CN117204574B

CN117204574B - Pectin-based dietary supplement for promoting intestinal flora fermentation balance and preparation method thereof

Info

Publication number: CN117204574B
Application number: CN202311481700.2A
Authority: CN
Inventors: 赵晓燕; 赵圆圆; 王丹; 王盼; 赵爽; 郑鄢燕; 马越
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2023-11-09
Filing date: 2023-11-09
Publication date: 2024-02-02
Anticipated expiration: 2043-11-09
Also published as: CN117204574A

Abstract

The invention discloses a preparation method of a pectin-based dietary supplement for promoting intestinal flora fermentation balance, which comprises the following steps: step one, taking fruits and vegetables and processing byproducts thereof as raw materials, and sequentially intercepting by a sequential extraction method to obtain water-soluble pectin and hemicellulose; step two, adopting complex enzyme to carry out synergistic enzymolysis on the obtained water-soluble pectin to obtain an enzymolysis product, inactivating enzyme, and screening to obtain water-soluble RG-II pectin; step three, placing the obtained water-soluble RG-II pectin and hemicellulose into a mixed solution of arabinose and galactose for re-dissolution and freeze-drying to obtain a compound pectin base; and step four, taking 620-640 parts of the obtained compound pectin base, mixing 20-30 parts of sugarcane fiber, 5-15 parts of citrus fiber and 7-20 parts of soybean fiber, and performing reverse molding and shaping to obtain a pectin-based dietary supplement primary product. The invention can screen and optimize the type of pectin in functional food, accurately regulate and control the structure, realize the balance of carbohydrate fermentation and protein fermentation, and maintain or improve the intestinal health level.

Description

Pectin-based dietary supplement for promoting intestinal flora fermentation balance and preparation method thereof

Technical Field

The invention relates to the field of finish machining of fruits and vegetables and processing byproducts thereof. More particularly, the present invention relates to a pectin-based dietary supplement for promoting intestinal flora fermentation balance and a method of preparing the same.

Background

The functional products of fruits and vegetables which can accurately interfere health by adjusting the colon fermentation characteristic through adjusting the intestinal flora structure are favored in the current society. In a broad sense, fermentation of nutrients in the large intestine can be divided into carbohydrate fermentation and protein fermentation. The metabolites after carbohydrate decomposition are mainly short chain fatty acids, wherein the content of acetic acid, propionic acid and butyric acid is high, and the metabolites have biological activity and participate in regulating the physiological process of organisms. While microbial metabolism of proteins in the large intestine can lead to the formation of deleterious metabolites including nitrosamines, branched fatty acids, amines, phenols, thiols, hydrogen disulfide, ammonia, and the like. Numerous studies have demonstrated the health effects of oligosaccharides, inulin and other prebiotics, however, due to the rapid fermentation characteristics of low molecular weight prebiotics, a series of deleterious metabolites may also be produced. The intestinal fermentation process starts from the cecum to the end of the transverse colon, and rapidly fermented prebiotics may cause the far-end colon microorganisms to lack fermentable carbohydrates, which in turn selectively degrade proteins, peptides or fats, producing harmful metabolites. In contrast, pectin or other higher molecular mass polysaccharides can be degraded and utilized by the intestinal flora in the cecum, proximal colon, distal colon, but the extent of utilization depends on the fine structure of the pectin.

Pectic polysaccharide is a potential prebiotic, is beneficial to enhancing the activity of intestinal flora, and is involved in improving physiological activities such as organism immunity, obesity, hyperlipidemia, colonitis and the like. The probiotics effect of pectin is closely related to the microbial fermentation process of pectin in intestinal tracts, and the invention patent with publication number of CB106389378A and name of a pectin hollow capsule discloses a pectin hollow capsule which comprises the following components in parts by weight: 50-65 parts of pectin powder, 15-20 parts of polyacrylic resin, 15-20 parts of ethyl cellulose water dispersion, … … and 80-100 parts of purified water, and the health care function of protecting gastric mucosa and helping digestion is achieved by adding the pectin powder. However, the pectin is still widely and inaccurately utilized at present, the molecular weight of the pectin is too high, the fermentation speed of intestinal microorganisms is low, and especially the degradation ratio in cecum is low, so that the intestinal probiotics effect of the pectin is not fully exerted.

In order to achieve accurate intervention in health, the type of pectin in the functional food needs to be screened and optimized, the structure of the pectin needs to be accurately regulated and controlled, the balance of carbohydrate fermentation and protein fermentation is realized, and after the pectin-based functional food is fermented by intestinal flora at the front end and the rear end of colon, the proliferation of short chain fatty acid producing bacteria is promoted, and more beneficial metabolites are produced; inhibiting the activity of flora involved in protein fermentation, reducing the production and accumulation of harmful metabolites, and finally maintaining or improving the intestinal health level.

Disclosure of Invention

It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.

It is still another object of the present invention to provide a method for preparing pectin-based dietary supplements that can screen and optimize the type of pectin in functional foods, precisely control its structure, achieve a balance of carbohydrate fermentation and protein fermentation, and maintain or increase intestinal health levels.

To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a method for preparing a pectin-based dietary supplement for promoting fermentation balance of intestinal flora, comprising:

step one, taking fruits and vegetables and processing byproducts thereof as raw materials, sequentially intercepting by a sequential extraction method to obtain water-soluble pectin and hemicellulose, dialyzing, and freeze-drying to obtain solid water-soluble pectin and hemicellulose;

step two, adopting compound enzyme to carry out synergistic enzymolysis on the water-soluble pectin obtained in the step one to obtain an enzymolysis product, putting the enzymolysis product into steam with the temperature of 90-100 ℃ to inactivate enzyme for 3-8 min, and then screening to obtain water-soluble RG-II pectin;

step three, placing the water-soluble RG-II pectin and hemicellulose obtained in the step two into an arabinose and galactose mixed solution with the mass concentration of 60-80%, and re-dissolving and freeze-drying to obtain a compound pectin base for later use, wherein the mass ratio of the water-soluble RG-II pectin to the hemicellulose to the mixed solution is 25-40: 6-10: 80-100, wherein the mass ratio of solute arabinose to galactose in the mixed solution is 1-3: 1, a step of;

And step four, taking 620-640 parts by weight of the compound pectin base obtained in the step three, mixing with 20-30 parts by weight of sugarcane fiber, 5-15 parts by weight of citrus fiber and 7-20 parts by weight of soybean fiber, and carrying out reverse molding and shaping to obtain a primary pectin-based dietary supplement product.

In the above technical scheme, the fruits and vegetables and processing byproducts thereof comprise leaf vegetables, tuber vegetables, melon fruits, berries, melons, orange fruits, stone fruits, kernel fruits and processing byproducts thereof.

In the technical scheme, the water-soluble RG-II pectin and hemicellulose form a first-generation compound ligand, the first-generation compound ligand is dissolved in a mixed solution of arabinose and galactose, freeze-dried to form a second-generation compound ligand compound pectin base, and the second-generation compound ligand is mixed with sugarcane fiber, citrus fiber and soybean fiber to prepare a third-generation compound ligand pectin base dietary supplement primary product.

In the technical scheme, the specific operation of re-dissolving the water-soluble RG-II pectin and hemicellulose obtained in the step two in an arabinose and galactose mixed solution with the mass concentration of 60-80% is as follows: firstly, the mass ratio is 1-3: 1, arabinose and galactose are dissolved in water to form a mixed solution with the mass concentration of 60-80%, and then water-soluble RG-II pectin and hemicellulose are dissolved in the mixed solution, wherein the mass ratio of the water-soluble RG-II pectin to the hemicellulose to the mixed solution is 25-40: 6-10: 80-100.

Preferably, the preparation method of the pectin-based dietary supplement further comprises the following step five: spraying 0.05-0.1% calcium chloride solution on each side of the primary pectin-based dietary supplement product obtained in the step four, and placing the primary pectin-based dietary supplement product into a vacuum freeze drying bin for drying until the mass water content of the material is lower than 5%, grading, selecting, boxing, filling nitrogen and packaging to obtain the pectin-based dietary supplement.

In the technical scheme, the condition of vacuum freeze drying after the pectin-based dietary supplement initial product is sprayed with the calcium chloride solution is that the cold trap temperature is lower than-65 ℃ and the material temperature is 30-50 ℃.

Preferably, in the first step, fruits and vegetables and processing byproducts thereof are taken as raw materials, and the method for obtaining the water-soluble pectin by sequential interception through a sequential extraction method comprises the following steps: beating fruits and vegetables and processing byproducts thereof into fruit and vegetable pulp, adding 1-2 MNaOH into the fruit and vegetable pulp, homogenizing with 95% ethanol for 1-2 h, suction-filtering to collect first filter residues, freeze-drying the obtained first filter residues, stirring in boiling water for 20-60 min, cooling, suction-filtering to obtain second filtrate and second filter residues, placing the second filtrate in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing in water for 36-72 h, freeze-drying to obtain water-soluble pectin, and placing in a dryer for standby.

In the above technical solution, preferably, the method for treating the second filtrate may further include: mixing the second filtrate and the flushing liquid, performing rotary evaporation concentration to 400-600 mL, placing the concentrated solution in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing in water for 36-72 h, and freeze-drying to obtain water-soluble pectin, and placing in a dryer for standby.

In the above technical scheme, the conditions for freeze-drying the first filter residue are as follows: the vacuum degree is 5-10 Pa; the temperature of the cold trap is minus 65 to minus 40 ℃; the temperature of the tray is 60-90 ℃; the lyophilization conditions of the contents in the dialysis bag were: the vacuum degree is 5-10 Pa; the temperature of the cold trap is minus 65 to minus 40 ℃; the temperature of the tray is 60-90 ℃.

Preferably, in the first step, fruits and vegetables and processing byproducts thereof are taken as raw materials, and the method for obtaining hemicellulose by sequential interception through a sequential extraction method comprises the following steps: dissolving the second filter residue in 0.1-0.5M potassium acetate solution containing 0.05-0.5M CDTA, filtering to obtain a third filtrate and a third filter residue, and dissolving the third filter residue in solution containing 0.02-0.1 MNaBH ₄ Collecting insoluble residues in 0.1-0.5M NaOH solution, and adding a solution containing 4-6M KOH and 0.02-0.1M NaBH ₄ In a borate solution of N ₂ Stirring at room temperature for 10-20 h, filtering to obtain a fourth filtrate, adjusting the pH value of the fourth filtrate to 2-5 by using an HCl solution, adding 1-5 mL of ethanol, placing the precipitate in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing in water for 36-72 h, and freeze-drying to obtain hemicellulose for later use.

In the above technical solution, the method for obtaining the third filtrate and the third filter residue may further be: and dissolving the second filter residue in 0.1-0.5M potassium acetate solution containing 0.05-0.5M CDTA, regulating the pH value to 6.5, stirring at 24 ℃ for 10-20 min, then placing in a constant-temperature shaking incubator at 28 ℃ for 6-8 h, and respectively collecting supernatant as a third filtrate and lower residues as a third filter residue.

In the technical scheme, the specific operation mode of the method for extracting the fruit and vegetable processing byproducts and intercepting to obtain hemicellulose is as follows: and (3) filling the third residue and residue into a Schott bottle, adding the mixed solution, putting into a rotor, filling nitrogen into the bottle, immediately covering a bottle cap after about 30 seconds, and stirring for 10-20 hours at room temperature. Filtering the solution by using a filter sintered by glass, wherein the pore diameter of the filter element is between 16 and 40 mu m at most; the Schott bottle and filter were rinsed with a small amount of deionized water and the filtrates were combined. The obtained filtrate was adjusted to pH 5 with HCl (placed in an ice bath, hydrochloric acid was added dropwise), 30mL of 37% HCl was added, after a rapid drop in pH was observed, HCl was slowly added dropwise to pH 5, and after pH adjustment, a few drops of 75% ethanol were added to reduce foaming. And (3) placing the precipitate in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing in water for 36-72 h, and freeze-drying to obtain hemicellulose for later use.

Preferably, the method for preparing the water-soluble pectin by the complex enzyme in the second step and the enzymolysis step comprises the following steps: setting the final concentration of the water-soluble pectin as 2-5wt% for the water-soluble pectin, adjusting the pH of the system to 4-4.6, adding polygalacturonase and polygalacturonase exonuclease, and carrying out enzymolysis for 1-4 hours at 35-55 ℃; and then regulating the pH value of the system to 5.5-6.5, continuously adding pectin lyase and pectin methylesterase, and carrying out enzymolysis for 1-4 hours at 50-70 ℃ to obtain an enzymolysis product containing water-soluble RG-I pectin and water-soluble RG-II pectin, wherein the final concentration of polygalacturonic acid endoenzyme, polygalacturonic acid exoenzyme pectin lyase and pectin methylesterase in the water-soluble pectin is 0.2-1U/mL.

Preferably, the method for screening to obtain the water-soluble RG-II pectin in the second step comprises the following steps: and (3) performing DEAE-cellulose column chromatography, eluting the enzyme-inactivated zymolyte with distilled water for 2-5 h to obtain a neutral sugar fraction, continuing eluting with 0.5-2.0M NaCl solution to obtain an acidic sugar fraction, eluting the obtained acidic sugar fraction with 0.5-2.0M NaCl solution in high-resolution size exclusion chromatography, collecting the acidic sugar fraction with the elution time in the range of 10-15 min, and screening and separating by using a thiobarbituric acid method to obtain the water-soluble RG-II pectin.

Preferably, in the second step, the enzyme deactivation treatment is to wrap the zymolyte with a PTFE filtering ventilation sealing film, and deactivate the enzyme for 3-8 min under the steam at the temperature of 90-100 ℃.

Preferably, vegetable oil is coated in the die before the die is inverted in the fourth step.

In the above technical scheme, the vegetable oil is preferably peony seed oil, olive oil and rapeseed oil, and the most preferred vegetable oil is peony seed oil.

The invention further claims a pectin-based dietary supplement prepared using the method of preparing the pectin-based dietary supplement.

The invention at least comprises the following beneficial effects:

1. the preparation method of the pectin-based dietary supplement for promoting the fermentation balance of intestinal flora, provided by the invention, can screen and optimize the type of pectin in functional food, accurately regulate and control the structure of the pectin, realize the balance of carbohydrate fermentation and protein fermentation, and promote the proliferation of short-chain fatty acid producing bacteria after the pectin-based functional food is fermented by the intestinal flora at the front end and the rear end of colon, so as to produce more beneficial metabolites; inhibiting the activity of the flora involved in protein fermentation, reducing the production and accumulation of harmful metabolites, maintaining or increasing the intestinal health level;

2. The preparation method of the pectin-based dietary supplement for promoting the fermentation balance of intestinal flora fills the technical blank of screening water-soluble RG-II pectin, and accurately compounds the water-soluble RG-II pectin and insoluble dietary fibers in a certain proportion, so that the material structure of pectin-based functional food is changed, the fermentation speed of the pectin-based functional food by large intestine microorganisms is influenced, and the microbial metabolites are accurately regulated and controlled;

3. according to the preparation method of the pectin-based dietary supplement for promoting intestinal flora fermentation balance, alkali liquor is added into the extracting solution of fruits and vegetables and processing byproducts thereof, so that on one hand, the structure of cell walls is damaged, pectin is easier to extract, on the other hand, the cross-linking among pectin, hemicellulose and cellulose is damaged, more pectin with various structures is dissolved out, and then RG-II structural domains with smaller molecular weight and higher proportion are prepared by continuously separating water-soluble pectin through directional secondary enzymolysis;

4. according to the preparation method of the pectin-based dietary supplement for promoting the fermentation balance of intestinal flora, disclosed by the invention, neutral sugar is added by a method of re-dissolving pectin and insoluble dietary fiber compound in neutral sugar solution and then freeze-drying, and specific neutral sugar is added into insoluble RG-II pectin and insoluble dietary fiber compound, so that the intestinal microbial diversity in a fermentation stage is obviously increased, and meanwhile, the production amount of beneficial metabolites is increased;

5. The pectin-based dietary supplement prepared by the preparation method provided by the invention has the pectin-based yield of 21.95g/100 g d.b., the molecular weight of 223.47kg/mol, and the contents of galacturonic acid, rhamnose, arabinose, galactose, xylose, glucose and fucose of 533.88, 20.52, 48.40, 49.06, 19.14, 6.35 and 1.17mg/g respectively, and moderate viscosity, can effectively reach the far-end colon, and can generate a large amount of beneficial metabolite acetic acid (36.75 mmol/L) and a small amount of harmful metabolite free ammonia (3.28 mmol/L);

6. the pectin-based dietary supplement prepared by the preparation method for promoting the fermentation balance of intestinal flora provided by the invention has the advantages that the degradation rate of the pectin-based dietary supplement in the intestinal tract exceeds 80% within 24 hours, the total short-chain fatty acid production rate is higher than 200%, and meanwhile, the acetic acid and propionic acid production rates are controlled to be stable at 75-80% within the fermentation periods of 3, 6, 12 and 24 hours, so that the protein fermentation is effectively inhibited, the occurrence time of the pectin-based dietary supplement at the tail end of the colon is later, and the fermentation speed is slower;

7. the pectin-based dietary supplement prepared by the preparation method for promoting the fermentation balance of intestinal flora provided by the invention is rich in arabinose and galactose, and is a good carbon source of the phylum firmicutes. After the product is fermented by polysaccharide degrading bacteria, the diversity of bacteria in intestinal tracts can be obviously increased, the richness, uniformity and coverage of flora are improved, short chain fatty acid producing bacteria (helicobacter, bacteroides, actinomycetes, candida, bifidobacteria, megamonas, clostridium praecox and ruminococcus) are obviously enriched, and meanwhile, the abundance of flora (propionibacterium sclerotium and prasuvorexa) related to protein fermentation is reduced. Short chain fatty acids play a critical role in maintaining intestinal balance and host health. Wherein the helicobacter, bacteroides, ruminococcus, bifidobacterium and actinomycetes can convert glucose into acetic acid, and can inhibit appetite and regulate fat absorption; the chaetomium and the ruminococcus can produce propionic acid and butyric acid, wherein the butyric acid is an important energy source of intestinal cells, can regulate the growth and apoptosis of epithelial cells and lymphocytes and promote the growth and differentiation of the epithelial cells and the lymphocytes, thereby being beneficial to preventing ulcerative colitis and colon cancer; the megamonas produces valeric acid and caproic acid simultaneously, and bacteroides, praecox, candida and the like can utilize pentose or hexose to synthesize propionic acid, which can participate in lipid metabolism and inhibit synthesis of cholesterol;

8. The invention provides the method for representing the fermentation speed of the carbohydrate by the generation rate of short-chain fatty acid (acetic acid, propionic acid and butyric acid) and the fermentation speed of the protein by the generation rate of free ammonia, indole and hydrogen sulfide for the first time, realizes the relative characterization of the fermentation speed of the carbohydrate and the fermentation speed of the protein, and lays a foundation for the accurate research of intestinal health.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a flow chart of the preparation of a pectin-based dietary supplement in accordance with one embodiment of the present invention;

FIG. 2 is a Fourier infrared spectrum of pectin-based materials in the dietary supplements of example 1 and comparative examples 1-5 of the present invention;

FIG. 3 is an Atomic Force Scanning Electron Microscope (AFSEM) of pectin-based dietary supplements of example 1 and comparative examples 1-5 of the present invention;

FIG. 4 is a graph showing the dissolution effect of pectin-based on the dietary supplements of example 1 and comparative examples 1-5 of the present invention;

FIG. 5 is a graph showing the apparent viscosity of pectin in the dietary supplements of examples 1 and comparative examples 1-5 of the present invention;

FIG. 6 is a graph showing the content of acetic acid, propionic acid and butyric acid after fermentation of the dietary supplements of example 1 and comparative examples 1 to 7 of the present invention;

FIG. 7 is a graph showing the content of isopropyl acid and isobutyric acid after fermentation of the dietary supplements of example 1 and comparative examples 1-7 of the present invention;

FIG. 8 is a graph showing the content of free ammonia after fermentation of the dietary supplements of example 1 and comparative examples 1-7 of the present invention;

figure 9 is a graph of intestinal flora abundance after fermentation of the dietary supplement of example 1 of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments so that those skilled in the art can practice the same by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

Example 1 (Using beet root as a raw material)

Step one, extracting water-soluble pectin: beating beet root into beet pulp, adding 2MNaOH into beet pulp, homogenizing with 95% ethanol for 2 hr, vacuum filtering to collect first residue, freeze drying the first residue, stirring in boiling water for 20min to obtain suspension, cooling the suspension, vacuum filtering to obtain second filtrate and second residue, placing the second filtrate in dialysis bag with molecular weight cutoff of 3500Da, dialyzing in water for 72 hr, lyophilizing to obtain water-soluble pectin, and placing in a dryer for use;

Extraction of hemicellulose: dissolving the second filter residue in 0.1-0.5M potassium acetate solution containing 0.05M CDTA, filtering to obtain a third filtrate and a third filter residue, and dissolving the third filter residue in solution containing 0.02M NaBH ₄ In a 0.5M NaOH solution, collecting insoluble residues and adding a solution containing 4M KOH, 0.02M NaBH ₄ In a 3.5% borate solution in N ₂ Stirring at room temperature under environment for 20 h, filtering to obtain fourth filtrate, adjusting pH to 2 with HCl solution, adding 2.5 mL ethanol, and standing the precipitate with molecular weight cut-off of 3500DaDialyzing in water for 72h, and lyophilizing to obtain hemicellulose;

setting the final concentration of the water-soluble pectin as 5 wt%, regulating the pH value of the system to 4.5, adding the polygalacturonic acid endoenzyme with the final concentration of 0.2U/mL and the polygalacturonic acid exoenzyme with the final concentration of 0.2U/mL, and performing enzymolysis for 2 hours at 45 ℃; then regulating the pH value of the system to 6, continuously adding pectin lyase with the final concentration of 0.2U/mL and pectin methylesterase with the final concentration of 0.2U/mL, and carrying out enzymolysis for 4 hours at 60 ℃ to obtain an enzymolysis product containing water-soluble RG-I pectin and water-soluble RG-II pectin, wrapping the enzymolysis product by adopting a PTFE filtering ventilation sealing film, and putting the enzymolysis product under steam at 100 ℃ for enzyme deactivation for 5 minutes;

Through DEAE-cellulose column chromatography, the enzymatic hydrolysate 5 h after enzyme deactivation is eluted with distilled water to obtain a neutral sugar fraction, the neutral sugar fraction is continuously eluted with 0.5M NaCl solution to obtain an acidic sugar fraction, the obtained acidic sugar fraction is eluted in high-resolution molecular exclusion chromatography through 0.5M NaCl solution, the acidic sugar fraction with the elution time within the range of 12.5min is collected, and the water-soluble RG-II pectin is obtained through screening and separation by using a thiobarbituric acid method.

Step three, placing the water-soluble RG-II pectin and hemicellulose obtained in the step two into an arabinose and galactose mixed solution with the mass concentration of 60%, and re-dissolving and freeze-drying to obtain a compound pectin base for later use, wherein the mass ratio of the water-soluble RG-II pectin to the hemicellulose to the mixed solution is 25:6: 80, wherein the mass ratio of solute arabinose to galactose in the mixed solution is 1:1, a step of;

step four, taking 620 parts of the compound pectin base obtained in the step three, mixing 20 parts of sugarcane fiber, 5 parts of citrus fiber and 7 parts of soybean fiber, adding the compound ingredients, uniformly mixing, pouring the mixture into a die with the length, width and height dimensions of 5cm multiplied by 1cm multiplied by 0.5cm, and quick-freezing and shaping to obtain a pectin-based dietary supplement primary product;

and fifthly, spraying 0.05% calcium chloride solution on each side of the primary pectin-based dietary supplement product obtained in the step four, placing the primary pectin-based dietary supplement product into a vacuum freeze drying bin for drying until the mass water content of the material is lower than 5%, grading, selecting, boxing, filling nitrogen and packaging to obtain the pectin-based dietary supplement.

Example 2

Step one, extracting water-soluble pectin: beating apples into apple pulp, adding 1MNaOH into the apple pulp, homogenizing with 95% ethanol for 1h, collecting first filter residues through a Buchner funnel, freeze-drying the obtained first filter residues, stirring in boiling water for 60min to obtain a suspension, cooling the suspension, performing suction filtration to obtain second filtrate and second filter residues, placing the second filtrate in a dialysis bag with 3500Da molecular weight cutoff, dialyzing in water for 48h, lyophilizing to obtain water-soluble pectin, and placing in a dryer for standby;

extraction of hemicellulose: dissolving the second filter residue in 0.5M potassium acetate solution containing 0.5MCDTA, vacuum filtering to obtain a third filtrate and a third filter residue, dissolving the third filter residue in 0.1M NaBH solution ₄ In a 0.1M NaOH solution, collecting insoluble residues and adding a solution containing 6M KOH, 0.1M NaBH ₄ In a 3.5% borate solution in N ₂ Stirring at room temperature for 10h under the environment, filtering to obtain a fourth filtrate, adjusting the pH value of the fourth filtrate to 3 by using HCl solution, adding 5mL of ethanol, placing the precipitate in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing in water for 48h, and freeze-drying to obtain hemicellulose for later use;

setting the final concentration of the water-soluble pectin as 2 wt%, regulating the pH value of a system to 4.6, adding the polygalacturonic acid endoenzyme with the final concentration of 0.5U/mL and the polygalacturonic acid exoenzyme with the final concentration of 0.5U/mL, and performing enzymolysis for 4 hours at 45 ℃; then regulating the pH value of the system to 6.5, continuously adding pectin lyase with the final concentration of 0.5U/mL and pectin methylesterase with the final concentration of 0.5U/mL, and carrying out enzymolysis for 3 hours at 70 ℃ to obtain an enzymolysis product containing water-soluble RG-I pectin and water-soluble RG-II pectin, wrapping the enzymolysis product by adopting a PTFE filtering ventilation sealing film, and putting the enzymolysis product under steam at 100 ℃ for inactivating enzymes for 8 minutes;

Through DEAE-cellulose column chromatography, the enzymatic hydrolysate after enzyme deactivation is eluted with distilled water for 5h to obtain a neutral sugar fraction, the neutral sugar fraction is eluted with 2.0M NaCl solution, the obtained acidic sugar fraction is eluted in high-resolution molecular exclusion chromatography through 2.0M NaCl solution, the acidic sugar fraction with the elution time within 15min is collected, and the water-soluble RG-II pectin is obtained through screening and separation by using a thiobarbituric acid method.

Step three, placing the water-soluble RG-II pectin and hemicellulose obtained in the step two into an arabinose and galactose mixed solution with the mass concentration of 80%, and re-dissolving and freeze-drying to obtain a compound pectin base for later use, wherein the mass ratio of the water-soluble RG-II pectin to the hemicellulose to the mixed solution is 40:10: 100, wherein the mass ratio of solute arabinose to galactose in the mixed solution is 3:1, a step of;

mixing 630 parts of the compound pectin base obtained in the step three, 25 parts of sugarcane fiber, 10 parts of citrus fiber and 15 parts of soybean fiber, adding the compound ingredients, uniformly mixing, pouring the mixture into a die with the length, width and height dimensions of 5cm multiplied by 1cm multiplied by 0.5cm, and quick-freezing and shaping to obtain a pectin-based dietary supplement primary product;

and fifthly, spraying 0.08% calcium chloride solution on each side of the primary pectin-based dietary supplement product obtained in the step four, placing the primary pectin-based dietary supplement product into a vacuum freeze drying bin for drying until the mass water content of the material is lower than 5%, grading, selecting, boxing, filling nitrogen and packaging to obtain the pectin-based dietary supplement.

Example 3

Step one, extracting water-soluble pectin: beating sunflower meal into sunflower meal pulp, adding 1.5MNaOH into the sunflower meal pulp, homogenizing with 95% ethanol for 1.5h, collecting first filter residue by suction filtration through a Buchner funnel, freeze-drying the obtained first filter residue, stirring in boiling water for 40min to obtain suspension, cooling the suspension, suction-filtering to obtain second filtrate and second filter residue, placing the second filtrate in a dialysis bag with molecular weight cutoff of 3500Da, dialyzing in water for 36h, lyophilizing to obtain water-soluble pectin, and placing in a dryer for standby;

extraction of hemicellulose: dissolving the second filter residue in 0.3M potassium acetate solution containing 0.25M CDTA, vacuum filtering to obtain third filtrate and third filter residue, and dissolving the third filter residue in solution containing 0.08M NaBH ₄ In a 0.4M NaOH solution, collecting insoluble residues and adding a solution containing 5M KOH, 0.06M NaBH ₄ In a 3.5% borate solution in N ₂ Stirring at room temperature for 15 hr, filtering to obtain fourth filtrate, and adjusting pH of the fourth filtrate to be the same as that of the fourth filtrate by HCl solution5, adding 5mL of ethanol, placing the precipitate in a dialysis bag with molecular weight cut-off of 3500Da, dialyzing in water for 36h, and freeze-drying to obtain hemicellulose for later use;

setting the final concentration of the water-soluble pectin as 3 wt%, regulating the pH value of a system to 4.6, adding the polygalacturonase with the final concentration of 1U/mL and the polygalacturonase with the final concentration of 1U/mL, and performing enzymolysis for 4 hours at 55 ℃; then regulating the pH value of the system to 6.5, continuously adding pectin lyase with the final concentration of 1U/mL and pectin methylesterase with the final concentration of 1U/mL, and placing the mixture at 70 ℃ for enzymolysis for 3 h to obtain an enzymolysis product containing water-soluble RG-I pectin and water-soluble RG-II pectin, wrapping the enzymolysis product by adopting a PTFE filtering ventilation sealing film, placing the enzymolysis product at 100 ℃ for inactivating enzymes for 3min;

Through DEAE-cellulose column chromatography, the enzymatic hydrolysate after enzyme deactivation is eluted with distilled water for 4 hours to obtain a neutral sugar fraction, the neutral sugar fraction is continuously eluted with 1.0M NaCl solution to obtain an acidic sugar fraction, the obtained acidic sugar fraction is eluted in high-resolution molecular exclusion chromatography through 1.0M NaCl solution, the acidic sugar fraction with the elution time within 15 minutes is collected, and the water-soluble RG-II pectin is obtained through screening and separation by using a thiobarbituric acid method.

Step three, placing the water-soluble RG-II pectin and hemicellulose obtained in the step two into an arabinose and galactose mixed solution with the mass concentration of 70%, and re-dissolving and freeze-drying to obtain a compound pectin base for later use, wherein the mass ratio of the water-soluble RG-II pectin to the hemicellulose to the mixed solution is 30:8:90, the mass ratio of solute arabinose to galactose in the mixed solution is 2:1, a step of;

mixing 630 parts of the compound pectin base obtained in the step three, 25 parts of sugarcane fiber, 10 parts of citrus fiber and 20 parts of soybean fiber, adding the compound ingredients, uniformly mixing, pouring the mixture into a die with the length, width and height dimensions of 5 cm multiplied by 1 multiplied by cm multiplied by 0.5 cm, and quick-freezing and shaping to obtain a primary pectin-based dietary supplement product;

and fifthly, spraying 0.06% calcium chloride solution on each side of the primary pectin-based dietary supplement product obtained in the step four, placing the primary pectin-based dietary supplement product into a vacuum freeze drying bin for drying until the mass water content of the material is lower than 5%, grading, selecting, boxing, filling nitrogen and packaging to obtain the pectin-based dietary supplement.

Example 4

Step one, extracting water-soluble pectin: beating apples, hawthorns, beetroots and sunflower meal into mixed fruit and vegetable pulp, adding 2M NaOH into the mixed fruit and vegetable pulp, homogenizing with 95% ethanol for 2h, collecting first filter residues through suction filtration of a Buchner funnel, freeze-drying the obtained first filter residues, stirring in boiling water for 20min to obtain suspension, cooling the suspension, suction-filtering to obtain second filtrate and second filter residues, placing the second filtrate in a dialysis bag with 3500Da molecular weight cutoff, dialyzing in water for 72h, freeze-drying to obtain water-soluble pectin, and placing in a dryer for standby;

extraction of hemicellulose: dissolving the second filter residue in 0.1-0.5M potassium acetate solution containing 0.05M CDTA, filtering to obtain a third filtrate and a third filter residue, and dissolving the third filter residue in solution containing 0.02M NaBH ₄ In a 0.5M NaOH solution, collecting insoluble residues and adding a solution containing 4M KOH, 0.02M NaBH ₄ In a 3.5% borate solution in N ₂ Stirring at room temperature for 20h under the environment, filtering to obtain a fourth filtrate, adjusting the pH value of the fourth filtrate to 2 by using HCl solution, adding 2.5mL of ethanol, placing the precipitate in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing in water for 72h, and freeze-drying to obtain hemicellulose for later use;

and (3) performing DEAE-cellulose column chromatography, eluting the enzyme-inactivated zymolyte with distilled water for 2-5 h to obtain a neutral sugar fraction, continuing eluting with 0.5-2.0M NaCl solution to obtain an acidic sugar fraction, eluting the obtained acidic sugar fraction with 0.5-2.0M NaCl solution in high-resolution size exclusion chromatography, collecting the acidic sugar fraction with the elution time in the range of 10-15 min, and screening and separating by using a thiobarbituric acid method to obtain the water-soluble RG-II pectin.

Step three, placing the water-soluble RG-II pectin and hemicellulose obtained in the step two into an arabinose and galactose mixed solution with the mass concentration of 65%, and re-dissolving and freeze-drying to obtain a compound pectin base for later use, wherein the mass ratio of the water-soluble RG-II pectin to the hemicellulose to the mixed solution is 350:7:85, the mass ratio of solute arabinose to galactose in the mixed solution is 3:1, a step of;

Mixing 640 parts of the compound pectin base obtained in the step three, 30 parts of sugarcane fiber, 15 parts of citrus fiber and 20 parts of soybean fiber, adding the compound ingredients, uniformly mixing, pouring the mixture into a die with the length, width and height dimensions of 5cm multiplied by 1cm multiplied by 0.5cm, and quick-freezing and shaping to obtain a pectin-based dietary supplement primary product;

and fifthly, spraying 0.1% calcium chloride solution on each side of the primary pectin-based dietary supplement product obtained in the step four, placing the primary pectin-based dietary supplement product into a vacuum freeze drying bin for drying until the mass water content of the material is lower than 5%, grading, selecting, boxing, filling nitrogen and packaging to obtain the pectin-based dietary supplement.

Comparative example 1

A pectin-based dietary supplement was prepared using the method of example 1, with the difference that: in the step 2, the water-soluble pectin obtained in the step 1 is not subjected to synergistic enzymolysis by using a complex enzyme, but is subjected to enzymolysis by using a pectin lyase and a single enzyme, and other steps are the same as those in the example 1.

The dietary supplement obtained in comparative example 1 is rich in HG units, has a linear molecular weight, a large molecular weight, a low polydispersity index, a high galacturonic acid content, a high fermentation speed, and a high content of free ammonia produced after 24 hours of fermentation.

Comparative example 2

A pectin-based dietary supplement was prepared using the method of example 1, with the difference that: in the step 1, 2MNaOH is added into beet pulp, and after the beet pulp is homogenized with 95% ethanol 2 h, the beet pulp is subjected to suction filtration by a Buchner funnel, filter residues are collected, and the filter residues are subjected to freeze drying to obtain alcohol insoluble substances, so that the extraction of hemicellulose is consistent with that of the example, the alcohol insoluble substances are used as objects to prepare water-soluble RG-II pectin according to the step 2, the water-soluble RG-II pectin is compounded with the hemicellulose according to the step 3, and other steps are the same as those of the example 1.

The dietary supplement obtained in comparative example 2 has higher galacturonic acid content, low polydispersity, better solubility in water, dense network structure of molecular chains and more aggregation. The fermentation speed is slower, and the content of acetic acid produced after 24 hours of fermentation is lower.

Comparative example 3

A pectin-based dietary supplement was prepared using the method of example 1, with the difference that: in the step 3, water-soluble RG-II pectin is not separated, and is directly compounded with hemicellulose, and the step 4 is referred to the compounding of sugarcane fiber, citrus fiber and soybean fiber, wherein the proportion is 3:1:0.5:0.5, the other steps are the same as in example 1.

The dietary supplement obtained in comparative example 3 has lower neutral sugar content, higher galacturonic acid content, rich insoluble dietary fiber, poor solubility, higher viscosity, and fibrous molecular chain. The fermentation is slow, the relative abundance of short-chain fatty acid producing bacteria is low after 24 and h, and the content of the produced short-chain fatty acid is low.

Comparative example 4

A pectin-based dietary supplement was prepared using the method of example 1, with the difference that: in the step 3, the step of re-dissolving in the mixed solution of arabinose and galactose was omitted, and the other steps were the same as in example 1.

The dietary supplement of comparative example 4 had lower arabinose and galactose content, more short chains, fewer micellar aggregates, and less acetic acid and butyric acid after fermentation 24 h without reconstitution.

Comparative example 5

A pectin-based dietary supplement was prepared using the method of example 1, with the difference that: the first generation complex is re-dissolved in the mixed solution of arabinose and galactose, freeze-dried to form a second generation complex, the step of compounding with sugarcane fiber, citrus fiber and soybean fiber is omitted in the step 4, and other steps are the same as in the example 1.

The obtained dietary supplement has the highest fermentation speed, and the relative abundance of the floras participating in protein fermentation such as propionibacterium sclerotium, prevotella and the like is higher, so that more harmful metabolites are produced.

Comparative example 6

Beet pectin prepared according to the commercial acid extraction and alcohol precipitation process is not subjected to any structural modification treatment.

Comparative example 7

Fructooligosaccharides are directly used as dietary supplements.

The pectin-based structures in the pectin-based dietary supplements obtained in example 1 and comparative examples 1-7 were compared and analyzed as follows:

characterization of the fine structure of the pectin domain fragment:

(1) The molecular weight of the pectin group was determined using high resolution size exclusion chromatography in combination with an eighteen-angle laser scatterometer (HPSEC-MALLS-RI) using NaCl solution as eluent, as follows: accurately weighing 5.0mg pectin sample, dissolving in 0.1 mM NaCl solution (mobile phase), filtering with 0.22 μm MF-Millipore filtration membrane, manually feeding 200 μl through quantitative loop, and setting flow rate to 0.5mL/min. The pectin was calculated and analyzed for weight average molecular mass (Mw), number average molecular weight (Mn) and polydispersity index (Mw/Mn) using ASTRA 5.3.4 software (Wyatt Technology, santa Barbara, calif., USA) to set the refractive index delta (dn/dc) to 0.135mL/g.

(2) The content of galacturonic acid in pectin base is determined by p-hydroxyphenol sulfuric acid method using a spectrophotometer, and the experimental method is as follows: accurately weighing 10.0mg pectin sample, sequentially adding 8.0mL concentrated sulfuric acid and 4.0mL distilled water, and hydrolyzing for 1h. 0.6mL of the hydrolysate was pipetted into a stoppered glass tube placed in an ice bath and 3.6mL of sodium tetraborate solution in concentrated sulfuric acid (98%) was added. Heating the glass tube with the plug in an oil bath at 100 ℃ for 5min, and then flushing the glass tube with flowing water to quickly cool. Adding 60.0 μl of p-phenylphenol solution (0.166. 0.166 g of 3-phenylphenol dissolved in 0.5% NaOH, constant volume to 100mL, and preserving in dark place) to pectin sample; the blank sample was added with 60.0. Mu.l of 0.5% NaOH and mixed well on a vortex for 1min for color development. The absorbance values of the standard, blank and pectin samples were determined using a UV-1800 UV spectrophotometer at 520 nm. And (3) preparing a standard curve by adopting a galacturonic acid standard with a concentration gradient of 40-240 mg/L.

(3) The neutral sugar composition and content in the pectin base were measured using a high performance anion chromatograph (HPAEC-PAD) as follows: accurately weighing 10.0mg of pectin, adding 4.0mL of 2.0 mM TFA, hydrolyzing at 110deg.C for 1.5h, drying the sample with nitrogen, and fixing the volume to 10mL with distilled water. Then the pectin neutral sugar is quantitatively analyzed by a 0.45 mu m MF-Millipore filter membrane and by adopting high performance anion exchange chromatography combined with pulse amperometric detection (HPAEC-PAD). The chromatographic conditions were as follows: the system was equilibrated with 100 mM NaOH for 5min, followed by 4.0mM NaOH for 5min, and the eluate was separated by using a CarboPac PA20 column (Dionex) with 4.0mM NaOH eluent, set at 30℃at a flow rate of 0.5mL, and the sample was introduced at 10.0. Mu.L. And (3) preparing a standard curve by adopting a neutral sugar mixed standard (D-uronic acid, rhamnose, arabinose, galactose, glucose and xylose) with a concentration gradient of 0.01-5.0 mg/L.

(4) Scanning was performed using a fourier transform infrared spectrometer (FT-IR) to analyze the type of functional groups of the pectin base, as follows: a spectrum grade potassium bromide powder was used, and pectin samples were prepared at 99:1, mixing and grinding the materials according to the proportion, and tabletting by a tabletting machine. Spectral scans were performed with potassium bromide powder tablets as background blanks. Fourier transform infrared spectrum is utilized, and the frequency is 4000-400 cm ^-1 Within a range of 4cm ^-1 The transmittance of the pectic polysaccharide was determined.

(5) By passing through ¹ H nuclear magnetic resonance spectrum [ ] ¹ H-NMR) in combination with hydrophilic interaction liquid chromatography (UHPLC) to resolve the sugar residues and glycosidic linkages of the pectic group, the NMR experimental procedure is as follows: pectin (10 mg/mL) was dissolved in D containing 0.1. Mu.L acetone ₂ O (99.96%) and 600.0. Mu.L was transferred to a 5mm NMR tube. After 5min of temperature equilibration, a standard pulse sequence presaturated with water signal was used to measure at 55 °c ¹ H-NMR spectrum. At a pulse angle of 9After 1024 scans at 0 °, the spectral data was collected into 16.4K data points, setting the mixing time to 10ms, the scan width to 10504.2Hz, the acquisition time to 3.0s, and the relaxation delay to 4.0s. Automatic phase, baseline correction, and alien peak identification were performed in Topspin software (version 3.2, bruker) and peak areas were quantified using mestenova (version 10.0.1, mestreLab Research). The UHPLC experimental method is as follows: pectin was diluted with 50% (v/v) acetonitrile and analyzed through a Vanquish UHPLC system (Thermo Scientific) equipped with an Acquiy amide column (1.7 μm,2.1 mm X150 mm), set at a flow rate of 0.4mL, and at a temperature of 40℃and process the data using Xcalibur 2.2 software (Thermo Scientific).

The flow chart of the preparation of pectin-based dietary supplements prepared in examples 1-4 is shown in FIG. 1. The yields and molecular weight distributions of the pectin groups in the different dietary supplements prepared in example 1 and comparative examples 1 to 5 are shown in table 1, the monosaccharide compositions and the saccharide ratios of the pectin groups in the different dietary supplements prepared in example 1 and comparative examples 1 to 5 are shown in table 2, the fourier infrared spectrograms of the pectin groups in the different dietary supplements prepared in example 1 and comparative examples 1 to 5 are shown in fig. 2, and the atomic force scanning electron microscope (afsem) charts of the pectin groups in the different dietary supplements prepared in example 1 and comparative examples 1 to 5 are shown in fig. 3.

TABLE 1 pectin based yields and molecular weight distribution in different dietary supplements

Note that: mw, weight average molecular weight; mn, number average molecular weight; mw/Mn, polydispersity.

As can be seen from Table 1, the dietary supplement of example 1 had a higher pectin content (21.95 g/100 g), and was inferior to comparative example 2, demonstrating that the procedure used in example 1 maximizes pectin extraction. The dietary supplement prepared in comparative example 2 was highest because the plant-based food was rich in alcohol insoluble materials and therefore had higher yields. In addition, the dietary supplements prepared in example 1 had moderate Mw (223.47 kg/mol) and Mn (20.20 kg/mol) but a larger polydispersity (11.063), indicating a broader molecular mass distribution, which would facilitate colonization of intestinal microorganisms and thus degradation of pectin.

TABLE 2 pectin-based monosaccharide composition and sugar ratios in different dietary supplements

Note that: galA, galacturonic acid; rha, rhamnose; ara, arabinose; gal, galactose; xyl, xylose; glu, glucose; fuc, fucose. Monosaccharide content unit: mg/g. Values are shown as mean ± standard error (n=3). The average value of different letters in the same column has obvious differencep<0.05）。

As can be seen from the neutral sugar composition of the dietary supplements in Table 2, the pectin content of arabinose (48.40 mg/g) and galactose (49.06 mg/g) in the dietary supplement prepared in example 1 was highest compared to comparative examples 1-5, which is related to the reconstitution of the pectin base in the arabinose and galactose mixed solution. The conventional acid thermal extraction method of pectin can lead pectin to have beta-elimination reaction, so that rhamnose, galactose and arabinose are easier to remove, thereby reducing the length of branched chains and increasing the linearity of molecules, but the pectin is redissolved in a mixed solution containing galactose and arabinose and then freeze-dried, so that galactose and arabinose can be effectively connected to a pectin chain in a sugar residue mode, and a secondary pectin-based compound rich in galactose and arabinose is obtained.

In addition, it can be seen from Table 2 that the dietary supplement prepared in comparative example 1 has a higher content of GalA (688.17 mg/g) in the pectin base, followed by comparative example 3 (570.50 mg/g) and example 1 (533.88 mg/g), indicating a higher proportion of pectin HG domains. This is because the raw material in comparative example 1 was not digested by pectase, and the HG domain was retained; comparative example 3 HG pectin base was selected for compounding; example 1 selects water-soluble pectin rich in HG domains. Comparative example 2 AIR was chosen as the pectin-based starting material, resulting in the highest xylose and glucose content. The dietary supplement prepared in comparative example 3 contained more insoluble dietary fiber than the other examples, so the xylose (24.49 mg/g) and glucose (15.53 mg/g) contents were the greatest, followed by comparative example 2 (15.47 mg/g,12.13 mg/g) because it selected AIR as the pectin-based starting material. However, comparative example 5 was not formulated with sugar cane fiber, citrus fiber and soybean fiber, so that the xylose (3.91 mg/g) and glucose (4.97 mg/g) contents were the lowest, but the xylose (19.14 mg/g) and glucose (6.35 mg/g) contents in this example were moderate, which increased the proportion of insoluble dietary fiber, delayed the fermentation rate, and the content of monosaccharides that could be fermented by microorganisms was less, thereby reducing the content of active ingredients, affecting the dietary supplement to exert its intestinal physiological activity.

As can be seen from the Fourier infrared spectra of FIG. 2, the pectin in the dietary supplements prepared in example 1 and comparative examples 1-5 have a spectrum of 3421-3400 cm ⁻¹ Region sum 2922-2933 cm ⁻¹ Similarly, the above dietary supplements were described as being rich in pectic polysaccharides. 534-618 cm ⁻¹ The absorption peak at this point was attributed to the stretching vibration of c=o in pectin GalA, and pectin in the dietary supplement prepared in example 1 had a pectin characteristic peak. However, comparative examples 4 and 5 did not find a narrow peak of strong absorption in v (c=o), confirming the lower GalA content of both. Located at 1047 and 1047 cm ⁻¹ The absorption peak at which represents the C-C stretch of cellulose, and at 941cm ⁻¹ The absorption peak at this point represents the hemicellulose ring oscillation, other than comparative example 5, at 1047 and 1047 cm ⁻¹ And 941cm ⁻¹ Absorption peaks were seen in the vicinity. Wherein, comparative 2 and comparative 3 are at 1047 cm ⁻¹ The strong absorption in the vicinity further explains the greater cellulose enrichment in both.

The molecular chain conformation of pectin was studied, see figure 3. The results show that pectin in the dietary supplement was observed by atomic force microscopy. The pectin in the dietary supplements prepared in example 1, comparative example 2, and comparative example 3 were found to have a highly crosslinked network structure with abundant sugar side chains. In contrast, comparative example 3, among others, is rich in insoluble dietary fibers, and the molecular chain takes on a fibrous form. In addition, the dietary supplement prepared in example 1 showed more long-chain cross-linking in the pectin due to van der Waals forces and hydrogen bonding that loosely bound RG-II pectin to cellulose and hemicellulose. At the same time, the side chains of the "smooth region" and the large number of "hair regions" induce their molecules to form a network structure with intramolecular and intermolecular crosslinks or entanglement. Whereas a shorter linear structure was observed in comparative example 1, this may be due to a higher HG domain ratio.

The pectin-based dietary supplements obtained in example 1 and comparative examples 1-7 were compared for in vitro simulated intestinal fermentation characteristics as follows:

intestinal microbial fermentation characterization:

(1) In vitro intestinal microbial fermentation model: adopting a fermentation enrichment medium to simulate a static colon environment in a sterile fermentation tank, adding experimental samples serving as carbon sources into the sterilized medium, respectively collecting 6 healthy adult feces under the sterile condition, diluting with a phosphoric acid buffer solution, inoculating into the medium, and fully homogenizing. Placing the fermentation bottle in a fermentation tank, vacuumizing, flushing nitrogen to maintain an anaerobic environment, and placing the fermentation bottle in a constant-temperature incubator to perform anaerobic culture at 37 ℃.

(2) Environmental factors: measuring the pH change of the in vitro fermentation liquid during fermentation 3, 6, 12 and 24 and h respectively; and analyzing the intestinal fermentation gas production amount by analyzing the piston position change of the fermentation system at different culture times.

(3) Carbohydrate metabolite content: centrifuging to obtain fermentation supernatant, and detecting the composition and content change of short chain fatty acid and branched chain fatty acid in fermentation broth by metaphosphoric acid acidification with Gas Chromatograph (GC).

(4) Protein metabolite content: an ammonia nitrogen test kit is adopted, ammonium chloride is used as a standard substance, and spectrophotometry is used for measuring the content change of free ammonia in fermentation liquor; measuring the content of dimethyl mercaptan and hydrogen sulfide by adopting a silver nitrate potentiometric titration method according to the volume of silver nitrate added during potential mutation; and (3) using diethyl ether as a solvent, using sodium hydroxide solution as an extractant, and adopting high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) to determine the content of trimethylamine oxide.

(5) Intestinal flora diversity analysis and dominant bacteria screening: sample DNA was extracted using QIAamp DNA Stool Mini kit and PCR amplification was performed using 338F and 806R primer pairs V3-V4 variable region and 16SrRNA sequencing was performed on IlluminaMiSeq platform. Comparing alpha diversity and beta diversity of intestinal flora, and researching species composition and species difference among groups; and screening dominant pectin degrading bacteria by utilizing LEfSe multistage species difference discriminant analysis and random forest analysis, and analyzing the absolute abundance of dominant pectin degrading bacteria and short-chain fatty acid producing bacteria by a 16S microbial diversity absolute quantitative sequencing technology.

The dissolution effect of the pectin base in the culture medium after fermentation of the different dietary supplements prepared in example 1 and comparative examples 1-5 is shown in fig. 4, the apparent viscosity curve of the pectin base in the culture medium after fermentation of the different dietary supplements prepared in example 1-5 is shown in fig. 5, the short chain fatty acid content after fermentation of the different dietary supplements prepared in example 1-7 is shown in fig. 6-7, the free ammonia content after fermentation of the different dietary supplements prepared in example 1-7 is shown in fig. 8, and the species abundance histogram of the intestinal flora after fermentation of the dietary supplements prepared in example 1 is shown in fig. 9.

As shown in fig. 4, the dietary supplement prepared in example 1 was fermented in vitro by 24h, and floc was still visible, indicating that pectin was not completely degraded and utilized by microorganisms, demonstrating moderate fermentation rate. As can be seen from fig. 5, the dietary supplement prepared in example 1 has a moderate apparent viscosity and a high shear resistance as the shear force increases. The dietary supplements prepared in comparative examples 7 and 8 had minimal viscosity and a linear structure with a large Mw because they did not contain hemicellulose and cellulose, and they were easily broken when high shear forces were applied, so the viscosity profile dropped most rapidly. And in the comparative example 3 which is added with more dietary fibers, the molecular chains have strong cross-linking and the viscosity is maximum, so that the compound has larger shearing resistance, but the compound is not easy to be utilized by intestinal flora in the intestinal tract due to the characteristics. In contrast, the random arrangement of short chain molecules in comparative example 4 was more easily aligned with the flow direction, thereby reducing the interaction between adjacent pectin chains and eventually decreasing the viscosity, but the short chain molecules were degraded by microorganisms very rapidly in the intestinal tract, which could lead to adverse reactions such as gas production.

As shown in fig. 6-8, all dietary supplements had slightly increased concentrations of acetic acid, propionic acid, butyric acid and total SCFA at the initial stage of fermentation (3-6 h); subsequently, the dietary supplements prepared in example 1 and comparative examples 3-5 significantly increased from 6 h to 24h in acetic acid, propionic acid, butyric acid and isovaleric acid after in vitro simulated fermentation. In addition, the acetic acid produced in example 1 increased from 4.89mmol/L to 36.75mmol/L over a fermentation period of 3-24 hours, followed by comparative example 5 (from 5.68mmol/L to 24.19 mmol/L), comparative example 1 (from 6.75mmol/L to 25.18 mmol/L) and comparative example 3 (from 7.40mmol/L to 20.29 mmol/L). In contrast, the dietary supplements produced in example 1 produced the highest levels of propionic acid and butyric acid, 7.65 and 0.99mmol/L respectively, after 24h fermentation, significantly higher than comparative example 5 (5.43 and 0.81mmol/L respectively) and comparative example 3 (5.62 and 0.52mmol/L respectively). Among these, the higher propionic acid concentration produced after fermentation of the dietary supplement of example 1 was attributable to its higher arabinose and rhamnose content. On the other hand, the compact conformation may increase the surface area of attachment of intestinal microorganisms, promote their colonization around pectin, and further promote the depolymerization of pectin glycosidic bonds by pectin hydrolases, whereby the resulting monosaccharides may be further utilized by the intestinal microbiota to produce short chain fatty acids.

In addition, the dietary supplement of example 1 has the property of slow fermentation, allowing it to reach the far end of the colon and promote the growth of beneficial intestinal microorganisms, thereby inhibiting protein fermentation and reducing the production of the harmful metabolite free ammonia. Branched chain fatty acids, such as isobutyric acid and isovaleric acid, are also important indicators of protein fermentation, along with free ammonia, are generally regarded as deleterious metabolites, which are associated with increased risk of rectal cancer and ulcerative colitis. As shown in FIG. 7, after 24 hours of fermentation, a large amount of isobutyric acid was detected in comparative example 3 and comparative example 4, and comparative example 3 contained a large amount of isobutyric acid and isovaleric acid at the same time. As shown in FIG. 8, after 24 hours of fermentation, the highest concentration of free ammonia was detected in comparative examples 6 and 7, and a large amount of isobutyric acid was detected in comparative example 3 and comparative example 4, and comparative example 3 contained a large amount of isovaleric acid at the same time, probably due to insufficient carbon source consumption in the latter stage of fermentation, and protein fermentation was dominant, which indicated that the fermentation rates of comparative examples 3, 4, 6 and 7 were faster.

As can be seen from the abundance of the flora after fermentation of the dietary supplement in FIG. 9, the pectin-based dietary supplement prepared in example 1 of the present invention is rich in arabinose and galactose, is a good carbon source for SCFA-producing bacteria of the phylum Thick-walled bacteria, and can significantly increase the diversity of bacteria in the intestinal tract after fermentation of the intestinal flora, and improve the richness, uniformity and coverage of the flora, so that the abundance of short chain fatty acid-producing bacteria (Maospira, bacteroides, actinomycetes, candida, bifidobacteria, megamonas, praecox and ruminococci) is significantly enriched, and the abundance of the flora (propionibacterium sclerotium and Prevotella) related to protein fermentation is reduced.

Reagents used in the above examples:

calcium chloride, sodium hydroxide, sodium borohydride, potassium acetate, cyclohexane trans-1, 2-diamine tetraacetic acid (CDTA), ethanol, and the like are purchased from Shanghai Aba Ding Shenghua technologies Co., ltd; polygalacturonic acid exonuclease (exo-PG), pectin Lyase (PL) are available from Ireland Megazyme; endo-polygalacturonase (endo-PG), pectin Methyl Esterase (PME), and sugar standards: d-galacturonic acid, rhamnose, arabinose, galactose, glucose, xylose, etc. were purchased from Sigma-Aldrich (China); hydrochloric acid, sodium acetate, absolute ethanol, trifluoroacetic acid, etc. are available from national pharmaceutical group chemical reagent company, inc. 3500Da dialysis bags were purchased from United states Combined carbonization (Viskase).

Other reagents and drugs used in the above examples are those commonly used in laboratory as obtained from commercial sources unless otherwise specified

The main instrumentation used in the above embodiments:

JYL-C010 grinder (Jiuyang Co., ltd.), ULT1386-3-V41 ultra-low temperature refrigerator (Siemens Feisher technology, china Co., ltd.), ALPHA1-4Lplus vacuum freeze-drying equipment (CHRIST, germany), BK-B26 thermostatic water bath (Shanghai macrolaboratory equipment, inc.), RCT basic heating magnetic stirring (IKA, germany), 3K15 high-speed refrigerated centrifuge (Sigma, germany), high performance size exclusion chromatography (Wyatt technology, united states), ICS-3000 high performance anion chromatography (Siemens, fei.e., siemens, japan), ALPCL-40L UV-ultraviolet visible spectrophotometer (Shimadzu, japan), TENSOR-27 Fourier transform infrared spectrometer (Bruker, germany), 0 ultra-high performance liquid chromatography (An Dongpa, sharpa, shaker, inc.), bruker Avance III-500 nuclear magnetic resonance spectrometer (Bruker, germany).

The number of equipment and the scale of processing described herein are intended to simplify the description of the present invention. The use, modification and variation of the method of preparing a pectin-based dietary supplement of the present invention will be apparent to those skilled in the art.

As described above, according to the present invention, the present invention includes at least the following advantageous effects:

4. according to the preparation method of the pectin-based dietary supplement for promoting the fermentation balance of intestinal flora, provided by the invention, neutral sugar is added by a method of re-dissolving pectin and insoluble dietary fiber compound in neutral sugar solution and then freeze-drying, and specific neutral sugar is added in insoluble RG-II pectin and insoluble dietary fiber compound, so that the intestinal microbial diversity in the fermentation stage is obviously increased, and the production amount of beneficial metabolites is increased;

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention may be suited, and further modifications may be readily made by one skilled in the art, and the invention is therefore not limited to the particular details and embodiments shown and described herein, without departing from the general concepts defined in the claims and the equivalents thereof.

Claims

1. A method of preparing a pectin-based dietary supplement for promoting fermentation balance of an intestinal flora, comprising:

step four, taking 620-640 parts by weight of the compound pectin base obtained in the step three, mixing with 20-30 parts by weight of sugarcane fiber, 5-15 parts by weight of citrus fiber and 7-20 parts by weight of soybean fiber, and carrying out reverse molding and shaping to obtain a primary pectin-based dietary supplement product;

the method for obtaining the water-soluble pectin by sequentially intercepting fruits and vegetables and processing byproducts thereof as raw materials through a sequential extraction method in the first step comprises the following steps of: beating fruits and vegetables and processing byproducts thereof into fruit and vegetable pulp, adding 1-2M NaOH into the fruit and vegetable pulp, homogenizing with 95% ethanol for 1-2 h, suction-filtering to collect first filter residues, freeze-drying the obtained first filter residues, stirring in boiling water for 20-60 min, cooling, suction-filtering to obtain second filtrate and second filter residues, placing the second filtrate in a dialysis bag with molecular weight cutoff of 3500Da, dialyzing in water for 36-72 h, freeze-drying to obtain water-soluble pectin, and placing in a dryer for standby;

The method for the water-soluble pectin obtained in the first step of the synergistic enzymolysis of the compound enzyme in the second step comprises the following steps: taking water-soluble pectin as an object, adjusting the pH of a system to 4-4.6, adding polygalacturonase endoenzyme and polygalacturonase exoenzyme, and carrying out enzymolysis for 1-4 hours at 35-55 ℃; and then regulating the pH value of the system to 5.5-6.5, continuously adding pectin lyase and pectin methylesterase, and carrying out enzymolysis for 1-4 hours at 50-70 ℃ to obtain an enzymolysis product containing water-soluble RG-I pectin and water-soluble RG-II pectin.

2. The method of preparing a pectin-based dietary supplement for promoting fermentation balance of intestinal flora as in claim 1, further comprising the step of: spraying 0.05-0.1% calcium chloride solution on each side of the primary pectin-based dietary supplement product obtained in the step four, and placing the primary pectin-based dietary supplement product into a vacuum freeze drying bin for drying until the mass water content of the material is lower than 5%, grading, selecting, boxing, filling nitrogen and packaging to obtain the pectin-based dietary supplement.

3. The method for preparing pectin-based dietary supplement for promoting intestinal flora fermentation balance of claim 2, wherein in the first step, fruits and vegetables and processing byproducts thereof are taken as raw materials, and hemicellulose is obtained by sequential interception through a sequential extraction method, which comprises the following steps: dissolving the second filter residue in 0.1-0.5M potassium acetate solution containing 0.05-0.5M CDTA, filtering to obtain a third filtrate and a third filter residue, and dissolving the third filter residue in solution containing 0.02-0.1M NaBH ₄ Collecting insoluble residues in 0.1-0.5M NaOH solution, and adding a solution containing 4-6M KOH and 0.02-0.1M NaBH ₄ In a borate solution of N ₂ Stirring at room temperature for 10-20 h, filtering to obtain a fourth filtrate, adjusting the pH value of the fourth filtrate to 2-5 by using an HCl solution, adding 1-5 mL of ethanol, placing the precipitate in a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing in water for 36-72 h, and freeze-drying to obtain hemicellulose for later use.

4. The method for preparing a pectin-based dietary supplement for promoting fermentation balance of intestinal flora according to claim 1, wherein in the method for preparing the water-soluble pectin by the complex enzyme in the second step, the complex enzyme is cooperated with the water-soluble pectin obtained in the first step, the final concentration of the water-soluble pectin is set to be 2-5 wt%, and the final concentrations of the polygalacturonase, the polygalacturonase exoenzyme, the pectin lyase and the pectin methylesterase in the water-soluble pectin are all 0.2-1U/mL.

5. The method of claim 1, wherein the step of screening to obtain water-soluble RG-II pectin comprises: and (3) performing DEAE-cellulose column chromatography, eluting the enzyme-inactivated zymolyte with distilled water for 2-5 h to obtain a neutral sugar fraction, continuing eluting with 0.5-2.0M NaCl solution to obtain an acidic sugar fraction, eluting the obtained acidic sugar fraction with 0.5-2.0M NaCl solution in high-resolution size exclusion chromatography, collecting the acidic sugar fraction with the elution time in the range of 10-15 min, and screening and separating by using a thiobarbituric acid method to obtain the water-soluble RG-II pectin.

6. The method for preparing the pectin-based dietary supplement for promoting the fermentation balance of intestinal flora according to claim 1, wherein in the second step, enzyme deactivation treatment is to wrap an enzymolysis product with a PTFE filtering and ventilation sealing film, and then placing the enzymolysis product under steam at 90-100 ℃ for 3-8 min.

7. The method of claim 1, wherein vegetable oil is applied to the mold prior to the step four.

8. A pectin-based dietary supplement characterized in that it is prepared by the preparation method of any one of claims 1-7.