Preparation method and application of blackberry oligosaccharide

Abstract

The invention discloses a preparation method of blackberry oligosaccharide, which comprises the following steps: dehydrating blackberry, grinding into powder, dissolving in water, mixing with ethanol, carrying out rotary evaporation and freeze-drying, sequentially carrying out protein removal and pigment removal, respectively carrying out elution by using DEAE-Sepharose FF ion layer coefficient and G100 chromatographic column, collecting the eluent, and carrying out freeze-drying to obtain the blackberry oligosaccharide. The blackberry oligosaccharide prepared by the preparation method is complete in component preservation, clear in structure and controllable in quality, and meanwhile, the prepared blackberry oligosaccharide also has the functions of promoting the growth of probiotics and improving the activity of the probiotics. Therefore, the blackberry oligosaccharide with a brand new effect is extracted from the blackberries, and more basis is provided for application of the blackberries in the food field.

Description

Technical Field

[0001] The present invention relates to the technical field of oligosaccharide synthesis, and specifically, to a preparation method and application of blackberry oligosaccharides. Background Art

[0002] Blackberry belongs to the subgenus Eubatus of the genus Rubus in the family Rosaceae, also known as the subgenus Rubus. As the blackberry fruit matures, its color gradually changes from green to red, and when it is fully mature, the fruit is purple-black or purplish-red, and its fruit shape presents a conical, cylindrical or oblong spherical shape due to different qualities.

[0003] Blackberry fruits mainly contain a large number of bioactive components such as polyphenols, flavonoids, anthocyanins, polysaccharides, oligosaccharides, volatile oils and trace elements, and have high nutritional and medicinal values; polysaccharides, as the main active substances in blackberry fruits, have been reported to have effects such as oxidation, anticoagulation, antithrombosis and immune regulation. Polysaccharides are usually long-chain macromolecular polymers formed by more than 10 monosaccharide molecules connected by glycosidic bonds, and the molecular weight is usually between several thousand and several million daltons; oligosaccharides are usually formed by 2-10 monosaccharide molecules connected by glycosidic bonds, and the molecular weight is mainly concentrated between 200 and 2000 daltons.

[0004] Currently, blackberry oligosaccharides are usually extracted from pectin or polysaccharides by enzymatic hydrolysis or fractional alcohol precipitation techniques. Currently, blackberry oligosaccharides have been verified to have antioxidant and anti-inflammatory activities, but polysaccharides or oligosaccharides with different molecular weights obtained by different extraction methods also have completely different functions. Therefore, the research on blackberry oligosaccharides mainly focuses on providing blackberry oligosaccharides with new effects and providing corresponding extraction methods. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above deficiencies of the prior art and provide a preparation method and application of blackberry oligosaccharides.

[0006] The first object of the present invention is to provide a preparation method of blackberry oligosaccharides.

[0007] The second object of the present invention is to provide the blackberry oligosaccharides prepared by the above preparation method.

[0008] The third object of the present invention is to provide the application of the above blackberry oligosaccharides in the preparation of products for promoting the growth and / or enhancing the activity of probiotics.

[0009] In order to achieve the above object, the present invention is realized by the following solutions:

[0010] The present invention claims to protect a preparation method of blackberry oligosaccharides, comprising the following steps:

[0011] S1. Dehydrate blackberries and grind them into powder, then dissolve the powder in water. After thorough mixing, separate the solid from the liquid and collect the supernatant.

[0012] S2. Mix the supernatant obtained in step S1 with ethanol, react thoroughly, rotary evaporate the ethanol, collect the liquid and freeze-dry it to obtain a freeze-dried sample.

[0013] S3. Remove protein and pigment from the freeze-dried sample obtained in step S2 in sequence to obtain crude blackberry oligosaccharides.

[0014] S4. Dissolve the crude blackberry polysaccharides obtained in step S3 in water, then load them onto a DEAE-Sepharose FF ion chromatography column for elution until no sugar is detected in the eluate, and collect the eluate to obtain fraction 1.

[0015] S5. Dissolve fraction 1 obtained in step S4 in water, then load it onto a G100 chromatography column, elute it thoroughly with deionized water, collect the eluate, and freeze-dry it to obtain blackberry oligosaccharides.

[0016] Preferably, in step S1, the blackberries are dehydrated and ground into powder with a mesh size of ≤40.

[0017] Preferably, after thorough mixing in step S1, ultrasonic treatment is also required.

[0018] More preferably, the conditions for ultrasonic treatment are 40KHZ and 150W.

[0019] Preferably, in step S2, the ethanol is ethanol with a volume concentration of 70%.

[0020] Preferably, in step S2, the supernatant and ethanol are mixed evenly at a volume ratio of 2 - 3:10.

[0021] More preferably, the supernatant and ethanol are mixed evenly at a volume ratio of 1:4.

[0022] Preferably, the protein removal in step S3 is specifically as follows:

[0023] The precipitate obtained in step S2 is freeze-dried and mixed evenly with Sevage reagent at a volume ratio of 2.5 - 3.5:1. After thorough reaction, centrifuge and collect the aqueous phase to obtain a protein-free mixture; the Sevage reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 3.5 - 4.5:1.

[0024] More preferably, the precipitate obtained in step S2 is freeze-dried and mixed evenly with Sevage reagent at a volume ratio of 3:1.

[0025] More preferably, the Sevage reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 4:1.

[0026] More preferably, the centrifugation is carried out at 4000 r / min for 10 min.

[0027] Preferably, in step S3, the decolorization is to remove pigments by macroporous resin adsorption.

[0028] More preferably, the macroporous resin is AB-8 type macroporous resin.

[0029] Further preferably, the AB-8 type macroporous resin is the activated AB-8 type macroporous resin.

[0030] Preferably, the elution in step S4 is specifically: eluting successively with deionized water, 0.2 M NaCl and 0.4 M NaCl.

[0031] More preferably, eluting successively with deionized water, 0.2 M NaCl and 0.4 M NaCl according to 2 - 3 BV (2 - 3 times the volume flux).

[0032] More preferably, the flow rate during the elution in step S4 is 0.8 - 1.2 mL / min.

[0033] Further preferably, the flow rate is 1 mL / min.

[0034] Preferably, the flow rate during the elution in step S5 is 0.8 - 1.2 mL / min.

[0035] More preferably, the flow rate is 1 mL / min.

[0036] The present invention purifies and separates blackberries, and then obtains blackberry oligosaccharides, and conducts structural identification on the extracted blackberry oligosaccharides, showing that the molecular weight of the shown blackberry oligosaccharides is 626 Da. At the same time, the extracted blackberry oligosaccharides also show the effects of promoting the growth of probiotics and improving the activity of probiotics.

[0037] Therefore, the present invention also requests protection for the blackberry oligosaccharides prepared by any of the above - mentioned preparation methods.

[0038] Among them, the blackberry oligosaccharides prepared by any of the above - mentioned preparation methods are composed of glucose, galactose and arabinose, and the molar mass ratio of glucose, galactose and arabinose is 98.64:0.96:0.40.

[0039] The present invention also requests protection for the application of the above - mentioned blackberry oligosaccharides in the preparation of products for promoting the growth of probiotics and / or improving the activity of probiotics.

[0040] Preferably, the product is food.

[0041] Preferably, the probiotics are lactic acid bacteria and / or bifidobacteria.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention provides a preparation method of blackberry oligosaccharides. After dehydrating blackberries and grinding them into powder, the powder is dissolved in water, then mixed with ethanol, rotary evaporated and freeze-dried. Protein and pigment removal are carried out in sequence. Then, elution is carried out using DEAE-Sepharose FF ion layer coefficient and G100 chromatography column respectively. The eluate is collected and freeze-dried to obtain blackberry oligosaccharides. The blackberry oligosaccharides prepared by the preparation method have complete components, clear structure and controllable quality. At the same time, the prepared blackberry oligosaccharides also show the functions of promoting the growth of probiotics and improving the activity of probiotics. Therefore, the present invention extracts blackberry oligosaccharides with new effects from blackberries, providing more basis for the application of blackberries in the food field. Description of the Drawings

[0044] Figure 1 For DEAE-Sepharose FF column chromatography (A) and G100 gel column elution curve (B) in Example 2

[0045] Figure 2 For the monosaccharide component determination results in Example 2; A is the chromatogram of 13 monosaccharide standards; B is the chromatogram of blackberry oligosaccharides;

[0046] Figure 3 For the molecular weight determination results in Example 2;

[0047] Figure 4 For the spectral detection results in Example 2; A is the infrared spectrum of blackberry oligosaccharides; B is the ultraviolet spectrum detection results of blackberry oligosaccharides;

[0048] Figure 5 For the OD600 test results of each culture group at each culture time in Example 3;

[0049] Figure 6 For the total sugar content test results of each culture group at each culture time in Example 3;

[0050] Figure 7 For the short-chain fatty acid content test results of each culture group at each culture time in Example 3; A is the acetic acid content test result; B is the propionic acid content test result; C is the butyric acid content test result;

[0051] Figure 8Graph showing the results of quantitative PCR detection for each culture group in Example 3; A is the graph of the fluorescence quantitative PCR detection results for all bacteria in each culture group; B is the graph of the fluorescence quantitative PCR detection results for lactic acid bacteria in each culture group; C is the graph of the fluorescence quantitative PCR detection results for Bifidobacterium in each culture group. Detailed implementation method

[0052] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0053] Example 1 Preparation of blackberry oligosaccharides

[0054] I. Experimental method

[0055] The preparation method of blackberry oligosaccharides includes the following steps:

[0056] S1. Dehydrate blackberries and grind them into blackberry powder with a particle size of ≤40 mesh. Mix the blackberry powder and water at a ratio of 1 g:10 mL to obtain mixture 1. Place mixture 1 in an ultrasonic machine and control the ultrasonic conditions to be 40 KHZ and 150 W for ultrasonic treatment to break the cell walls in the blackberry powder and obtain mixture 2;

[0057] Centrifuge mixture 2 at 4000 rpm for 10 min and collect the supernatant;

[0058] S2. Mix the supernatant obtained in step S1 with ethanol with a volume concentration of 70% at a volume ratio of 1:4, place it at 4°C for 12 h, perform solid-liquid separation, rotary evaporate the ethanol from the supernatant, collect the aqueous solution and freeze-dry it to obtain a freeze-dried sample;

[0059] S3. Mix the precipitate freeze-dried in step S2 and Sevage reagent (prepared by mixing chloroform and n-butanol at a volume ratio of 4:1) at a volume ratio of 4:1, place it on a shaker and shake at 120 rpm for 25 min, then centrifuge at 4000 r / min for 10 min to separate aqueous phase 1 and remaining liquid 1;

[0060] Repeat the following steps for remaining liquid 1 until only the aqueous phase remains after centrifugation: Mix remaining liquid 1 and Sevage reagent at a volume ratio of 4:1, place it on a shaker and shake at 120 rpm for 25 min, then centrifuge at 4000 r / min for 10 min to separate aqueous phase 2 and remaining liquid 2;

[0061] Combine aqueous phase 1 and aqueous phase 2 to obtain mixture 3;

[0062] S4. Activate the AB-8 macroporous resin with HCl and NaOH according to its instructions to obtain an activated macroporous resin; mix the mixed solution 3 obtained in step S3 and the activated AB-8 macroporous resin in a volume ratio of 7:1, place on a shaker and shake at 120 rpm for 3 hours, then filter, collect the mixed solution 4 and freeze-dry to obtain blackberry crude oligosaccharides;

[0063] S5. 0.5 g of the crude blackberry polysaccharide obtained in step S4 was dissolved in 5 mL of ultrapure water, filtered using a 0.45 μm microporous filter membrane, and then applied to a DEAE-Sepharose FF ion chromatography column. 3 BV (3 times the flux) of deionized water, 0.2 M NaCl and 0.4 M NaCl were used for elution in sequence. During the elution process, the flow rate was controlled to be 1 mL / min, and one tube of eluate was collected for every 10 mL until no sugar was detected in the eluate. The eluate was collected and freeze-dried to obtain component 1; at the same time, the elution curve of the DEAE-Sepharose FF ion chromatography column was recorded;

[0064] S6. Dissolve 0.5 g of component 1 obtained in step S5 in 5 mL of ultrapure water, filter with a 0.45 μm microporous filter membrane, apply it to a G100 chromatography column, and elute with 3 BV of deionized water. Collect one tube for every 10 mL of elution until no sugar is detected in the eluate. Collect the eluate, freeze-dry it, and obtain blackberry oligosaccharides; at the same time, record the elution curve of the G100 chromatography column.

[0065] 2. Experimental Results

[0066] The elution curve is shown in Figure 1 As shown, Figure 1 A in the figure is the elution curve of DEAE-SepharoseFF ion chromatography column. Figure 1 B in the figure is the elution curve of the G100 column. The results show that blackberry oligosaccharides are eluted from the 11th to 23rd tubes during the elution process of the G100 column.

[0067] Example 2 Structural Identification and Analysis of Blackberry Oligosaccharides

[0068] 1. Experimental Methods

[0069] According to the preparation method shown in Example 1, blackberry oligosaccharide was prepared and the following identification and analysis were performed:

[0070] 1. Determination of monosaccharide components

[0071] Take 2 mg of blackberry oligosaccharide and add it to 1 mL of TFA solution with a concentration of 2 M. Place it in an oven at 121 °C and heat for 2 h. Then, blow it dry with nitrogen, wash it with 99.99% (w / w) methanol, blow it dry again, repeat the washing process 3 times, dissolve it in 0.5 mL of sterile water after drying, and transfer it to a clean chromatographic vial for chromatographic detection. Record the chromatographic results;

[0072] Among them, the chromatographic conditions are as follows: Use a Thermo ICS5000 ion chromatographic system (ICS5000, Thermo Fisher Scientific, USA) to analyze and detect the monosaccharide components by electrochemical detection; use Dionex TM CarboPac TM PA20 (150*3.0 mm, 10 μm) liquid chromatographic column; the injection volume is 5 μL; mobile phase A is H2O, mobile phase B is 0.1 M NaOH, and mobile phase C is 0.1 M NaOH and 0.2 M NaAc; the elution gradient is: by volume percentage, at 0 min, 95% mobile phase A and 5% mobile phase B; at 26 min, 85% mobile phase A, 5% mobile phase B, and 10% mobile phase C; at 42 min, 85% mobile phase A, 5% mobile phase B, and 10% mobile phase C; at 42.1 min, 60% mobile phase A and 40% mobile phase C; at 52 min, 60% mobile phase A and 40% mobile phase B; at 52.1 min, 95% mobile phase A and 5% mobile phase B; at 60 min, 95% mobile phase A and 5% mobile phase B.

[0073] Then, perform chromatographic detection on 13 monosaccharide standards according to the above method and record the chromatographic results of each monosaccharide standard; the information of the 13 monosaccharide standards is shown in Table 1.

[0074] Table 1 Information of 13 Monosaccharide Standards

[0075] Name English Name Abbreviation CAS No. Molecular Formula Fucose Fucose Fuc 2438-80-4 <![CDATA[C6H 12 O5]]> Rhamnose Rhamnose Rha 10030-85-0 <![CDATA[C6H 14 O6]]> Arabinose Arabinose Ara 5328-37-0 <![CDATA[C5H 10 O5]]> Galactose Galactose Gal 26566-61-0 <![CDATA[C6H 12 O6]]> Glucose Glucose Glc 50-99-7 <![CDATA[C6H 12 O6]]> Xylose Xylose Xyl 58-86-6 <![CDATA[C5H 10 O5]]> Mannose Mannose Man 3458-28-4 <![CDATA[C6H 14 O6 <!-- 4 -->]]> Fructose Fructose Fru 57-48-7 <![CDATA[C6H 12 O6]]> Ribose Ribose Rib 50-69-1 <![CDATA[C5H 10 O5]]> Galacturonic Acid Galacturonic Acid Gal-UA 14982-50-4 <![CDATA[C6H 10 O7]]> Glucuronic Acid Glucuronic Acid Glc-UA 6556-12-3 <![CDATA[C6H 10 O7]]> Mannuronic Acid Mannuronic Acid Man-UA 6814-36-4 <![CDATA[C6H 10 O7]]> Guluronic Acid Guluronic Acid Gul-UA 15769-56-9 <![CDATA[C6H 10 O7]]>

[0076] 2. Molecular Weight Determination

[0077] Prepare an aqueous solution of blackberry oligosaccharide at 2 mg / mL (i.e., blackberry oligosaccharide solution), then filter it through a 0.22 μm microporous filter, and then through a 0.45 μm filter membrane, and use high performance liquid gel permeation chromatography (HPGPC) to test the molecular weight.

[0078] The HPGPC test conditions are as follows: High performance liquid gel permeation chromatography (waters 2695 type); differential refractive index detector (waters 2414); Ultrahydrogel IMLiner gel column; column temperature 35 °C; differential detector temperature 35 °C; injection volume 10 μL; mobile phase is deionized water, and the flow rate is 0.6 mL / min.

[0079] 3. Spectral Detection

[0080] After mixing 3 g of blackberry oligosaccharides evenly with 600 mg of KBr, a tablet was pressed and then infrared spectral detection was carried out. The infrared spectral detection was to measure the transmittance in the range of 400 - 4000 cm -1 using FTIP (Bruker TENSOR 27, Germany);

[0081] An aqueous solution of blackberry oligosaccharides with a concentration of 0.1 mg / mL was prepared, and then ultraviolet spectral detection was carried out in the range of 200 - 800 nm using an ultraviolet scanner.

[0082] 4. Methylation / GC-MS Analysis

[0083] 3 mg of blackberry oligosaccharides was added to 500 μL of DMSO solution, then 1 mg of NaOH was added. After incubation for 30 min, 50 μL of methyl iodide solution was added and reacted for 1 h. Then 1 mL of water and 2 mL of dichloromethane were added. After vortex mixing, the aqueous phase was discarded by centrifugation. After washing with water three times, the lower dichloromethane phase was aspirated and dried with nitrogen.

[0084] 100 μL of 2 M TFA was added to the dichloromethane phase, and the reaction was carried out at 121 °C for 90 min. Then it was evaporated to dryness at 30 °C. 50 μL of 2 M ammonia water and 50 μL of 1 M NaBD4 were added, and after mixing evenly, the reaction was carried out at 26 °C for 2.5 h. 20 μL of acetic acid was added to terminate the reaction, and it was dried with nitrogen. It was washed twice with 250 μL of methanol and dried with nitrogen. Then 250 μL of acetic anhydride was added, and after vortex mixing, the reaction was carried out at 100 °C for 2.5 h. 1 mL of water was added and left standing for 10 min, then 500 μL of dichloromethane was added. After vortex mixing, the aqueous phase was discarded by centrifugation. After washing with water three times, the lower dichloromethane phase was collected and subjected to GC-MS analysis on the instrument.

[0085] The analytical instrument used for GC-MS analysis was the 7890A - 5977B gas chromatograph - mass spectrometer of Agilent Technologies Inc. (CA, USA), and the model of the automatic sampler was G4567A;

[0086] The chromatographic parameters were as follows: Using an Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), the chromatographic column was BOX70 (30 m × 0.25 mm × 0.25 μm, SGE, Australia), the injection volume was 1 μL, the split ratio was 10:1, and the carrier gas was helium; the initial temperature of the column oven was 140 °C, held for 2 min, then heated to 230 °C at a rate of 3 °C / min and held for 3 min.

[0087] The mass spectrometry parameters were as follows: a quadrupole mass spectrometry detection system (Agilent 5977B; Agilent Technologies, USA) equipped with an electron impact ionization source (EI) and a MassHunter workstation was used; the electron impact ionization source (EI) was adopted, and detection was carried out in the full scan (SCAN) mode, with the mass scan range (m / z) being 50 - 350.

[0088] II. Experimental Results

[0089] 1. The results of the monosaccharide composition determination are shown in Figure 2 as follows. Figure 2 In Figure 2 A is the chromatogram of 13 monosaccharide standards, and

[0090] In Figure 2 B is the chromatogram of blackberry oligosaccharide. The results showed that based on the concentration of 13 monosaccharide standards as the abscissa and the peak area of the standards as the ordinate, the linear relationship between each monosaccharide and its peak area was determined from Figure 2 A in

[0091] Based on the linear relationship and combined with Figure 3 B in

[0092] it was found that blackberry oligosaccharide was composed of glucose, galactose, and arabinose with a molar ratio of 98.64:0.96:0.40.

[0092] 3. The results of spectral detection are shown in Figure 4 as follows. Figure 4 In Figure 4 A is the infrared spectrum of blackberry oligosaccharide, and

[0093] In -1 B is the ultraviolet spectrum detection result of blackberry oligosaccharide. The results showed that the strong absorption peak of blackberry oligosaccharide at 3383.08 cm -1 was attributed to the O - H stretching vibration of the sugar ring, the absorption peaks at 2931.74 cm -1 and 1404.58 cm -1 were attributed to the C - H stretching vibration, the absorption peak at 1616.32 cm -1 was attributed to the characteristic peak of the bound water adsorbed by the oligosaccharide, the absorption peak at 1055.04 cm -1 was attributed to the C - O - C stretching vibration on the pyranose ring, the absorption peak at 916.17 cm -1There is an absorption peak at the position of α-glycosidic bond. And the ultraviolet spectrum results show that there is no obvious absorption peak in the range of 200-800 mm of ultraviolet light, indicating that the blackberry oligosaccharides do not contain proteins.

[0094] 4. The GC-MS analysis results are shown in Table 2.

[0095] Table 2 GC-MS analysis results

[0096]

[0097]

[0098] The results show that the blackberry oligosaccharides prepared in Example 1, after hydrolysis, reduction and acetylation, contain sugar residues such as t-Glc(p), 3-Glc(p), 2-Glc(p), 6-Glc(p), 4-Glc(p), 3,4-Glc(p) and 4,6-Glc(p), indicating that the connection mode of carbon atoms in the α-D-glucose molecule of blackberry oligosaccharides is 3→4 connection, and the connection mode of carbon atoms in the β-D-glucose molecule is 4→6 connection.

[0099] Effect of blackberry oligosaccharides in Example 3 on the proliferation and growth of probiotics

[0100] I. Experimental method

[0101] Take fresh fecal samples from healthy c57 mice that are normally fed. After weighing, mix them with PBS buffer at a ratio of 1 g:5 mL to obtain fecal homogenate and store it at -80 °C for later use.

[0102] Preparation of growth medium: Dissolve 1.825 g of brain heart infusion broth (BHI) medium without glucose, 25 μL of resazurin and 0.05 g (0.1%, w / w) of L-cysteine hydrochloride in 50 mL of deionized water, boil in a water bath for 20 min. After the medium cools to 26 °C, add 0.1 mL of the mixed solution of hemin and vitamin K1;

[0103] The mixed solution of hemin and vitamin K1 is as follows: Dissolve 5 mg of hemin in 5 mL of 0.1 mol / L NaOH to obtain a hemin solution, dissolve 5 μL of vitamin K1 in 5 mL of absolute ethanol to obtain a vitamin K1 solution, and mix the hemin solution and the vitamin K1 solution in a volume ratio of 1:1 and filter through a 0.22 μm filter membrane to obtain the mixed solution of hemin and vitamin K1.

[0104] BO group medium (experimental medium): Add 10 mg of the blackberry oligosaccharides prepared in Example 1 and 5 mL of the growth medium to an anaerobic tube, and sterilize it in an autoclave at 121 °C for 20 min to obtain the BO group medium.

[0105] The difference between the Glu group medium (positive medium) and the BO group medium is that the blackberry oligosaccharide is replaced with an equal amount of glucose, and the other treatments are the same, thus obtaining the Glu group medium.

[0106] The difference between the Control group medium and the BO group medium is that the blackberry oligosaccharide prepared in Example 1 is not added, and the other treatments are the same, thus obtaining the blank group medium.

[0107] The fecal homogenate was inoculated into the media of different groups at an inoculation amount of 0.5% (v / v), and cultured in an anaerobic environment at 37°C for 48 h. Each medium group was inoculated in parallel 3 times.

[0108] At 0 h, 6 h, 12 h, 24 h, and 48 h of culture, 0.2 mL of the culture solution was sampled from each culture group to measure the OD600 value of each culture group at each culture time. At the same time, the total sugar content in the culture solution was measured by the phenol-sulfuric acid method. Then, the content of short-chain fatty acids in the culture solution was measured by gas chromatography (GC), and real-time fluorescence quantitative PCR (qPCR) was performed to measure the gene expression of lactic acid bacteria and bifidobacteria.

[0109] The specific method for measuring the content of short-chain fatty acids is as follows:

[0110] 0.3 mL of the culture solution was centrifuged at 4500 r / min for 10 min, and the supernatant was collected. 25 μL of 50% (w / w) sulfuric acid and 1 mL of ether were added, vortexed and mixed evenly for 3 min. After passing through a 0.22 μm filter membrane, it was measured by a gas chromatography-mass spectrometry instrument;

[0111] The chromatographic parameters were as follows: Agilent DB-WAX capillary column (30 m × 0.25 mm × 0.25 μm), the carrier gas was high-purity helium (purity ≥ 99.999%), the flow rate was 1.0 mL / min, the inlet temperature was 220°C, the injection volume was 1 μL, splitless injection, and the solvent delay time was 2.5 min;

[0112] The mass spectrometry parameters were as follows: electron impact ionization source (EI), the ion source temperature was 230°C, and the interface temperature was 220°C.

[0113] Among them, the qPCR process was carried out according to the instructions of the SYBR Green Pro Taq HS Premixed qPCR Kit (purchased from Aikrui Biotechnology Co., Ltd., AG11702); the primers involved in the qPCR process and the corresponding annealing temperatures are shown in Table 2. The 16S rDNA V4 region primers were used to perform qPCR detection on all bacteria in the culture medium, the lactic acid bacteria primers were used to perform qPCR detection on lactic acid bacteria in the culture medium, and the bifidobacterium primers were used to perform qPCR detection on bifidobacteria in the culture medium, and relative quantitative detection was carried out based on the qPCR detection results respectively.

[0114] Table 2 PCR primer sequences and annealing temperatures

[0115]

[0116] II. Experimental results

[0117] The OD600 test results of each culture group at each culture time are shown as Figure 5 follows. The results show that: at 0 h of culture, the OD600 in the media of the Control group, Glu group and BO group were 0.25857 ± 0.0179, 0.2479 ± 0.0244, and 0.2747 ± 0.0099 respectively, and there was no significant difference in OD600 among the media of the three groups (p < 0.05); after 6 h of culture, the OD600 in each group of media gradually increased, and the OD600 in the BO group of media increased the most; after 12 h of culture, the OD600 in the BO group of media was significantly higher than that in the Glu group of media and the Control group of media; after 24 h of culture, the OD600 in the BO group of media gradually decreased to 0.5326 ± 0.0133, and there was no significant difference from the Glu group and the Control group.

[0118] The results indicate that: the blackberry oligosaccharides prepared in Example 1 not only have no toxic effect on the growth of intestinal flora, but also can promote the growth of intestinal flora, and the number of intestinal flora at 6 h of culture is significantly higher than that of other groups.

[0119] The total sugar content test results of each culture group at each culture time are shown as Figure 6 follows. The results show that: the total sugar content in each group of media continuously decreased during the culture process from 0 to 48 h, indicating that it was degraded and utilized by the microorganisms in the fecal homogenate during the culture process, resulting in a continuous decrease in the total sugar content.

[0120] The short-chain fatty acid content test results of each culture group at each culture time are shown as Figure 7 follows, Figure 7 where A in Figure 7 is the test result graph of acetic acid content,Figure 7 In [figure], C is the test result graph of butyric acid content.

[0121] The results showed that: when cultured for 0 - 24 h, the acetic acid concentration in the medium of the BO group increased from 14.35048 ± 0.2174 μg / mL to 16.97816 ± 0.57612 μg / mL, and the acetic acid concentration in the medium of the Glu group increased from 14.55156 ± 0.21726 μg / mL to 17.67565 ± 0.52801 μg / mL; while the acetic acid concentration in the medium of the Control group increased from 14.20302 ± 0.42882 μg / mL to 15.49526 ± 0.27277 μg / mL, which was significantly lower than the acetic acid content in the medium of the BO group and the Glu group; indicating that the blackberry oligosaccharide prepared in Example 1 can effectively promote the production of acetic acid by anaerobic flora represented by Bifidobacterium.

[0122] When cultured for 48 h, the propionic acid concentration in the medium of the BO group reached the highest, which was 10.80648 ± 0.57995 μg / mL, showing a significant increase compared with the propionic acid concentration in the medium of the Control group (5.993 ± 1.21667 μg / mL); during the culture process of 0 - 48 h, the butyric acid content in the medium of each group gradually increased, but there was no significant difference.

[0123] It shows that blackberry oligosaccharide has very significant probiotic activity and can be a potential source of prebiotics, playing a beneficial role in maintaining human health through the production of short-chain fatty acids.

[0124] The fluorescence quantitative PCR detection result graphs of each culture group are as Figure 8 shown. Figure 8 In [figure], A is the relative quantitative result graph of fluorescence quantitative PCR detection for all bacteria in each culture group, Figure 8 in [figure], B is the relative quantitative result graph of fluorescence quantitative PCR detection for lactic acid bacteria in each culture group, Figure 8 and in [figure], C is the relative quantitative result graph of fluorescence quantitative PCR detection for Bifidobacterium in each culture group.

[0125] The results showed that: after 48 h of cultivation, the gene expression levels of the microorganisms (total bacteria) in the medium of the BO group reached the maximum, and were significantly higher than those of the microorganisms in the media of the Glu group and the Control group; and at 48 h of cultivation, the gene expression levels of the lactic acid bacteria in the medium of the BO group also reached the maximum, and were significantly higher than those of the lactic acid bacteria in the media of the Glu group and the Control group; at 12 h of cultivation, the gene expression levels of the bifidobacteria in the medium of the BO group reached the maximum, which was significantly higher than that of the bifidobacteria in the media of the Glu group and the Control group. With the increase of the cultivation time, the gene expression levels of the bifidobacteria in the medium of the BO group gradually decreased, but were still significantly higher than those of the bifidobacteria in the media of the Glu group and the Control group.

[0126] The results indicated that: the blackberry oligosaccharide prepared in Example 1 of the present invention could effectively promote the growth of lactic acid bacteria and bifidobacteria.

[0127] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description and ideas. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing blackberry oligosaccharides, characterized in that: The following steps are involved: S1. Dehydrate the blackberries, grind them into powder and dissolve them in water. After fully mixing, separate the solid and liquid and collect the supernatant; S2. The supernatant obtained in step S1 is mixed with ethanol, and the mixture is fully reacted. After the ethanol is evaporated, the liquid is collected and freeze-dried to obtain a freeze-dried sample; S3. The freeze-dried sample obtained in step S2 is subjected to protein removal and pigment removal to obtain crude blackberry oligosaccharides; S4. The crude blackberry oligosaccharide obtained in step S3 is dissolved in water, and then eluted on a DEAE-SepharoseFF ion chromatography column until no sugar is detected in the eluate, and the eluate is collected to obtain component 1; The elution is carried out using deionized water and NaCl; S5. The component 1 obtained in step S4 is dissolved in water, and then applied to a G100 chromatography column, fully eluted with deionized water, and the eluate is collected and freeze-dried to obtain blackberry oligosaccharides.

2. The preparation method according to claim 1, characterized in that: In step S2, the supernatant and ethanol are evenly mixed in a volume ratio of 2 to 3:

10.

3. The preparation method according to claim 1, characterized in that: The protein removal in step S3 is specifically as follows: The freeze-dried precipitate obtained in step S2 is mixed evenly with the Sevage reagent at a volume ratio of 2.5 to 3.5:

1. After sufficient reaction, the mixture is centrifuged and the aqueous phase is collected to obtain a protein-free mixed solution; the Sevage reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 3.5 to 4.5:

1.

4. The preparation method according to claim 1, characterized in that: The pigment removal in step S3 is to remove the pigment by adsorption using a macroporous resin.

5. The preparation method according to claim 4, characterized in that: The macroporous resin is AB-8 type macroporous resin.

6. The preparation method according to claim 1, characterized in that: The elution in step S4 is specifically: eluting with deionized water, 0.2M NaCl and 0.4M NaCl in sequence.

7. The preparation method according to claim 6, characterized in that: The flow rate during the elution process in step S4 is 0.8-1.2 mL / min.

8. Blackberry oligosaccharide prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the blackberry oligosaccharide according to claim 8 in the preparation of a product for promoting the growth of probiotics and / or improving the activity of probiotics.

10. The use according to claim 9, characterized in that: The probiotics are lactic acid bacteria and / or bifidobacteria.

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