阅读说明：本技术 一种具有调节结肠内菌群功效的荔枝多糖-多酚加合物及其制备方法与应用 (Litchi polysaccharide-polyphenol adduct with effect of regulating colonic flora and preparation method and application thereof ) 是由 苏东晓 罗楠 舒彬 杨新泉 曾庆祝 于 2021-08-13 设计创作，主要内容包括：本发明公开了一种具有调节结肠内菌群功效的荔枝多糖-多酚加合物及其制备方法与应用。本发明方案中,荔枝多糖-多酚加合物可以通过促进结肠内菌群增殖,降低pH以维持结肠微环境健康,还有利于促进肠道系统中对人体有益的乙酸、丙酸、丁酸等短链脂肪酸的产生；本发明方案制备的荔枝多糖-多酚加合物能显著改善肠道微环境健康,可用于添加在食品或药品中,具有广阔的应用前景。(The invention discloses a litchi polysaccharide-polyphenol adduct with an effect of regulating colonic flora and a preparation method and application thereof. In the scheme of the invention, the litchi polysaccharide-polyphenol adduct can maintain the health of a colon microenvironment by promoting the proliferation of flora in the colon and reducing the pH, and is also beneficial to promoting the generation of short-chain fatty acids such as acetic acid, propionic acid, butyric acid and the like which are beneficial to a human body in an intestinal tract system; the litchi polysaccharide-polyphenol adduct prepared by the scheme of the invention can obviously improve the intestinal microenvironment health, can be used for being added into food or medicines, and has wide application prospect.)

1. Application of litchi polysaccharide-polyphenol adduct in preparation of food or medicine with effect of regulating colonic flora.

2. The use according to claim 1, wherein said modulating the efficacy of the colonic flora comprises: promoting proliferation of flora in the colon, lowering pH and/or increasing short chain fatty acids in the intestinal system.

3. The use of claim 1, wherein the monosaccharides of the litchi polysaccharide-polyphenol adduct comprise mannose and galactose.

4. The use as claimed in claim 1, wherein the polyphenol in the lychee polysaccharide-polyphenol adduct comprises gallic acid, gallocatechin, caffeic acid and vanillin.

5. Use according to claim 1, wherein the short chain fatty acids comprise acetic acid, propionic acid and butyric acid.

6. The method for extracting litchi polysaccharide-polyphenol adduct of claim 1, wherein the method comprises the steps of:

s1, adding ethanol into the litchi pulp, carrying out alcohol precipitation, carrying out solid-liquid separation, and collecting a solid phase A;

s2, dissolving the solid phase A obtained in the step S1, then carrying out water extraction, and collecting a liquid phase A and a solid phase B;

s3, removing protein from the liquid phase A obtained in the step S2, adding ethanol, and precipitating with ethanol to obtain a precipitate, namely the water-soluble polysaccharide-polyphenol adduct;

s4, adding a sodium carbonate solution into the solid phase B obtained in the step S2, carrying out solid-liquid separation, and collecting a solid phase C and a liquid phase B, wherein the liquid phase B is an alkali-soluble polysaccharide-polyphenol adduct extracting solution; the solid phase C is an insoluble polysaccharide-polyphenol adduct.

7. The method of claim 6, wherein in step S3, the removing protein is performed by using a protein precipitation reagent; preferably, the protein precipitation reagent is Sevag reagent.

8. The method according to claim 1, wherein in step S4, the mass-to-volume ratio of the solid phase B to the added sodium carbonate solution is 1: 4 to 8.

9. A lychee polysaccharide-polyphenol adduct prepared according to the process of any one of claims 6 to 8.

10. A food product for regulating colon health comprising the lychee polysaccharide-polyphenol adduct of claim 9.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a lychee polysaccharide-polyphenol adduct with an effect of regulating colonic flora, and a preparation method and application thereof.

Background

Along with the social development, the living standard of people is continuously increased. The demand for health is also increasing. There are tens of thousands of microorganisms in the colon, and these microbial flora play an indispensable role in the immune regulation and nutritional metabolism of the human body.

Litchi is a plant of litchi of Sapindaceae, and the mature litchi has bright red pulp and peel, white and crystal pulp, and is fragrant, sweet and tasty. The litchi has high nutritive value, and the pulp contains a large amount of sugar, microorganism C, amino acid, mineral substances and the like. It also contains abundant active substances such as polysaccharide and polyphenol. The related technology shows that the polysaccharide has the function of regulating the intestinal flora, but the pure plant polysaccharide has low efficiency of regulating the intestinal flora and cannot regulate the beneficial flora with the specific polyphenol metabolism function.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of the lychee polysaccharide-polyphenol adduct in preparing food or medicines with the effect of regulating the flora in colon.

The invention also provides a preparation method of the litchi polysaccharide-polyphenol adduct.

The invention also provides a litchi polysaccharide-polyphenol adduct prepared by the method.

According to one aspect of the invention, the application of the litchi polysaccharide-polyphenol adduct in preparing medicines and/or foods for regulating intestinal flora disorder is provided.

In some embodiments of the invention, the modulated efficacy of colonic flora comprises: in the intestinal system, proliferation of flora in the colon is promoted, pH is lowered and/or short chain fatty acids are increased.

In some embodiments of the present invention, the monosaccharides of the lychee polysaccharide-polyphenol adduct comprise mannose and galactose.

In some embodiments of the present invention, the polyphenol in the lychee polysaccharide-polyphenol adduct comprises gallic acid, gallocatechin, caffeic acid, and vanillin.

In some embodiments of the present invention, the litchi polysaccharide-polyphenol adduct is a complex of a polysaccharide and a polyphenol.

In some embodiments of the invention, the short chain fatty acids include acetic acid, propionic acid, and butyric acid.

According to a second aspect of the present invention, a method for extracting litchi polysaccharide-polyphenol adduct is provided, the method comprising the following steps:

s1, adding ethanol into the fruit pulp, carrying out alcohol precipitation, carrying out solid-liquid separation, and collecting a solid phase A;

s2, dissolving the solid phase A obtained in the step S1, then carrying out water extraction, and collecting a liquid phase A and a solid phase B;

s3, removing protein from the liquid phase A obtained in the step S2, adding ethanol, and precipitating with ethanol to obtain a precipitate, namely the water-soluble polyphenol adduct;

s4, adding a sodium carbonate solution into the solid phase B obtained in the step S2, carrying out solid-liquid separation, and collecting a solid phase C and a liquid phase B, wherein the liquid phase B is an alkali-soluble polyphenol adduct extracting solution; the solid phase C is an insoluble polyphenol adduct.

In some embodiments of the invention, in step S1, the mass-to-volume ratio of pulp to ethanol is 1:1 to 10.

In some embodiments of the invention, in step S1, the mass fraction of ethanol is 95% to 100%.

In some embodiments of the present invention, in step S1, the temperature of the alcohol precipitation is 1-4 ℃ and the time is 8-12 hours.

In some embodiments of the present invention, in step S1, the solid-liquid separation method is filtration.

In some embodiments of the invention, in step S2, the water extraction is performed by heating in a water bath at 85-95 ℃ for 4-6 hours.

In some embodiments of the present invention, in step S2, the solid-liquid separation method is centrifugation, the centrifugation rate is 4000-6000 rpm, and the time is 10-15 min.

In some embodiments of the present invention, in step S3, the protein removing means employs a protein precipitating agent; preferably, the protein precipitation reagent is Sevag reagent.

In some embodiments of the invention, the composition of the Sevag reagent comprises chloroform and n-butanol; the volume ratio of the trichloromethane to the n-butanol is 4: 1.

in some embodiments of the invention, the ratio of liquid phase a to Sevag reagent is 1: 2-6 times of 10-20 min and 9-15 times of extraction. The extraction temperature is 55-65 ℃.

In some embodiments of the present invention, the step of adding Sevag reagent in step S3 further comprises a stirring step, wherein the stirring speed is 100 to 500rpm, and the stirring time is 1 to 5 min.

In some embodiments of the present invention, the step of removing the organic solvent by using a rotary evaporator at 60-70 ℃ is further included after the Sevag reagent is added in step S3.

In some embodiments of the present invention, the protein precipitate is removed by centrifugation at 4000-6000 rpm for 10-15 min.

In some embodiments of the invention, in step S3, the mass fraction of ethanol is 95% to 100%.

In some embodiments of the present invention, in step S3, the water-soluble polyphenol adduct further comprises a drying step of drying at 50-75 ℃ under vacuum to a constant weight.

In some embodiments of the present invention, in step S3, the temperature of the alcohol precipitation is 1-4 ℃ and the time is 8-12 hours.

In some embodiments of the invention, in step S4, the mass-to-volume ratio of the solid phase B to the added sodium carbonate solution is 1: 4 to 8.

In some embodiments of the invention, in step S4, the concentration of the sodium carbonate solution is 0.05-0.10 mol/L.

In some embodiments of the invention, in step S4, the sodium carbonate solution further includes 20 to 40mmol/L sodium borohydride.

In some embodiments of the present invention, in step S4, the solid-liquid separation method is centrifugation, the centrifugation rate is 4000-6000 rpm, and the time is 10-15 min.

In some embodiments of the present invention, the step S4 further includes a stirring step after the sodium carbonate solution is added, wherein the stirring speed is 100-500 rpm and the stirring time is 1-5 min.

According to a third aspect of the present invention, a litchi polysaccharide-polyphenol adduct is provided, which is prepared by the above method.

A food for regulating colon health comprises the above litchi polysaccharide-polyphenol adduct.

A medicine for regulating colonic homeostasis comprises the litchi polysaccharide-polyphenol adduct.

According to a preferred embodiment of the present invention, at least the following advantages are provided: in the application of the lychee polysaccharide-polyphenol adduct in preparing food or medicine with the effect of regulating colonic flora, the lychee polysaccharide-polyphenol adduct can promote the proliferation of colonic flora and reduce the pH value so as to maintain the health of colonic microenvironment and is also beneficial to promoting the generation of short-chain fatty acids such as acetic acid, propionic acid and butyric acid which are beneficial to human bodies in an intestinal system; the litchi polysaccharide-polyphenol adduct prepared by the scheme of the invention can obviously improve the intestinal microenvironment health, has better effect compared with single litchi polysaccharide or polyphenol, can be effectively added into food or medicine, and has wide application prospect.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a high performance liquid chromatogram of three litchi polysaccharide-polyphenol adducts in test examples of the present invention;

FIG. 2 is a graph showing the bound phenol content in various digests in a fermentation experiment according to the test example of the present invention;

FIG. 3 is a graph showing the results of pH measurement with time in a fermentation test in the test example of the present invention;

FIG. 4 is a graph showing the results of measurement of acetic acid, propionic acid and butyric acid produced in the fermentation test in the test example of the present invention.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Example 1

The embodiment provides an extraction method of a litchi polysaccharide-polyphenol adduct, which comprises the following specific processes:

(1) soaking 200g of litchi pulp in absolute ethyl alcohol, and standing overnight at 4 ℃ to remove small molecular substances such as fat, pigment and the like;

(2) centrifuging at 4500r/min for 10min, and collecting precipitate;

(3) adding the precipitate obtained in the step (2) into 1000mL of distilled water, and extracting for 4h in a water bath at 90 ℃. Extracting the filter residue again and combining the filtrates;

(4) vacuum concentrating the filtrate obtained in the step (3) at 60 ℃, deproteinizing for 8 times by a Sevag method, adding 4 times of volume of absolute ethyl alcohol, and standing overnight at 4 ℃; filtering, collecting the precipitate and drying to obtain the water-soluble polyphenol adduct.

(5) Adding 500mL of 0.05mol/L Na into the filter residue obtained in the step (3)₂CO₃Shaking and extracting the solution (containing 20mmol/L sodium borohydride) for 4h, and centrifuging at 4000rpm for 10min to obtain supernatant and precipitate; freeze-drying the supernatant to obtain alkali-soluble polyphenol adduct; the precipitate was freeze-dried to obtain an insoluble polyphenol adduct.

Test examples

1. Detection of three polyphenol adducts

High Performance Liquid Chromatography (HPLC) -Diode Array Detector (DAD) was used to identify the bound phenol content in the 3 polyphenol adducts separately; identifying the phenolic compositions of the three polyphenol adducts by adopting a TOF 5600+ liquid chromatography high-resolution tandem mass spectrometer; and (3) identifying the phenolic compounds in the three polyphenol adducts by adopting a reversed phase high performance liquid chromatography. The method comprises the following steps:

the 3 polyphenol adducts prepared in example 1 were chromatographed using YMC-Pack ODS-A column (250X 4.6mml. D, 5 μm particle size). The mobile phase consisted of 0.4% aqueous acetic acid (solution a) and acetonitrile (solvent B). The gradient curves are as follows: 0-40min, 5-25% B; 40-45min, 25-35% B; the total running time is 50min, the balance is 5min, the flow rate is 1.0mL/min, the sample injection amount is 20 mu L, and the column incubator is 30 ℃. All samples were filtered through a 0.45 μm filter prior to analysis. (as shown in fig. 1).

The phenolic compounds of the three polyphenol adducts prepared in example 1 were mixed in a ratio of 1:1:1 and identified using a mass spectrometer using a TOF 5600+ liquid chromatography high resolution tandem mass spectrometer equipped with a turbine ion spray ionization source operating in negative ion ESI mode using an eclipse plus C18 column (2.1 × 100mm, 1.8 μm, agilent, germany). The capillary voltage was set at 4500v (negative) and the mobile phase was 0.4% formic acid in water (a) and acetonitrile (B). Analysis was performed with an elution gradient: 0-16min, 5-25% B; 16-18min, 25-35% B; 18-20min, 35-50% B. The flow rate was 0.4mL/min, the column temperature was 35 deg.C, the ion source temperature was 500 deg.C, and the sample volume was 4. mu.L. Before analysis, all samples were filtered through a 0.22 μm filter. Scanning within the range of 100-1000 m/z by 10 times of multi-channel acquisition. (as shown in tables 1-3).

TABLE 1 Water-soluble polyphenol adducts

TABLE 2 alkali soluble polyphenol adducts

TABLE 3 insoluble polyphenol adducts

And (3) identifying the bound phenol content in the three polyphenol adducts by adopting a reversed phase high performance liquid chromatography. The high performance liquid chromatography adopts YMC-Pack ODS-A column (250X 4.6mml.D., 5 μm particle diameter); a column oven 30 ℃; the sample volume is 20 mu L; a detector: DAD; performing high performance liquid chromatography analysis on Agilent 1260 liquid chromatograph at a mobile phase flow rate of 1.0 mL/min. The mobile phase was solvent a (water/acetic acid, 996: 4) and solvent B (acetonitrile). The elution gradient was as follows: 0-40min of solvent B: 5% -25%; 40-45min of solvent B: 25 to 35 percent; 45-50 min of solvent B: 35% -55%; the total operation is carried out for 50min, and the balance is carried out for 5 min. Chromatographic data were collected under a 280nm uv detector and the content of phenolic compounds in the samples was analyzed according to the retention time of the reference standards (gallic acid, (+) -catechin, caffeic acid, procyanidin B2, plateau catechin, quercetin, syringic acid) as the average of three replicates obtained from the three samples.

TABLE 4

The data result of the high performance liquid chromatography is shown in fig. 1, and it can be seen from the figure that 3 polyphenol-polysaccharide adducts were successfully prepared by the scheme of the present invention, the mass spectrum data is shown in tables 1-3, the content of bound phenol in the 3 polyphenol adducts is shown in table 4, and it can be seen from the table that 3 polyphenol adducts were successfully prepared by the scheme of the present invention, and the polyphenol-polysaccharide adducts prepared by the scheme of the present invention were identified to contain gallic acid, catechin, syringaldehyde, protocatechin, protocatechualdehyde, rutin, (+) -gallocatechin, caffeic acid, procyanidin B2, plateau catechin, and the content of polyphenol is high.

2. Application of litchi polysaccharide-polyphenol adduct in improvement of colonic flora

The experimental method comprises the following steps: the water-soluble polyphenol adduct, insoluble polyphenol adduct and alkali-soluble polyphenol adduct prepared in example 1 were mixed in a ratio of 1:1: 10 of 250.00mg in mass ratio is placed in a 100mL centrifuge tube, and 0.1mL0.3 mol/L CaCl is added₂Solution, 14mL SSF electrolyte stock, 2mL alpha-amylase solution (1500U/mL using SSF electrolyte stock), and 3.9mL distilled water was added to make a total volume of 20 mL. Adjusting pH to neutral with 1mol/L NaOH, placing the centrifuge tube in a shaking water bath kettle at 37 deg.C, shaking in water bath for 30min, and performing simulated oral digestion; then 0.01mL0.3mol/L CaCl is added₂Solution, 15mL SGF electrolyte stock, 3.2mL pepsin solution (25000U/mL, enzyme solution prepared by digesting electrolyte stock with gastric juice), distilled water was added to a total volume of 20 mL. Adjusting the pH value to 3.0 by using 6mol/L HCl, and then placing the centrifugal tube in a shaking water bath kettle at 37 ℃ for water bath shaking for 120min to simulate gastric juice digestion; then 0.04mL of 0.3mol/L CaCl was added₂11mL SIF electrolyte stock solution, 5mL pancreatin solution (800U/mL, enzyme solution prepared by digesting electrolyte stock solution with intestinal juice), and distilled water were added to a total volume of 20 mL. After pH was adjusted to neutral with 1mol/L NaOH, the centrifuge tube was placed in a 37 ℃ shaking water bath and shaken in a water bath for 120min to simulate intestinal fluid digestion. The formulation of the electrolyte stock in simulated oral gastrointestinal digestion is shown in table 2.

TABLE 2 preparation of electrolyte stock solutions for simulated oral gastrointestinal digestion

SSF: simulating saliva; SGF: simulating gastric juice; SIF: simulated intestinal fluid

The method for testing the simulated colon fermentation experiment comprises the following steps:

preparing a growth medium: 1L of growth medium contained: peptone 2.00g, yeast extract 2.00g, sodium chloride 0.10g, K₂HPO₄ 0.04g，KH₂PO₄ 0.04g，MgSO₄·7H₂O 0.01g，CaCl₂·6H₂O 0.01g(CaCl₂5.1mg)，NaHCO₃2.00g, heme 0.02g, cysteine hydrochloride 0.50g, bile salt 0.50g, resazurin 1mg, Tween 802 mL and vitamin K110 muL. One part is firstly subpackaged in a sample tube according to the quantity requirement of the sample, and the rest is directly sterilized for 15min at 121 ℃.

Weighing 100mg of lychee polysaccharide-polyphenol adduct digested by oral cavity-gastric juice-intestinal juice respectively into 15mL centrifuge tubes, adding 9mL growth medium, and sterilizing at 121 ℃ for 15 min. Adding 1mL of colonic microbial extract into an anaerobic incubator (10% CO)₂，10％H₂And 80% N₂) And performing fermentation culture at 37 ℃. For the blank Control group (Control), only growth medium and colonic microbial extract were added. The centrifuge tubes were removed at different time points of 0.25h, 6h, 12h, 24h and 48h of fermentation for analysis. 3 replicates were performed per fermentation time point.

After fermentation, centrifuging the centrifuge tube at 4 deg.C and 10000rpm for 10min, storing the supernatant at-20 deg.C, and storing the precipitate at-80 deg.C.

(1) Determination of bound phenol content

Extraction of bound phenol: the supernatants obtained by colon fermentation after simulated oral digestion, gastric juice digestion, intestinal juice digestion and oral-gastric juice-intestinal juice digestion are respectively added into 2M sodium hydroxide solution and stirred for 18h at room temperature by a magnetic stirrer under the atmosphere of nitrogen. The mixture was then neutralized with 6M hydrochloric acid and extracted 6 times with ethyl acetate. The combined organic phases were evaporated to dryness in vacuo at 35 ℃ and redissolved in 85% methanol and then made to volume of 10mL to give bound phenol which was stored in a refrigerator at-20 ℃.

And (3) detection: 125. mu.L of the above bound phenol sample diluted 20 times was added with 125. mu.L of the forskolin phenol reagent and distilled water (0.50mL), respectively. After vortex shaking uniformly and light-shielding reaction for 6min, sequentially adding 1.25mL of Na₂CO₃Solution (7%, m/v) and 1mL of distilled water. Vortex and shake evenly, and then react for 90min in dark. The absorbance was measured at 760nm using a Shimadzu UV-1800 spectrophotometer. The total phenol content is expressed as Gallic Acid Equivalent (GAE) content per 100 g Dry Weight (DW) of litchi pulp, using gallic acid as a standard.

The experimental results are shown in fig. 2, from which it can be seen that the total phenol content of the polysaccharide-polyphenol adduct is increasing in the process of simulating in vitro digestion and colonic fermentation, probably because the polysaccharide-polyphenol adduct is hydrolyzed by various digestive enzymes, but the polyphenol content reaches the highest after colonic fermentation, probably through the fermentation of beneficial intestinal flora, more polyphenol is released, and also that the polyphenol existing in the bound form can be protected by dietary polysaccharide, transported to the colon, exert better probiotic effect, and regulate intestinal flora.

(2) pH change during colonic fermentation

And (3) placing the centrifuged fermentation tube on an ice box, and measuring the change of the pH values of the blank control and the digested litchi polysaccharide-polyphenol adduct at different time points in the fermentation process by using a pH meter.

The experimental result is shown in fig. 3, and it can be seen from the figure that the lychee polysaccharide-polyphenol adduct prepared by the scheme of the invention regulates intestinal flora by reducing the pH value, promotes the proliferation of the intestinal flora and maintains the health of the microenvironment of the colon.

(3) Short chain fatty acid changes during colonic fermentation

And respectively taking 1mL of the blank control group after centrifugation, fermenting for 48h and fermentation supernatant after fermenting for 48h by using the digested litchi polysaccharide-polyphenol adduct, filtering by using a 0.22 mu m aqueous phase membrane, and determining the content change conditions of acetic acid, propionic acid and butyric acid in the fermentation process by using a GC-2010plus gas chromatograph. The gas chromatography conditions were as follows: using DB-FEAP chromatographic column and detector FID, wherein the detection temperature and the injection port temperature are both 240 ℃, and the temperature rise program is 70-240 ℃. The carrier gas is nitrogen, the flow rate is 30mL/min, and the split ratio is 1: 9, the air flow rate is 400mL/min, and the hydrogen flow rate is 30 mL/min. The sample amount was 1. mu.L, and the measurement time was 42.47 min.

The experimental result is shown in fig. 4, and it can be seen from the figure that the lychee polysaccharide-polyphenol adduct prepared by the invention can regulate and control the generation of beneficial short-chain fatty acids formic acid, acetic acid and propionic acid in an intestinal system, regulate intestinal flora, and promote the proliferation of the intestinal flora, so as to improve the health of the microenvironment of the colon.

In conclusion, the litchi polysaccharide-polyphenol adduct prepared by the scheme of the invention can effectively improve the microenvironment of the colon, and has better effect compared with dried litchi, litchi polysaccharide or litchi polyphenol.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.