CN110551231B - Preparation method of carboxymethylated xylan, carboxymethylated xylan product and application of carboxymethylated xylan product - Google Patents
Preparation method of carboxymethylated xylan, carboxymethylated xylan product and application of carboxymethylated xylan product Download PDFInfo
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- 229920001221 xylan Polymers 0.000 title claims abstract description 113
- 150000004823 xylans Chemical class 0.000 title claims abstract description 104
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 239000006041 probiotic Substances 0.000 claims abstract description 36
- 235000018291 probiotics Nutrition 0.000 claims abstract description 35
- 230000035755 proliferation Effects 0.000 claims abstract description 27
- 241000894006 Bacteria Species 0.000 claims abstract description 16
- 230000000529 probiotic effect Effects 0.000 claims abstract description 14
- 240000001929 Lactobacillus brevis Species 0.000 claims abstract description 11
- 235000013957 Lactobacillus brevis Nutrition 0.000 claims abstract description 11
- 238000000338 in vitro Methods 0.000 claims abstract description 11
- 230000001737 promoting effect Effects 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 39
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 24
- 238000006467 substitution reaction Methods 0.000 claims description 21
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 15
- 239000011259 mixed solution Substances 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 10
- FOCAUTSVDIKZOP-UHFFFAOYSA-N chloroacetic acid Chemical compound OC(=O)CCl FOCAUTSVDIKZOP-UHFFFAOYSA-N 0.000 claims description 9
- 229940106681 chloroacetic acid Drugs 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
- 239000012670 alkaline solution Substances 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 230000003472 neutralizing effect Effects 0.000 claims description 5
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- 230000007935 neutral effect Effects 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 2
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- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 35
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- 238000012986 modification Methods 0.000 abstract description 12
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- 241000186673 Lactobacillus delbrueckii Species 0.000 abstract description 7
- KHQHQJDNEISKKS-UHFFFAOYSA-M sodium;2-chloroacetic acid;hydroxide Chemical compound [OH-].[Na+].OC(=O)CCl KHQHQJDNEISKKS-UHFFFAOYSA-M 0.000 abstract description 5
- 229920001282 polysaccharide Polymers 0.000 description 25
- 239000005017 polysaccharide Substances 0.000 description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 21
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- 239000000126 substance Substances 0.000 description 13
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- 239000002253 acid Substances 0.000 description 6
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- DFQICHCWIIJABH-UHFFFAOYSA-N naphthalene-2,7-diol Chemical compound C1=CC(O)=CC2=CC(O)=CC=C21 DFQICHCWIIJABH-UHFFFAOYSA-N 0.000 description 3
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- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 2
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- 150000001875 compounds Chemical class 0.000 description 2
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 2
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0057—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Xylans, i.e. xylosaccharide, e.g. arabinoxylan, arabinofuronan, pentosans; (beta-1,3)(beta-1,4)-D-Xylans, e.g. rhodymenans; Hemicellulose; Derivatives thereof
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention provides a preparation method of carboxymethylated xylan, a product and application thereof, wherein carboxymethylated xylan is subjected to carboxymethylation modification by a sodium hydroxide-chloroacetic acid method, the obtained carboxymethylated xylan is used for probiotic culture, the in-vitro proliferation effect of carboxymethylated xylan with different concentrations on probiotics (Lactobacillus delbrueckii subsp bulgaricus and Lactobacillus brevis) is examined by taking an OD value and a pH value as indexes, and the result shows that the carboxymethylated xylan subjected to carboxymethylation modification by the sodium hydroxide-chloroacetic acid method has a remarkable effect of promoting the proliferation of the outside of probiotic bacteria, so that a basis is provided for the development of the carboxymethylated xylan and the research of prebiotics.
Description
Technical Field
The invention relates to the technical field of modification of polysaccharides, and particularly relates to a preparation method of carboxymethylated xylan, a carboxymethylated xylan product and application of the carboxymethylated xylan product.
Background
Prebiotics are defined as a non-digestible or indigestible food ingredient of the human body, which selectively stimulates the growth and activity of colon physiologically active bacteria, thus producing a health effect on the host, and the physiological function of prebiotics is mainly realized by promoting the reproduction of beneficial bacteria in the human body in the intestine and optimizing the balance of flora, which is shown in the improvement of intestinal function and the enhancement of immunity of the organism; therefore, the selection of prebiotics needs not only to have the property of not being digested or difficult to be digested by human body, but also to have good effect of promoting the proliferation of the prebiotics outside the cells, and substances which are beneficial to the prebiotic function in the prior art mainly comprise some non (or difficult) digestible oligosaccharides, such as fructo-oligosaccharide, xylo-oligosaccharide, chitosan oligosaccharide and the like, and in addition, polyols such as xylitol, lactitol and the like;
xylan is the main component of hemicellulose in plant cells, is a complex poly-pentose, accounts for one third to two fifths of the dry weight of plant cells, and is the polysaccharide with the most abundant content except cellulose in nature. The xylan has wide sources and is an abundant biomass resource, and the biological activity of the xylan as plant polysaccharide is mainly reflected in the aspects of oxidation resistance, tumor resistance, immunoregulation and the like. Xylan can not be digested and absorbed by gastrointestinal tract, but can be fermented by intestinal microorganisms to generate short-chain fatty acids, the short-chain fatty acids have the effects of protecting the intestinal tract and improving diabetes, cardiovascular diseases and the like, but xylan does not have the effect of promoting the proliferation of probiotics outside the bacterial cells, so xylan is processed and produced into xylose, xylitol, xylooligosaccharide and the like serving as prebiotics of the intestinal microorganisms in the prior art.
The chemical structure modification means that the structure and configuration of a molecule are properly modified by a physical and chemical means, and the xylan polysaccharide is subjected to chemical structure modification, so that the molecular structure and configuration of the xylan polysaccharide are modified, and the xylan polysaccharide is changed in physical and chemical properties or becomes a derivative of another new compound. Many experiments have shown that appropriate chemical structure modifications can significantly enhance the biological activity of polysaccharides. For xylan, hydroxyl in xylan molecules is used as a main modification group, and an active functional group is introduced by chemical methods such as carboxymethylation, sulfation, alkylation, oxidation and the like, and physical methods such as mixing, loading and the like, so that the physicochemical function of xylan can be improved. Therefore, if the chemical structure modification of xylan can be carried out to prepare modified xylan with the function of promoting the intestinal probiotic extracellular proliferation, the method has important significance.
Disclosure of Invention
The invention aims to provide a preparation method of carboxymethylated xylan, which is used for carrying out carboxymethylation modification on xylan, determining the structural characteristics of the xylan and researching the in-vitro proliferation effect of the carboxymethylated xylan on probiotics.
Another object of the present invention is to provide carboxymethylated xylan produced by the above method for producing carboxymethylated xylan.
The invention also aims to provide the application of the carboxymethylated xylan in the process of promoting the proliferation of probiotic bacteria outside, wherein the carboxymethylated modified xylan is directly used for culturing the probiotic bacteria to promote the proliferation of the probiotic bacteria outside, so that the technical problem that the xylan does not have the function of promoting the proliferation of the probiotic bacteria outside is solved.
One of the technical schemes of the invention is as follows: a preparation method of carboxymethylated xylan comprises the following steps of dissolving xylan in isopropanol and 20% alkaline solution, stirring in ice bath to obtain solution A, dissolving chloroacetic acid in the isopropanol solution, uniformly stirring, mixing with the 20% alkaline solution to obtain mixed solution B, slowly dropping the mixed solution B into the solution A, heating and stirring until the reaction is finished, neutralizing with hydrochloric acid to be neutral, dialyzing with running water, evaporating, concentrating, and freeze-drying to obtain carboxymethylated xylan.
Preferably, the addition amount of xylan in solution A is 1-6g/100ml of the mixed solution, and the addition amount of chloroacetic acid in solution B is 5-20g/100 ml.
Preferably, the volume ratio of isopropanol to 20% sodium hydroxide solution in solution A and solution B is 2: 1, and solution A and solution B are mixed in equal volumes.
Preferably, the ice bath is stirred for 1-5 h.
Preferably, the reaction temperature is heated to 30-70 ℃, and the stirring time is 1-5 h.
Preferably, the hydrochloric acid is 1mol · L-1The running water dialysis time of the hydrochloric acid is 24 hours.
The second technical scheme of the invention is as follows: the carboxymethylated xylan prepared by the preparation method of the carboxymethylated xylan has the substitution degree of 0.4-1.2.
The third technical scheme of the invention is as follows: the application of the carboxymethylated xylan in promoting the proliferation process of the probiotic bacteria outside the bacteria.
Preferably, the probiotic is lactobacillus brevis or lactobacillus bulgaricus.
The invention at least comprises the following beneficial effects:
in the preparation process of carboxymethylated xylan, the addition amount of xylan in the solution A and the addition amount of chloroacetic acid in the solution B are key factors influencing the carboxymethylated substitution degree of xylan. The carboxymethylation substitution degree of xylan shows a trend of increasing and then decreasing with the addition of xylan and chloroacetic acid. Different reaction times and different reaction temperatures also have an effect on the degree of carboxymethylated substitution of the xylan. The tendency to increase and then decrease with increasing reaction time and the degree of substitution to increase with increasing reaction temperature but to decrease beyond a certain temperature. The carboxymethylation substitution degree has influence on the in vitro proliferation of the probiotics, and the prepared carboxymethylation xylan has the in vitro proliferation effect of the probiotics only when the substitution degree is 0.4-1.2, so the carboxymethylation xylan with different substitution degrees can be prepared according to the control of the addition amount of raw materials, the reaction temperature and the time.
The invention changes the molecular structure and configuration of xylan polysaccharide by proper carboxymethylation modification, changes the physicochemical property of xylan polysaccharide into a new derivative of a compound, introduces active functional groups, thereby enhancing the biological activity of xylan, and can be used for the in vitro proliferation of probiotics. The polysaccharide as carbon source substance can be decomposed and utilized by the probiotics. The solubility of the polysaccharide can be improved after the polysaccharide is chemically modified, so that the polysaccharide can better participate in the metabolic process of probiotics, is decomposed and utilized by the probiotics, and promotes the proliferation of the probiotics. Therefore, the proliferation effect of the chemically modified product on the probiotics is improved compared with that of unmodified xylan. The result of the invention shows that the xylan modified by carboxymethylation has obvious effect of promoting the in vitro proliferation of the probiotics.
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 graph of infrared spectroscopic analysis before and after carboxymethylation modification of xylan, wherein X and Y represent xylan and carboxymethylated xylan;
FIG. 2 is a graph showing the effect of different concentrations of carboxymethylated xylan on the in vitro proliferation of Lactobacillus brevis;
FIG. 3 is a graph showing the effect of different concentrations of carboxymethylated xylan on the in vitro proliferation of Lactobacillus delbrueckii subspecies bulgaricus;
FIG. 4 is a graph showing the effect of 3% carboxymethylated xylan on the growth rate of Lactobacillus brevis;
FIG. 5 is a graph showing the effect of 3% carboxymethylated xylan on the growth rate of Lactobacillus delbrueckii subspecies bulgaricus;
FIG. 6 shows the effect of different concentrations of carboxymethylated xylan on acid production by Lactobacillus brevis;
FIG. 7 shows the effect of different concentrations of carboxymethylated xylan on acid production by Lactobacillus delbrueckii subsp.bulgaricus;
FIG. 8 is a graph showing the effect of carboxymethylated xylans of varying degrees of substitution on the proliferation of Lactobacillus brevis;
FIG. 9 is a graph showing the effect of different degrees of substitution of carboxymethylated xylans on the proliferation of Lactobacillus delbrueckii subspecies bulgaricus;
Detailed Description
The present invention is described in further detail below to enable those skilled in the art to practice the invention with reference 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
Preparing carboxymethylated xylan by a sodium hydroxide-chloroacetic acid chemical method, weighing 6g of xylan, dissolving the xylan in 100mL of isopropanol and 50mL of 20% NaOH solution, stirring for 3h in ice bath to obtain a solution A, dissolving 30g of chloroacetic acid in 100mL of isopropanol, and mixing with 50mL of 20% NaOH solution to obtain a mixed solution B. Slowly dropping the mixed solution B into the solution A, stirring for 4h at 60 ℃, stopping reaction, cooling to room temperature, neutralizing to neutrality by using 1 mol. L-1 hydrochloric acid, dialyzing for 24h with running water, evaporating and concentrating the sample solution, and freeze-drying to obtain the carboxymethylated xylan.
Infrared spectroscopic analysis
Mixing 1mg of dried carboxymethylated xylan and 1mg of xylan polysaccharide with 100mg of KBr reference, grinding, tabletting at 4000-400 cm-1The infrared spectrum scanning is carried out by an infrared spectrometer to obtain an infrared spectrum scanning pattern (figure 1).
Measurement of degree of substitution of carboxymethylated xylan
Preparing carboxymethylated xylan solution of 0.4mg/mL by using secondary water, uniformly mixing 0.25mL with 0.25mL of concentrated sulfuric acid in a test tube, heating at 125 ℃ for 3h, sucking 2mL of 2, 7-dihydroxynaphthalene solution (the 2, 7-dihydroxynaphthalene is prepared into 0.1mg/mL solution by using 95% concentrated sulfuric acid and is stored in a dark place for 5-6h), adding the solution into the mixed solution, fully and uniformly mixing, stopping the reaction in a boiling water bath for 20min, cooling to room temperature, finally adding 2mL of distilled water, measuring the absorption value at 520nm by using the distilled water as a reference, and parallelly measuring three groups. Replacing the polysaccharide sample with glycolic acid dried overnight in calcium chloride, making a standard curve, calculating the gram of glycolic acid per gram of polysaccharide sample, recording as A, and calculating the value of the degree of carboxymethylation substitution of xylan according to the following formula:
DScm＝162A/(76-80A)
wherein DScm is the degree of substitution, A is the grams of glycolic acid per gram of sample, 162 is the molecular mass of 2, 7-dihydroxynaphthalene, 76 is the molecular mass of glycolic acid, 80 is SO4 2-Calculated as the degree of substitution of the carboxymethylated xylan prepared was 0.68.
Example 2
Preparing carboxymethylated xylan by a sodium hydroxide-chloroacetic acid chemical method, weighing 3g of xylan, dissolving the xylan in 100mL of isopropanol and 50mL of 20% NaOH solution, stirring for 3h in ice bath to obtain a solution A, dissolving 15g of chloroacetic acid in 100mL of isopropanol, and mixing with 50mL of 20% NaOH solution to obtain a mixed solution B. Slowly dropping the mixed solution B into the solution A, stirring for 2h at 30 ℃, stopping reaction, cooling to room temperature, neutralizing to neutrality by using 1 mol. L-1 hydrochloric acid, dialyzing for 24h with running water, evaporating and concentrating the sample solution, and freeze-drying to obtain the carboxymethylated xylan with the substitution degree of 0.43.
Example 3
Preparing carboxymethylated xylan by a sodium hydroxide-chloroacetic acid chemical method, weighing 6g of xylan, dissolving the xylan in 100mL of isopropanol and 50mL of 20% NaOH solution, stirring for 3h in ice bath to obtain a solution A, dissolving 20g of chloroacetic acid in 100mL of isopropanol, and mixing with 50mL of 20% NaOH solution to obtain a mixed solution B. Slowly dropping the mixed solution B into the solution A, stirring for 5h at 60 ℃, stopping reaction, cooling to room temperature, neutralizing to be neutral by using 1 mol. L-1 hydrochloric acid, dialyzing for 24h with running water, evaporating and concentrating the sample solution, and freeze-drying to obtain the carboxymethylated xylan with the substitution degree of 1.18.
Effect example 1:
according to the technical scheme, carboxymethylated xylan is used as a carbon source of a culture medium, so that the formula of the culture medium is not too much, a high-sugar culture medium is not suitable for fungus growth, and the influence of the concentration of the carboxymethylated xylan on the in-vitro proliferation of probiotics is detected to be the maximum amount by taking 3% in an experiment.
After 10mL of MRS basal medium containing carboxymethylated xylan polysaccharides prepared in example 1 at different concentrations (0.5, 1.0, 1.5, 2.0, 3.0,%, W/V) were added and dissolved by gentle heating, xylan at different concentrations (basal medium containing xylan as a carbon source) was used as a negative control, and fructo-oligosaccharide (FOS) at different concentrations was used as a positive control, and autoclaved at 121 ℃ for 20 min. 100 μ L of multiply activated Lactobacillus brevis and Lactobacillus delbrueckii subspecies Bulgaria were inoculated, respectively, and cultured at 37 ℃ for 48 hours, and samples were taken to determine the OD of each culture solution at a wavelength of 600nm and the pH of the culture medium, and 3 replicates of each experiment were repeated. As a result, the OD in the sample was shown in FIGS. 2 and 3600The nm value increases with the concentration of carboxymethylated xylan, which indicates that the number of strains in the sample to be tested tends to increase with the increase of the concentration. Within the set polysaccharide concentration range, the higher the concentration, the better the in vitro proliferation of the probiotics. Increase of probiotics in the set polysaccharide concentration range of the experimentReproductive effects all exhibited an ascending potential and reached a maximum at 3%.
Effect of carboxymethylated xylan polysaccharides on probiotic growth Rate
OD grown from probiotic as a result of the effect of different concentrations of carboxymethylated xylan on the activity of probiotic600And pH index can be found to be optimal when the polysaccharide concentration is 3%, so that carboxymethylated xylan, xylan and FOS are added at 3% concentration to prepare liquid culture medium, culture time is used as abscissa, and OD is OD600And the pH value is used as an ordinate, the index is measured, and the growth curve of the probiotics is drawn, as shown in figure 4 and figure 5. From the figure, the liquid culture medium added with carboxymethylated xylan according to the concentration of 3 percent has obviously better external proliferation effect on probiotics than xylan, and achieves the technical effect of promoting the external proliferation effect of probiotics.
Effect example 2:
when the bacteria are cultured, the culture medium is sour after being cultured for several days, the bacteria utilize a carbon source in a culture solution in the growth process, and acidic substances are generated after metabolism, so that the growth conditions of the bacteria can be visually and respectively observed by measuring the pH value of the liquid culture medium which is cultured for 48 hours after the bacteria are added.
As shown in fig. 6 and 7, with polysaccharide concentrations of 0, 0.5, 1.0, 1.5, 2.0, and 3.0% (W/V) as abscissa, XY, CXY, and FOS respectively represent the influence of xylan, carboxymethylated xylan, and fructo-oligosaccharide culture medium on acid production by probiotic bacteria, the pH of the experimental strain in the sample to be tested generally decreased and reached the minimum value when the polysaccharide concentration was 3%. As can be seen from the figure, carboxymethylated xylan polysaccharides are more capable of influencing the acid production of probiotics than xylan. Carboxymethylated xylan has a significant effect on acid production by Lactobacillus brevis and Lactobacillus delbrueckii subsp.
Effect example 3
As shown in FIGS. 8 and 9, the effect of carboxymethylated xylan with different degrees of substitution on the acid production of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus brevis was plotted for 12 hours on the abscissa and the ordinate on the pH. As is clear from the figure, the degree of substitution is from 0 to 1, and the pH value decreases with increasing degree of substitution, indicating that carboxymethylated xylan promotes the proliferation of Lactobacillus brevis and Lactobacillus delbrueckii subsp. However, when the degree of substitution is increased beyond a certain value, the effect is reduced. The reason may be that some groups may be substituted to affect their activity, or that a change in structure may affect their activity. It is also possible that certain polysaccharides are chemically modified to bind to enzymes required by probiotics, such as alpha-glucosidase or beta-glucosidase, to hydrolyze prebiotics, or to bind to enzyme-substrate complexes to inhibit their activity.
While the embodiments of the invention have been disclosed above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made thereto by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (2)
1. The application of carboxymethylated xylan in promoting the proliferation of probiotics outside a cell is characterized in that the carboxymethylated xylan is xylan with carboxymethyl groups, and the substitution degree is 0.4-1.2; the preparation method comprises the following steps:
dissolving xylan in a mixed solution of isopropanol and 20% alkaline solution, stirring in ice bath to obtain a solution A, dissolving chloroacetic acid in the isopropanol solution, uniformly stirring, mixing with the 20% alkaline solution to obtain a mixed solution B, slowly dropping the mixed solution B into the solution A, heating and stirring until the reaction is finished, neutralizing with hydrochloric acid to be neutral, dialyzing with running water, evaporating and concentrating, and freeze-drying to obtain carboxymethylated xylan;
the addition amount of the xylan in the solution A is 1-6g/100ml, and the addition amount of the chloroacetic acid in the solution B is 5-20g/100 ml;
the volume ratio of the isopropanol to the 20% alkaline solution in the solution A and the solution B is 2: 1, and the solution A and the solution B are mixed in equal volume;
stirring for 1-5h in ice bath;
heating to 30-70 deg.C, and stirring for 1-5 h;
the hydrochloric acid is 1 mol. L -1The running water dialysis time of the hydrochloric acid is 24 hours.
2. Use of carboxymethylated xylans according to claim 1 for the promotion of the proliferation of probiotic bacteria in vitro, wherein said probiotic bacteria are Lactobacillus brevis or Lactobacillus bulgaricus.
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Application publication date: 20191210 Assignee: Guilin Fanyi Technology Co.,Ltd. Assignor: GUILIN University OF TECHNOLOGY Contract record no.: X2023980044835 Denomination of invention: Preparation method, product, and application of carboxymethyl xylan Granted publication date: 20210615 License type: Common License Record date: 20231031 |
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