L, and FeSO4 of 0.001 g/L. The pH of the medium was adjusted to 7.0~7.2.

Table 2 showed the yields and the fucose contents of Enterobacter EPS prepared using different fermentation media in Embodiment 2. Table 2 Medium © ©@ © @ © © © 6 © EPS yield (g/L) 4.5 65 65 48 5.4 5.8 6.5 3 6.5 Fucose content 43.13 45 45 3874 4054 3994 4429 302 4265 (% mol) Table 3 showed the optimizing culture condition parameters of fermentation medium of Enterobacter: Table 3 Temperature C Rotation Speed r/min Time h © 27 150 48 © 32 150 48 ©) 38 150 48 © 32 120 48 © 32 180 48 © 32 150 24 D 32 150 72 Table 4 showed the yields and fucose content of Enterobacter EPS prepared under different fermentation and culture conditions in Embodiment 2 Table 4 Culture conditions © © ©) © © © © EPS yield (g/L) 5.9 65 55 6.3 6.3 4.1 6.4 Fucose content (%mol) 45 45 4327 4418 4416 4237 4428 Embodiment 3: Determination of basic properties of EPS According to Embodiment 2, conditions of Enterobacter fermentation medium were optimized, and culture conditions with the highest EPS yield and fucose content were selected. The final medium formula was set as follows: beef extract of 10 g/L, yeast extract of 6 g/L, peptone of 10 g/ L, glucose of 30 g/L, NH4Cl of

0.3 g/L, KCI of 1 g/L, (NH4)2SQO4 of 0.3 g/L, Na:HPO4 of 10 g/L, KH2PO4 of 3 g/L, K25SO4 of 1 g/L, NaCl of 1 g /L, MgSO4+7H20 of 0.2g/L, CaCl2*6H20 of 0.02 g/L, and FeSO, of 0.001 g/L. The pH of the medium was adjusted to 7.0~7.2. The medium was incubated at 32 °C and speed of 150 r/min for 48 h. As shown in Table 5, monosaccharide compositions of EPS prepared under optimal conditions included the fucose, glucose, galactose, glucuronic acid, galacturonic acid, mannose, and rhamnose, among which the fucose has mole ratio of about 45.02%.

Table 5 showed a molar ratio of monosaccharide compositions of EPS in Embodiment 3 Table 5 Monosaccharide Mol% Fucose 45.02% + 0.01 Glucose 20.91% + 0.01 Galactose 22.69% + 0.04 Glucuronic acid 4.09% + 0.04 Galacturonic acid 3.71% + 0.03 Mannose 1.86% + 0.03 Rhamnose 1.12% + 0.01 In this experiment, a KBr tablet method was used to measure the sample by infrared spectroscopy. The sample has the following processing method: based on the mass ratio 1:200 of the sample to KBr, about 2-3 Mg sample and 100-600 mg KBr were weighed, transferred to an agate mortar and ground evenly into a powder. The ground powder was transferred to a film-making mold and was placed under pressure of 10 tons for 2 min. After removing the pressure, the piece was taken for the next test. The prepared test piece was visually inspected to be transparent. The test piece was taken out and loaded into a sample holder for infrared spectroscopy analysis, and the model of used instrument was Nicolet Nexus 470 infrared spectrometer. As shown in FIG.2, FIG.2 showed a view of infrared spectrum of Enterobacter EPS. It can be seen that the sample had characteristic absorption peaks at 3427.69, 2926.56, 1639.69, and 1404.60 cm, respectively, which were characteristic absorption peaks of stretching and vibration of characteristic absorption peaks produced by stretching and vibration of O-H, C-H, C=0, and C-O of the polysaccharide. The absorption peaks at 1067.13 cm! and

1026.77 cm indicated the presence of pyranose in polysaccharide, which indicated that the polysaccharide has a carboxyl structure. This result was consistent with the results of HPLC which showed the presence of uronic acid.

The molecular weight of EPS was determined by HPLC system, a refractive index detector, and a TSK gel G4000 PWXL chromatographic column. The system temperature was set to 35°C. The mobile phase consists of 0.2 mol/L NaNO3 and

0.01 mol/L NaH2PO4 was used. The flow rate was 0.5 mL/min and the sampling volume was 20 pL. Different molecular weight dextran (80, 150, 270, 410 and 670 kDa) were used to draw standard curves. Results were shown in FIG. 3. FIG.3 showed a GPC spectrum of molecular weight of Enterobacter EPS with a peaked at 19.674 min. According to standard curve calculation, the EPS had a molecular weight of 5.2x10° Da.

Embodiment 4: Preparation of FCOs through acidolysis at high temperature and at high pressure Conditions of the acidolysis by high pressure were shown in Table 6. The EPS of Enterobacter F-CE2 was dissolved in ultrapure water to prepare a 1~5% polysaccharide solution. Hydrochloric acid was added to the polysaccharide solution. The pH was adjusted to 2~4, the temperature was 130~150 °C and the pressure was 0.1~0.3 Mpa., Treatment was performed for 2~5 h. The pH was adjusted to 7.0 with 2 mol/L NaOH. The obtained oligosaccharide solution was centrifuged at 5000 r/min for 10 min. Precipitates were dried and weighed, and the supernatant was collected. A dialysis bag with molecular weight cut-off of 500 Da was used to intercept the supernatant to obtain an oligosaccharide sugar solution with a molecular weight of less than 500 Da. The sugar solution was lyophilized into a powder and weighed. Yields of EPS oligosaccharide were shown in Table 7. Results showed that oligosaccharide with a molecular weight of less than 500 Da had a yield of 50%~70% after the acid treatment of Enterobacter EPS in an environment at high temperature and at high pressure. Optimal reaction conditions were as follows: polysaccharide concentration of 30 mg/ml, pH of 3, vacuum degree of 0.2 Mpa and treatment for 4~5 h.

Table 6 showed conditions for acidolysis of Enterobacter EPS at high- pressure in Embodiment 4.

Table 6 Serial Polysaccharide Vacuum Degree ; ; Number Concentration mg/mI pH Mpa Reaction Time h © 10 3 0.2 4 © 30 3 0.2 4 ©) 50 3 0.2 4 © 30 4 0.2 4 © 30 2 0.2 4 © 30 3 0.3 4 D 30 3 0.1 4 3 0.2 2 © 30 3 0.2 5 Table 7 showed yields of acidolysis of Enterobacter EPS oligosaccharide at high-pressure in Embodiment 4.

Table 7 Conditions ® @ ® @ ® ® @ © Yields of66.74% 70.24% 56.61% 68.75% 60.19% 57.24% 70.18% 53.25% 70.24% oligosaccharide Embodiment 5: Analysis of FCOs Structure (1) Analysis for monosaccharide compositions of FCOs Monosaccharide compositions of the FCOs were detected according to the method of HPLC analysis in Embodiment 2. Results were shown in FIG.4. FIG.4 showed a HPLC spectrum of the monosaccharide compositions of FCOs. It can be seen that FCOs consists of glucose, galactose, and fucose, with a ratio of 1:1:2.

(2) Mass spectrometry analysis of FCOs The analysis was performed using Agilent 1290 Infinity || system equipped with Agilent 6460 triple quadrupole mass spectrometer. Positive and negative ion ESI-MS and ESI-CID-MS/MS were used to identify the molecular weight of FCOs. The mobile phase was acetonitrile/water (1:1, V/V), the flow rate was 0.4 mL/min, the atomizer pressure was 40 psi, the capillary voltage was 3500 V, the collision gas was argon, and the collision energy was 10-55 eV. FIG.5 showed mass spectrum results of the oligosaccharides sample; FIG.5-A showed a positive ion mode ESI-MS, and FIG.5-B showed a positive ion mode ESI-CID-MS/MS. It can be seen that the FCO was a fucosyl-disaccharide with a molecular weight of 326 Da. Combined with results of monosaccharide composition, the fucosyl- disaccharide may be a disaccharide consisting of glucose and fucose, or a disaccharide consisting of galactose and fucose.

(3) NMR analysis of FCOs For NMR analysis of FCOs, 10 mg of a fucosyl-disaccharide sample was dissolved in 0.5 mL DO and lyophilized. The sample was then redissolved in 0.5 mL of D2O, and acetone was added to be used as an internal standard for the calibration of chemical shift. All one-dimensional (*H, ‘°C, DEPT90°, 135.) and two- dimensional ('H-'H COSY, "H-H TOCSY, 'H-'*C HSQC, 'H-*C HMBC) NMR spectra were recorded using Agilent DD2-500 spectrometer with frequency of 500MHz at 25°C. FIG.6 to 10 showed the NMR results of fucosyl-disaccharide. FIG.6 shows the 1D 'H NMR spectrum. FIG.7 shows the 1D "°C NMR spectrum.

FIG.8 shows the 2D NMR 'H -'H TOCSY spectrum.

FIG.9 shows the 2D NMR ‘H -13C HSQC spectrum.

FIG.10 shows 2D NMR 'H -*C HMBC spectrum; Table 8 showed chemical shifts of 'H and '*C of each monosaccharide residue of the fucosyl-disaccharide.

The results showed that the fucosyl-disaccharide had a structure of B-D-Glcp-(1—4)-B-L-Fucp and a-D-Galp-(1—3)-B-L-Fucp.

Table 8 showed the 'H and "°C chemical shifts of each monosaccharide residue of fucosyl-disaccharide in Embodiment 5. Table 8 1 2 3 4 5 6 a-D-Galp-(1—3)-B-L-Fucp (a) D-Gal ‘H 5.256 3.798 4.032 3.864 3.491 3.759/3.548 BC 104.01 73.512 70.059 70.896 72.546 62.678 (B)L-Fuc 'H 4.425 3.802 3.503 3.701 23.902 1.275 BC 94.259 70.592 77.896 71.643 68.514 17.321 B-D-Glcp-(1—4)-B-L-Fucp (B)D-Glc IH 4.542 3.526 3.773 3.996 3.543 3.891/3.589 BC 97.928 75.695 77.581 71.090 77.022 64.521 (B)L-Fuc H 4.603 3.512 3.586 3.799 3.856 1.213 13C 103.896 73.546 75.123 81.134 68.409 17.254 Embodiment 6: Probiotic effect of fucosyl-disaccharide In order to explore the probiotic effect of the fucosyl-disaccharide on intestinal microbiota, an experimental process simulated an intestinal anaerobic fermentation environment was used to explored the proliferation effects of the fucosyl-disaccharide on Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium bifidum, and Akkermansia muciniphila, and the substrate utilization efficiency of fucosyl-disaccharide.

Deoxygenated YCFA fermentation medium was used with a single bacteria inoculation amount of 0.5%. Fermentation was conducted at 37 °C for 48 h.

The fermentation products were taken at 0, 12, 24, 36 and 48 h, respectively, for subsequent analysis.

Results were shown in FIG.11. FIG.11 showed the proliferation-promoting effects of fucosyl-disaccharide and 2'-FL prebiotics on four probiotics strains and strain’s acid-producing capacity.

F1G.11-A showed the Bifidobacterium breve.

FIG.11-B showed the Bifidobacterium infantis.

FIG.11-C showed the Bifidobacterium bifidum, and F1G.11-D showed the Akkermansia muciniphila.

Compared with 2'-FL, the fucosyl-disaccharide significantly promoted proliferation of Bifidobacterium breve, Akkermansia muciniphila, Bifidobacterium infantis, and Bifidobacterium bifidum (p<0.05), and the pH value could be reduced within 12 h (reduction by 1 to 2). After the fermentation for 24 h, compared to 2'-FL, the fucosyl-disaccharide can promote bifidobacteria strains to produce more formic acid, acetic acid and lactic acid.

The fucosyl-disaccharide can promote the Akkermansia muciniphila to produce a low level of butyric acid, while 2'-FL cannot.

As shown in FIG.12, FIG.12 showed results of the fucosyl-disaccharide and 2'-FL prebiotics in promoting production of short-chain fatty acids of four probiotics strains.

FIG.12-A showed the Bifidobacterium breve.

FIG.12-B showed the Bifidobacterium infantis.

FIG. 12-C showed the Bifidobacterium bifidum, and FIG.12-D showed the Akkermansia muciniphila.

It should be understood that the person skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the protection scope of the appended claims of the present invention.

Claims (10)

1.Fucosyl-disaccharide with prebiotic effect, wherein the fucosyl-disaccharide has a structure of B-D-Glcp-(1—4)-B-L-Fucp and a-D-Galp- (1—3)-B-L-Fucp.

2. A method for preparing fucosyl-disaccharide with prebiotic effect according to claim 1, wherein the method specifically comprises the following steps: (1) fermenting Enterobacter F-CE2 to extract extracellular polysaccharide (EPS); (2) preparing a solution with the EPS obtained in step (1) and performing acidolysis for the EPS at high temperature and at high pressure to obtain degraded sugar solution; (3) performing ultrafiltration for the degraded sugar solution obtained in step (2) to remove undegraded macromolecular sugars, and then lyophilizing the obtained filtrate solution to obtain the fucosyl-disaccharide.

3. The method according to claim 2, wherein the Enterobacter F-CE2 of step (1) has a preservation number of CGMCC No. 20359, was preserved on July 14, 2020 in China General Microbiological Culture Collection Center, No. 3, 1% Yard, Beichen West Road, Chaoyang District, Beijing, the Enterobacter F-CE2 has mole ratio of 30%-45% of fucose in the EPS.

4. The method according to claim 2, wherein a specific preparation method of the EPS in step (1) comprises: after the third activation of Enterobacter F-CE2, the Enterobacter F-CE2 is inoculated into a fermentation medium, after shaking and fermentation, a resulting fermentation broth is centrifuged at 6000 r/min for 15 min to collect a supernatant, the resulting supernatant is rotated, evaporated and concentrated, after the concentration, the supernatant is precipitated with three times volume of 95% ethanol to precipitate the EPS, and the EPS is collected, then dissolved in ultrapure water, dialyzed in a dialysis bag for 48 h and then vacuum dried.

5. The method according to claim 4, wherein the fermentation medium has the following formula: beef extract of 5-15 g/L, yeast extract of 2-6 g/L, peptone of 10~20 g/L, glucose of 20- 40 g/L, NH4Cl of 0.3 g/L, KCI of 1 g/L, (NHa)2SO4 of 0.3 g/L, NazHPO4 of 10 g/L, KH2PO4 of 3 g/L, K2SO4 of 1 g/L, NaCl of 1 g/L, M9gSO4 *7H20 of 0.2 g/L, CaCl2*6H20 of 0.02 g/L, and FeSO4 of 0.001 g/L, pH is 7.0~7.2.

6. The method according to claim 4 or 5, wherein the fermentation medium has an inoculum volume of 0.1% to 1% (V/V), fermentation temperature of 27 °C to 38 °C, shaking speed of 120 r/min~180 r/min, and fermentation time of 24 h~72 h.

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

8. The method according to claim 2, wherein an EPS solution in step (2) has the following conditions for acidolysis: adding hydrochloric acid to the EPS solution, adjusting pH to 2~4, and having treating time of 2 to 5 h, and concentration of 10 to 50 mg/ml.

9. The method according to claim 2, wherein the molecular weight of the degraded sugar solution obtained by ultrafiltration in step (3) is less than or equal to 500 Da.

10. An application of fucosyl-disaccharide of claim 1 in promoting the proliferation of probiotics.