Preparation method of synbiotics for regulating and controlling glycolipid metabolic activity

文档序号：803046 发布日期：2021-03-26 浏览：28次中文

阅读说明：本技术 一种调控糖脂代谢活性合生元的制备方法 (Preparation method of synbiotics for regulating and controlling glycolipid metabolic activity ) 是由胡腾根邹宇晓廖森泰黎尔纳刘凡沈维治王思远于 2020-12-11 设计创作，主要内容包括：本发明公开了一种调控糖脂代谢活性合生元的制备方法,以桑叶为原料,经半纤维素酶水解,并通过纳滤、超滤及柱层析纯化得到桑叶低聚糖,然后以桑叶低聚糖为碳源富集具有调控糖脂代谢活性的肠道菌群,分离得到最显著富集的菌株,最后将纯化得到的桑叶低聚糖与分离得到的富集菌株共培养制备富含桑叶低聚糖和菌株的合生元。本发明调控糖脂代谢活性合生元的制备方法简单,并且制备得到的合生元能够有效调控糖脂代谢活性。(The invention discloses a preparation method of synbiotics for regulating glycolipid metabolism activity, which takes mulberry leaves as a raw material, hydrolyzes through hemicellulase, obtains mulberry leaf oligosaccharides through nanofiltration, ultrafiltration and column chromatography purification, then enriches intestinal flora with the glycolipid metabolism regulation activity by taking the mulberry leaf oligosaccharides as a carbon source, separates to obtain a most obviously enriched strain, and finally co-cultures the purified mulberry leaf oligosaccharides and the enriched strain obtained by separation to prepare the synbiotics rich in the mulberry leaf oligosaccharides and the strain. The preparation method of the synbiotics for regulating the glycolipid metabolic activity is simple, and the prepared synbiotics can effectively regulate the glycolipid metabolic activity.)

1. A preparation method of synbiotics for regulating and controlling glycolipid metabolic activity is characterized by comprising the following steps:

(1) taking dried mulberry leaves as a raw material, hydrolyzing polysaccharide and hemicellulase by water extraction, and performing nanofiltration and ultrafiltration to obtain a primarily purified mulberry leaf oligosaccharide solution;

(2) adding the primarily purified mulberry leaf oligosaccharide solution into a DEAE-52 chromatographic column, collecting eluent for 250-400min by taking 0.3mol/L NaCl as eluent, concentrating, then adding the concentrated solution into a sephadex G-15 column, collecting eluent for 70-130min by taking deionized water as eluent, and concentrating to obtain the purified mulberry leaf oligosaccharide solution;

(3) using purified mulberry leaf oligosaccharide solution as a single carbon source to replace glucose in an MRS culture medium, carrying out in-vitro anaerobic culture on mice fecal bacteria with glycolipid metabolic disturbance, enriching intestinal flora with glycolipid metabolic activity regulation and control, and obtaining lactobacillus murinus by a streaking separation method;

(4) inoculating the enriched lactobacillus murinus to a culture medium taking a mulberry leaf oligosaccharide solution as a carbon source for fermentation, and performing freeze spray drying to obtain the synbiotics with the glycolipid metabolism regulation activity.

2. The method for preparing synbiotics for regulating glycolipid metabolism activity according to claim 1, characterized in that step (1) comprises the following steps:

(1-1) taking dried mulberry leaves as a raw material, adding 10-15% (w/v) mulberry leaf powder into a phosphoric acid buffer solution with the pH value of 4.5-5.0, carrying out water extraction on polysaccharide for 6-8h at the temperature of 60-70 ℃, centrifuging for 5min at the speed of 5000 Xg, and concentrating the supernatant to obtain a mulberry leaf polysaccharide solution;

(1-2) taking 3-3.5% (w/v) mulberry leaf polysaccharide solution, adding 0.5-1% (w/v) hemicellulase, adjusting the pH value to 5.5-6.0, reacting for 6-8h at 45-50 ℃, centrifuging for 5min at 8000 Xg, and concentrating the supernatant to obtain oligosaccharide crude reaction liquid;

(1-3) sequentially carrying out nanofiltration and ultrafiltration on the oligosaccharide crude reaction solution, and removing monosaccharide and polysaccharide which is not degraded by enzyme and has a molecular weight of more than 2000Da in the oligosaccharide crude reaction solution to obtain a primarily purified mulberry leaf oligosaccharide solution.

3. The method as claimed in claim 1, wherein the concentration of the oligosaccharide solution of mulberry leaf primarily purified in step (2) is 10-20% by weight.

4. The method for preparing synbiotics for regulating glycolipid metabolism activity according to claim 1, wherein the content of the mulberry leaf oligosaccharide solution in the culture medium of step (3) is 10-20 g/L.

5. The method for preparing synbiotics for regulating glycolipid metabolism activity according to claim 1, wherein the total number of effective viable bacteria of said mouse fecal bacteria in step (3) is not less than 10⁸CFU/mL, and the culture time is 48-72 h.

6. The method for preparing synbiotics for regulating glycolipid metabolism activity according to claim 1, characterized in that the intestinal flora enriched in step (3) includes aerococcus urinaria, lactobacillus murinus and candidate saccharomonas monads.

7. The method for preparing synbiotics for regulating glycolipid metabolism according to claim 1, characterized in that the total number of effective live bacteria of lactobacillus murinus enriched and separated in step (3) is 10⁸-10¹⁰CFU/mL。

8. The method for preparing synbiotics for regulating glycolipid metabolism activity according to claim 1, wherein the content of the mulberry leaf oligosaccharide solution in the culture medium of step (4) is 20-30 g/L.

9. The method for preparing synbiotic for regulating glycolipid metabolism activity according to claim 1, characterized in that in step (4) the fermentation temperature is 30-40 ℃, the fermentation time is 48-96h, and the pH is controlled at 5.5-6.2.

10. A synbiotic produced by the method of any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of synbiotics, in particular to a preparation method of synbiotics for regulating and controlling glycolipid metabolic activity.

Background

The mulberry leaf polysaccharide has been reported to have biological activity of regulating glycolipid metabolism and the like, but the molecular weight and viscosity of the mulberry leaf polysaccharide are high, so that the bioavailability of the polysaccharide in a body is low, and researches show that the polysaccharide contains a large amount of glycosidic bonds with non-alpha-1, 4 configuration, can not be completely degraded by digestive juice of a human body and can be degraded into micromolecular functional oligosaccharide to regulate intestinal flora so as to play a role.

The functional oligosaccharide can not be degraded by gastrointestinal digestive juice of a human body and can not be directly absorbed and utilized by the human body, but can be used as a prebiotic which is an energy substance of intestinal microorganisms, regulates and controls intestinal flora, promotes the growth of probiotics, inhibits harmful bacteria proliferation and influences intestinal homeostasis, thereby exerting a health effect. The probiotics can synthesize digestive enzyme for degrading glycosidic bond with non-alpha-1, 4 configuration, thereby utilizing oligosaccharide, reducing the depth of crypt of small intestine, increasing villus height, increasing the surface area of small intestine, promoting the absorption of nutrient substances in intestinal tract by adjusting the host mucous membrane and system immunity function or by adjusting the flora balance in intestinal tract, and keeping the intestinal tract healthy. The synbiotics are formed by compounding prebiotics and probiotics, and the health effects of the prebiotics and the probiotics are exerted simultaneously through the combination of specific prebiotics and probiotics. The synbiotics is used as a novel functional substance, is widely applied to the fields of food, health care products, beverages, medicines, feed additives and the like, has good market prospect, and becomes a hotspot of current research.

Disclosure of Invention

In view of the above, the invention provides a mulberry leaf oligosaccharide with glycolipid metabolism regulation activity, and takes the mulberry leaf oligosaccharide as a carbon source, enriches probiotics with glycolipid metabolism regulation activity, and compounds the probiotics with glycolipid metabolism regulation activity and the probiotics with glycolipid metabolism regulation activity to finally obtain a synbiotic with stronger glycolipid metabolism regulation activity.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of synbiotics for regulating glycolipid metabolic activity comprises the following steps:

(3) using purified mulberry leaf oligosaccharide solution as a single carbon source to replace glucose in an MRS culture medium, carrying out in-vitro anaerobic culture on mouse fecal bacteria induced glycolipid metabolic disturbance by streptozotocin and high-fat feed, enriching intestinal flora with glycolipid metabolic activity regulation and control, and obtaining lactobacillus murinus by a streaking separation method;

Preferably, in the above method for preparing a synbiotic for regulating glycolipid metabolic activity, the step (1) specifically comprises the following steps:

Preferably, in the above method for preparing synbiotics for regulating glycolipid metabolism activity, the mass concentration of the mulberry leaf oligosaccharide solution primarily purified in step (2) is 10-20%.

The beneficial effects of the above technical scheme are: if the mass concentration of the primarily purified mulberry leaf oligosaccharide solution is too low, the nanofiltration and ultrafiltration effects can be influenced; if the concentration is too high, mass transfer resistance is easy to generate, so that the pressure is too high during nanofiltration and ultrafiltration, and the separation membrane is damaged.

Preferably, in the above method for preparing synbiotics for regulating glycolipid metabolism activity, the content of the mulberry leaf oligosaccharide solution in the culture medium of step (3) is 10-20 g/L.

The beneficial effects of the above technical scheme are: if the concentration of the mulberry leaf oligosaccharide solution in the culture medium is too low, the enrichment of probiotics is influenced; while too high a concentration can result in segregation of the probiotic plasmodium, inhibiting the growth of significant enrichment bacteria.

Preferably, in the above method for preparing synbiotics for regulating glycolipid metabolism activity, the total number of effective viable bacteria of said mouse fecal bacteria in step (3) is not less than 10⁸CFU/mL, and the culture time is 48-72 h.

The beneficial effects of the above technical scheme are: when the number of probiotics is too low, the separation of the enrichment bacteria is obviously influenced; the strain activity is influenced when the culture time is too short and the strain is in a lag phase and when the culture time is too long and the strain is in a decay phase, and the strain activity is highest when the culture time is 48-72 hours after a large number of tests prove that the strain activity is the highest.

Preferably, in the above method for preparing a synbiotic capable of regulating glycolipid metabolism activity, the intestinal flora enriched in step (3) includes Aerococcus urinaria, Lactobacillus murinus and candidate saccharomonas monads.

Preferably, in the above method for preparing synbiotics for regulating glycolipid metabolism activity, the total number of effective live bacteria of lactobacillus murinus enriched and separated in step (3) is 10⁸-10¹⁰CFU/mL。

The beneficial effects of the above technical scheme are: if the concentration is too low, the growth rate of probiotics can be reduced, and the probiotic effect is influenced; too high a concentration can lead to segregation of the probiotic walls.

Preferably, in the above method for preparing synbiotics for regulating glycolipid metabolism activity, the fermentation temperature in step (4) is 30-40 ℃, the fermentation time is 48-96h, and the pH is controlled at 5.5-6.2.

The beneficial effects of the above technical scheme are: under the condition, the growth rate of the probiotics is fastest, and the activity is highest.

The invention also provides the synbiotic prepared by the method.

According to the technical scheme, compared with the prior art, the invention discloses a preparation method of a synbiotic for regulating and controlling glycolipid metabolic activity, and the preparation method has the beneficial effects that:

(1) the invention combines nanofiltration and ultrafiltration technologies, greatly simplifies the purification steps of the mulberry leaf oligosaccharide, and combines ion exchange chromatography and sephadex chromatography to prepare the functional mulberry leaf oligosaccharide with the purity of more than 95 percent; the obtained mulberry leaf oligosaccharide has uniform molecular weight of 1427Da, and consists of mannose 10-15%, ribose 10-15%, rhamnose 20-25%, glucose 25-30% and arabinose 25-30% by mol mass;

(2) the mulberry leaf oligosaccharide obtained by separation can resist the degradation of digestive juice of a human body, enters an intestinal tract regulation flora and plays a role in regulating the metabolic activity of glycolipid in a manner depending on the intestinal tract flora;

(3) the lactobacillus murinus is obtained by separating the mulberry leaf oligosaccharide from the glycolipid metabolic disturbance mouse intestinal flora, the regulation and control of the glycolipid metabolic activity of the lactobacillus murinus is verified, and the lactobacillus murinus and the glycolipid metabolic disturbance mouse intestinal flora are finally compounded to prepare the synbiotics with stronger activity.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the separation and purification of (a) DEAE-52(b) G15 of the mulberry leaf oligosaccharide MLO2-1 in accordance with example 1 of the present invention;

FIG. 2 is a drawing showing the monosaccharide composition of the mulberry leaf oligosaccharide MLO2-1 in example 1 of the present invention;

FIG. 3 is a graph showing the changes of the molecular weight and the content of reducing sugar (d) in different digestive juices (a) saliva, (b) gastric juice, (c) intestinal juice of the mulberry leaf oligosaccharide MLO2-1 according to example 1 of the present invention;

FIG. 4 is a drawing showing how mulberry leaf oligosaccharide MLO2-1 regulates glycolipid metabolic activity (a) body weight according to example 1 of the present invention; (b) blood glucose;

FIG. 5 is a drawing showing that the mulberry leaf oligosaccharide MLO2-1 significantly enriches glycolipid metabolism disturbance mouse flora in example 1 of the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Adding folium Mori powder into distilled water to obtain 10% (w/v) solution, extracting polysaccharide with water at 80 deg.C and pH4.9 for 8 hr, centrifuging at 5000 × g for 5min, collecting supernatant, and concentrating to obtain folium Mori polysaccharide solution;

taking 3% mulberry leaf polysaccharide solution, adding 1% hemicellulase, reacting at 50 deg.C for 8h, reacting at pH 6.0, centrifuging at 8000 Xg for 5min, collecting supernatant, and concentrating to obtain crude reaction solution of mulberry leaf oligosaccharide;

removing monosaccharide and polysaccharide which is not degraded by enzyme and has molecular weight larger than 2000Da in the mulberry leaf oligosaccharide crude reaction solution by using a nanofiltration (200Da) membrane system and an ultrafiltration (2000Da) membrane system together to obtain a primarily purified oligosaccharide solution;

adding the oligosaccharide solution (20%, w/v) into DEAE-52 chromatographic column (20mm × 300mm), eluting with 0.3mol/L NaCl as eluent at flow rate of 1mL/min, collecting 250-400min eluate MLO2, and concentrating; then adding the 5% (w/v) concentrated solution into a sephadex G-15 column (20mm multiplied by 300mm), eluting with deionized water as an eluent at the flow rate of 1mL/min, collecting the eluent for 70-130min, and concentrating to obtain the separated and purified mulberry leaf oligosaccharide MLO2-1 solution.

20g/L of mulberry leaf oligosaccharide MLO2-1 is used as a single carbon source to replace glucose in an MRS culture medium, and is used for in vitro anaerobic culture of glycolipid metabolism disturbance mouse coprophila (10)⁸CFU/mL) for 72h, isolating lactobacillus murinus (l.murinus) by streaking;

adding 10g/L of the above folium Mori oligosaccharide MLO2-1 solution into 30g/L¹⁰Fermenting with Lactobacillus murinus (L.murinus) at 37 deg.C and pH of 6.0 for 72 hr, and freeze spray drying to obtain synbiotics rich in folium Mori oligosaccharide and Lactobacillus murinus.

Example 2

Adding folium Mori powder into distilled water to obtain 12.5% (w/v) solution, extracting polysaccharide with water at 70 deg.C and pH of 4.8 for 7 hr, centrifuging at 5000 × g for 5min, collecting supernatant, and concentrating to obtain folium Mori polysaccharide solution;

taking 3.25% mulberry leaf polysaccharide solution, adding 0.75% hemicellulase, reacting at 45 deg.C for 7h, reacting at pH 5.7, centrifuging at 8000 Xg for 5min, collecting supernatant, and concentrating to obtain crude reaction solution of mulberry leaf oligosaccharide;

adding the primarily purified oligosaccharide solution (15%, w/v) into DEAE-52 chromatographic column (20mm × 300mm), eluting with 0.3mol/L NaCl as eluent at flow rate of 1.5mL/min, collecting 250-400min eluate MLO2, and concentrating; then adding 2.5% (w/v) of the concentrated solution into a sephadex G-15 column (20mm multiplied by 300mm), eluting with deionized water as an eluent at the flow rate of 1.5mL/min, collecting 70-130min of eluent, and concentrating to obtain the separated and purified mulberry leaf oligosaccharide MLO2-1 solution.

15g/L mulberry leaf oligosaccharide MLO2-1 is used as a single carbon source to replace glucose in an MRS culture medium, and is used for in vitro anaerobic culture of glycolipid metabolism disturbance mouse fecal bacteria (10)⁸CFU/mL) for 60h, isolating lactobacillus murinus (l.murinus) by streaking;

adding 10g/L of the above folium Mori oligosaccharide MLO2-1 solution into 25g/L⁹Fermenting with CFU/mL Lactobacillus murinus (L.murinus) at 40 deg.C and pH 5.5 for 60 hr, and freeze spray drying to obtain synbiotics rich in folium Mori oligosaccharide and Lactobacillus murinus.

Example 3

Adding folium Mori powder into distilled water to obtain 15% (w/v) solution, extracting polysaccharide with water at pH4.5 and 60 deg.C for 6h, centrifuging at 5000 × g for 5min, collecting supernatant, and concentrating to obtain folium Mori polysaccharide solution;

taking 3.5% mulberry leaf polysaccharide solution, adding 0.5% hemicellulase, reacting at 45 deg.C for 6h, reacting at pH 5.5, centrifuging at 8000 Xg for 5min, collecting supernatant, and concentrating to obtain crude reaction solution of mulberry leaf oligosaccharide;

adding the primarily purified oligosaccharide solution (10%, w/v) into DEAE-52 chromatographic column (20mm × 300mm), eluting with 0.3mol/L NaCl as eluent at flow rate of 2mL/min, collecting 250-400min eluate MLO2, and concentrating; then adding 1% (w/v) of the concentrated solution into a sephadex G-15 column (20mm multiplied by 300mm), eluting with deionized water as an eluent at the flow rate of 2mL/min, collecting 70-130min of eluent, and concentrating to obtain separated and purified mulberry leaf oligosaccharide MLO2-1 solution;

uses 10g/L mulberry leaf oligosaccharide MLO2-1 as a single carbon source to replace MRS cultureGlucose in the medium and its use in the anaerobic culture of glycolipid metabolically disturbed mouse faecalis in vitro (10)⁸CFU/mL) for 48h, isolating lactobacillus murinus (l.murinus) by streaking;

adding 10g/L of the above folium Mori oligosaccharide MLO2-1 solution into 20g/L⁸Fermenting with Lactobacillus murinus (L.murinus) at 30 deg.C and pH of 6.2 for 48 hr, and freeze spray drying to obtain synbiotics rich in folium Mori oligosaccharide and Lactobacillus murinus.

Comparative example 1

Adding folium Mori powder into distilled water to obtain 10% (w/v) solution, extracting polysaccharide with water at 80 deg.C and pH of 4.9 for 8 hr, centrifuging at 5000 × g for 5min, collecting supernatant, and concentrating to obtain folium Mori polysaccharide solution;

taking 3% mulberry leaf polysaccharide solution, adding 1% hemicellulase, reacting at 50 deg.C for 8h, reacting at pH 6.0, centrifuging at 8000 Xg for 5min, and collecting supernatant to obtain folium Mori oligosaccharide crude reaction solution;

adding the primarily purified oligosaccharide solution (20%, w/v) into DEAE-52 chromatographic column (20mm × 300mm), eluting with 0.3mol/L NaCl as eluent at flow rate of 1mL/min, collecting the eluate of 250-; then adding 5% (w/v) of the concentrated solution into a sephadex G-15 column (20mm multiplied by 300mm), eluting with deionized water as an eluent at the flow rate of 1mL/min, collecting the eluent for 70-130min, and concentrating to obtain the separated and purified mulberry leaf oligosaccharide solution.

And (3) freezing, spraying and drying the mulberry leaf oligosaccharide solution with the concentration of 30g/L to obtain pure mulberry leaf oligosaccharide.

Comparative example 2

adding the primarily purified oligosaccharide solution (20%, w/v) into DEAE-52 chromatographic column (20mm × 300mm), eluting with 0.3mol/L NaCl as eluent at flow rate of 1mL/min, collecting the eluate of 250-; then adding 5% (w/v) of the concentrated solution into a sephadex G-15 column (20mm multiplied by 300mm), eluting with deionized water as an eluent at the flow rate of 1mL/min, collecting the eluent for 70-130min, and concentrating to obtain a mulberry leaf oligosaccharide solution after separation and purification;

20g/L mulberry leaf oligosaccharide is used as a single carbon source to replace glucose in an MRS culture medium, and is used for in-vitro anaerobic culture of glycolipid metabolism disturbance mouse fecal bacteria (10)⁸CFU/mL) for 72h, isolating lactobacillus murinus (l.murinus) by streaking;

adding 10g/L of the glucose solution into 30g/L¹⁰And (3) fermenting the lactobacillus murinus (L.murinus) for 72 hours at the temperature of 37 ℃ and under the condition of pH 6.0, and finally freezing and spray-drying to obtain the lactobacillus murinus powder.

The purity of the mulberry leaf oligosaccharides prepared in examples 1 to 3 and comparative example 1 was analyzed and tested, and the test results are shown in table 1.

TABLE 1 test results

	Example 1	Example 2	Example 3	Comparative example 1
					Purity (%)	95.6	94.5	92.8	95.3

To further illustrate the effects achieved by the present invention, the following experiments were performed:

70 male C57BL/6J mice (20 +/-2 g) with age of 8 weeks are divided into 7 groups, 10 mice are respectively taken as a normal group, a model group and an experimental group (examples 1-3 and comparative examples 1 and 2), the normal group is fed with common feed, the model group and the experimental group are fed with mice with 60% high-fat feed to induce glycolipid metabolic disorder models, after the models are established, the experimental group is fed with common feed for six weeks after being fed with the high-fat feed, and the normal group and the model group are fed with normal saline with equal doses, and the weight, the blood sugar and the blood fat changes of the mice are observed.

TABLE 1 Effect of examples 1-3 and comparative examples 1-2 on mouse body weight and blood glucose

Body weight

Blood sugar

HDL-C

LDL-C

Normal group

23.85±0.16a

4.98±0.46a

1.91±0.20a

6.11±0.92a

4.95±0.47d

0.29±0.08a

Model set

28.13±0.57d

7.32±0.12d

3.92±0.25c

8.59±0.71c

2.74±0.50a

4.06±0.10e

Example 1

24.42±0.17b

6.37±0.15c

1.91±0.15a

6.21±0.11a

4.85±0.22cd

0.49±0.10b

Example 2

24.56±0.13b

6.46±0.11bc

1.95±0.17a

6.26±0.15a

4.76±0.14c

0.61±0.09b

Example 3

24.65±0.16bc

6.55±0.13bc

2.06±0.21ab

6.31±0.12ab

4.70±0.12c

0.67±0.13b

Comparative example 1

25.00±0.26c

6.86±0.33b

1.91±0.31a

6.27±0.31ab

4.17±0.36b

1.23±0.28c

Comparative example 2

25.57±0.43c

6.93±0.15b

2.35±0.41b

6.58±0.23b

4.05±0.33b

1.46±0.15d

As can be seen from Table 1, examples 1-3 and comparative examples 1-2 can significantly reduce the body weight, blood sugar, TG, TC, HDL-C and LDL-C levels (P <0.05) of mice with glycolipid metabolism disorder, and examples 1-3 are significantly better than comparative examples 1-2 in the effects of reducing body weight, blood sugar, TC, LDL-C and increasing HDL-C (P <0.05), which indicates that the mulberry leaf oligosaccharide and Lactobacillus murinus have the function of synergistically regulating the glycolipid metabolism activity.

In addition, the invention also specially carries out test analysis of various performance indexes aiming at the prepared mulberry leaf oligosaccharide, and the test standard and the analysis result are as follows:

(1) method for measuring total sugar-phenol-sulfuric acid method:

2mL of glucose solution (0-400 mu g/mL) with different concentrations are taken, 1.0mL of 5% phenol solution and 5mL of concentrated sulfuric acid are sequentially added, the mixture is shaken and stirred evenly, the reaction is carried out for 15min at 40 ℃, distilled water is used as a blank control, the light absorption value is measured at 490nm, and a standard curve is drawn.

Sample treatment: 2mL of a sample with a certain concentration, 1mL of 5% phenol solution and 5mL of concentrated sulfuric acid, shaking up, reacting at 40 ℃ for 15min, measuring a light absorption value, and calculating the content of total sugar in the sample.

(2) The method for measuring reducing sugar comprises the following steps:

taking 2mL of glucose solution (0-500 mu g/mL) with different concentrations, adding 3.0mL of DNS solution, shaking up, reacting in boiling water bath for 10min, cooling, diluting to 15mL, taking distilled water as blank control, measuring absorbance at 540nm wavelength of a spectrophotometer, and drawing a standard curve.

Sample treatment: 2mL of a sample with a certain concentration and 3.0mL of DNS solution are shaken up, reacted in a boiling water bath for 10min, cooled and then the volume is determined to be 15mL, the light absorption value is measured, and the content of reducing sugar in the sample is calculated.

(3) And (3) determination of polysaccharide content: polysaccharide content-total sugar content of polysaccharide extract-reducing sugar content of polysaccharide extract.

(4) And (3) determining the content of oligosaccharide: the oligosaccharide content is the total sugar content in the hydrolysis reaction liquid-the reducing sugar content in the hydrolysis reaction liquid.

(5) Analysis of oligosaccharide purity:

filtering a sample to be detected by using a 0.22 mu m filter membrane, and analyzing the oligosaccharide purity by using high performance liquid chromatography, wherein the chromatographic conditions are as follows: the detector is an evaporative light detector, and the temperature of an ELSD drift tube is 45 ℃; column model Shodex Asahipak NH2P-504E (4.6 mm. times.250 mm,5 μm), column temperature 30 deg.C; the mobile phase is 75% acetonitrile, the flow rate is 1mL/min, the sample amount is 20 μ L, and dextran with the polymerization degree of 2-10 is used as a standard.

(6)16S rDNA method

A bacterial DNA extraction kit is used for extracting thallus 16S rDNA in fermentation liquor, a V3-V4 hypervariable region sequence primer is designed for PCR amplification, then a Miseq library is constructed, and Illumina PE300 is adopted for sequencing.

Referring to FIG. 5, the mulberry leaf oligosaccharide obtained in example 1 can be significantly enriched in Aerococcus urinaeequi, Lactobacillus murinus, Candidatus Saccharionins.

(7) Determination of molecular weight

The molecular weight of the oligosaccharide is measured by adopting a GPC gel permeation chromatograph of Waters company, a chromatographic column is an APC AQ450, an Acquity APC AQ 125 and an Acquity APC AQ45 series column, a mobile phase is deionized water, and the flow rate is 0.1 mol/L.

Referring to FIG. 1, the molecular weight of the mulberry leaf oligosaccharide obtained in example 1 is 1427 Da.

(8) Monosaccharide composition analysis

mu.L of trifluoroacetic acid (4mol/L) was added to 100. mu.L of 5mg/mL oligosaccharide solution, hydrolysis was carried out at 120 ℃ for 2h, excess trifluoroacetic acid was removed by a nitrogen blower, 125. mu.L of NaOH (0.12mol/L) was added to the hydrolysate solution, followed by derivatization with 50. mu.L of LPMP (0.5mol/L) at 70 ℃ for 100min, 1mL of chloroform was added after derivatization to extract excess PMP three times, the aqueous layer was subjected to monosaccharide composition analysis by 0.45 μm filtration, and the assay was carried out by Agilent 1260 liquid chromatography, equipped with an Eclipse Plus C18 column (4.6X 250mm,5 μm), mobile phase of 0.1mol/L ammonium acetate (pH 6.7) and acetonitrile 13:87(v/v), flow rate of 1.0mL/min, column temperature of 30 ℃ and detection wavelength of 245 nm.

Referring to the attached figure 2, the monosaccharide composition of the mulberry leaf oligosaccharide obtained in example 1 is mannose, ribose, rhamnose, glucose and arabinose, and the molar mass percentage content of each monosaccharide is 10%, 20%, 30% and 30% respectively.

(9) Simulating digestive juice digestion

5.0mL of oligosaccharide solution (2mg/mL) was digested in 5.0mL of simulated digest, and samples were taken at 0.5h, 1h, 2h, 4h and 6h to determine the molecular weight and reducing sugar content.

Preparation of simulated digestive juice:

saliva: saliva of healthy volunteers who did not take antibiotics within a month was collected, centrifuged at 5000 Xg for 10min, and the supernatant was collected.

Simulated gastric fluid: from 3.1mg/mL NaCl, 1.1mg/mL KCl, 0.25mg/mLCaCl₂And 0.6mg/mL NaHCO₃Composition, then 37.5mg gastric lipase, 35.4mg pepsin and 1.5mLCH are added into 150mL simulated gastric juice₃COONa (1mol/L, pH 5) solution, and finally adjusting the pH of the simulated solution to 3.0 with 0.1mol/L HCl.

Simulating intestinal juice: from 5.4mg/mL NaCl, 0.65mg/mL KCl and 0.33mg/mLCaCl₂Composition, pH adjusted to 7.0 with 0.1mol/L NaOH, followed by addition of 200g bile salt (4%, w/w), 100g pancreatin solution (7%, w/w) and 1.3mg trypsin to 100g of mock intestinal fluid, and finally pH adjusted to 7.5 with 0.1mol/L NaOH.

Referring to the attached figure 3, the molecular weight of the mulberry leaf oligosaccharide obtained in the example 1 is basically unchanged in saliva, artificial simulated gastric juice and intestinal juice, and the content of reducing sugar in the simulated juice is not obviously changed, and the result shows that the mulberry leaf oligosaccharide can resist the digestion and degradation of saliva, artificial simulated gastric juice and intestinal juice, and finally the intestinal tract is decomposed and utilized by intestinal flora to play a role in regulating and controlling the intestinal flora.

(9) Regulation and control of glycolipid metabolic activity by mulberry leaf oligosaccharide

The experimental animals C57BL/6J are divided into 8 groups, each group comprises 10 animals, namely a normal group, a model group, a mulberry leaf oligosaccharide MLO2-1 treatment group, an antibiotic treatment group and a mulberry leaf oligosaccharide MLO2-1+ antibiotic treatment group, a glycolipid metabolic disturbance model is constructed by using high-fat feed, the experimental groups are respectively subjected to intragastric gavage with 50mg/kg/d of mulberry leaf oligosaccharide MLO2-1 and 50mg/kg/d of mulberry leaf oligosaccharide MLO2-1+100 mu g/mL of antibiotic, the blank groups are subjected to intragastric gavage with the same dosage of physiological saline, and the experimental period is 2 months in total. Observing the blood sugar change and the body weight of the mouse; and taking visceral fat, fixing by 4% paraformaldehyde, washing and dehydrating, transparentizing, waxing, embedding, slicing and pasting, dewaxing, hematoxylin-eosin dyeing, dehydrating, transparentizing, sealing and the like, and then observing and photographing under an optical microscope.

Referring to fig. 4, the mulberry leaf oligosaccharide obtained in example 1 can significantly inhibit the increase of body weight and blood sugar of mice, and exert the activity of regulating glycolipid metabolism in a manner depending on intestinal flora.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. For the scheme disclosed by the embodiment, the scheme corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

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Preparation method of synbiotics for regulating and controlling glycolipid metabolic activity

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