PHGG with specific molecular weight as well as preparation method and application thereof

Abstract

The invention discloses PHGG with specific molecular weight as well as a preparation method and application thereof, and relates to the technical field of PHGG preparation. The method comprises the following steps: (1) mixing guar gum, cellulase and mannase in a phosphate buffer solution, and then stirring for enzymolysis to obtain a guar gum enzymolysis solution; (2) performing enzyme deactivation and sterilization on the obtained guar gum enzymolysis solution, and performing freeze drying after filtration to obtain PHGG; in the step (2), the molecular weight of the PHGG is 5 to 500 kDa. Compared with the prior art, the operation is simpler, and the molecular weight of the prepared PHGG is more accurate.

Description

Technical Field

[0001] The invention relates to the technical field of PHGG preparation, in particular to PHGG with a specific molecular weight and a preparation method and application thereof. Background Art

[0002] Guar gum is prepared from the endosperm of guar bean seeds, and its main component is galactomannan. Guar gum is composed of mannopyranose units as the backbone to form the main chain, and every two mannopyranose units have galactopyranose units connected by α-1,6 bonds as side chains. The large amount of galactose side groups distributed on guar gum galactomannan increases the steric hindrance between the galactomannan molecules and β-mannanase, resulting in high viscosity, low activity and hydrolysis efficiency, which hinders its application in the food industry.

[0003] Guar gum hydrolysis can produce low molecular weight galactomannan, i.e. partially hydrolyzed guar gum (PHGG). PHGG is a new type of soluble dietary fiber that has the functions of regulating blood sugar and blood lipids, relieving constipation and other irritable bowel syndromes. At the same time, PHGG can play a prebiotic role by regulating the abundance of intestinal flora and producing short-chain fatty acids.

[0004] Many methods have been developed to produce PHGG, such as ultrasound, acid hydrolysis and microwave treatment; enzymatic hydrolysis is a green and environmentally friendly biodegradation method, among which the most widely used enzyme preparation is mannanase. Cellulase, as a non-specific enzyme preparation, is also used in the preparation of PHGG, but there is little research on the synergistic preparation of the two.

[0005] Changes in PHGG molecular weight drive different fermentation results and may change the microbial composition in the human colon. Studies have found that low-molecular-weight PHGG, as a prebiotic, can promote the growth of probiotics and produce a large amount of short-chain fatty acids, among which the most prominent is that it can significantly increase the butyrate content. Short-chain fatty acids can lower intestinal pH and stimulate intestinal peristalsis, thereby effectively promoting small intestinal metabolism and promoting intestinal waste excretion. Therefore, it is necessary to prepare PHGG with different molecular weights in a targeted manner.

[0006] Chinese patent CN109811022A discloses a method for preparing galactomannan oligosaccharides and its application, the method comprising: (1) performing segmented composite enzymatic hydrolysis on galactomannan such as guar gum, sesbania gum, locust bean gum, fenugreek gum, tara gum, etc., characterized in that the enzyme is a composite enzyme of acid mannanase and cellulase; (2) utilizing segmented ultrafiltration classification to obtain a mixture of disaccharides, trisaccharides, tetrasaccharides, and pentasaccharides after degradation. The method optimizes the enzymatic hydrolysis process and improves the yield of oligosaccharides, but does not involve the preparation of galactomannan with various molecular weights.

[0007] In view of this, in order to solve the deficiencies of the prior art, the present invention provides a PHGG with a specific molecular weight and a preparation method and application thereof. Summary of the invention

[0008] The purpose of the present invention is to provide a PHGG with a specific molecular weight and a preparation method and application thereof, and to achieve the purpose of controlling the molecular weight of PHGG by limiting specific parameters in the preparation method.

[0009] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0010] In one aspect, the present invention provides a method for preparing PHGG of a specific molecular weight, comprising the following steps:

[0011] (1) guar gum, cellulase and mannanase are mixed in a phosphate buffer, and then stirred for enzymolysis to obtain a guar gum enzymolysis solution;

[0012] (2) sterilizing the obtained guar gum enzymatic hydrolysis solution, filtering it, and then freeze-drying it to obtain PHGG;

[0013] In step (2), the molecular weight of the PHGG is 5-500 kDa.

[0014] Preferably, in step (1), the concentration of guar gum is 0.001-0.01 g / mL.

[0015] Preferably, in step (1), the amount of cellulase added is 8000 U / g.

[0016] Preferably, in step (1), the added amount of the mannanase is 100 U / g.

[0017] Preferably, in step (1), the pH value of the phosphate buffer is 4-6.

[0018] Preferably, in step (1), the stirring speed is 200 rpm.

[0019] Preferably, in step (1), the temperature of the enzymatic hydrolysis is 40-60°C.

[0020] Preferably, in step (1), the enzymatic hydrolysis time is 2-14 hours.

[0021] Further preferably, in step (1), the concentration of the guar gum is 0.005 g / mL; in step (2), the molecular weight of the PHGG is 8.9-35 kDa.

[0022] More preferably, in step (1), the enzymatic hydrolysis time is 6-14 h; in step (2), the molecular weight of PHGG is 8.9-25 kDa.

[0023] More preferably, in step (1), the enzymatic hydrolysis time is 6-10 h; in step (2), the molecular weight of PHGG is 15-25 kDa.

[0024] Finally, preferably, in step (1), the enzymatic hydrolysis time is 6 h; in step (2), the molecular weight of PHGG is 25 kDa.

[0025] In another aspect, the present invention also provides PHGG prepared by the above preparation method.

[0026] In another aspect, the present invention also provides the use of the above PHGG in regulating human intestinal flora.

[0027] The beneficial effects of the present invention are:

[0028] (1) The present invention discloses a method for preparing PHGG with a specific molecular weight. Compared with the prior art, the operation of the present invention is simpler and the molecular weight of the prepared PHGG is more accurate.

[0029] (2) The present invention prepares PHGG with a molecular weight of 8.9-35 kDa. PHGG within this molecular weight range can reach the colon and be fermented by intestinal flora, stimulating the growth of beneficial intestinal bacteria; PHGG within this molecular weight range can effectively reduce the abundance of harmful bacteria and increase the abundance of beneficial bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The acid stability test results of Examples 1-4 are shown.

[0031] Figure 2 The pH changes during the in vitro fermentation of Examples 1-4.

[0032] Figure 3 The SCFAs content produced during the in vitro fermentation process of Examples 1-4.

[0033] Figure 4 This is the Venn diagram of intestinal microbial OTUs processed in Examples 1-4.

[0034] Figure 5 The PCA main component analysis results of the intestinal flora of Examples 1-4 are shown.

[0035] Figure 6 The intestinal microbial genus level distribution diagram of Examples 1-4.

[0036] Figure 7This is the differential species analysis of the intestinal flora at the genus level of Examples 1-4.

[0037] Figure 1-7 In the figure, group GG represents guar gum that has not been enzymatically hydrolyzed. DETAILED DESCRIPTION

[0038] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they should also fall within the scope of the present application.

[0039] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0040] Cellulase was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with the product number CAS: 9012-54-8.

[0041] Mannanase was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with the product number CAS: 37288-54-3.

[0042] Guar gum was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with the product number of CAS: 9000-30-0.

[0043] Example 1

[0044] Guar gum was hydrolyzed by cellulase and mannanase, with a guar gum concentration of 0.5%, a pH of 5, and a temperature of 50°C. First, 5 g of guar gum was dissolved in 1000 mL of phosphate buffer, and then 8000 U / g of cellulase and 100 U / g of mannanase were added simultaneously, and then stirred at 200 rpm for 2 h in a magnetic stirrer. After 2 h of enzymolysis, a guar gum enzymolysis solution was obtained. The obtained guar gum enzymolysis solution was sterilized by inactivating enzymes in a boiling water bath, and then freeze-dried after filtration to obtain PHGG.

[0045] Example 2

[0046] The stirring for 2 h in Example 1 was replaced by stirring for 6 h, and the rest of the preparation process was the same as in Example 1.

[0047] Example 3

[0048] The stirring for 2 h in Example 1 was replaced by stirring for 10 h, and the rest of the preparation process was the same as that in Example 1.

[0049] Example 4

[0050] The stirring for 2 h in Example 1 was replaced by stirring for 14 h, and the rest of the preparation process was the same as that in Example 1.

[0051] Example 5

[0052] Guar gum was hydrolyzed by cellulase and mannanase, with a guar gum concentration of 1%, a pH of 4, and a temperature of 40°C. First, 1 g of guar gum was dissolved in 100 mL of phosphate buffer, and then 8000 U / g of cellulase and 100 U / g of mannanase were added simultaneously, and then stirred at 200 rpm in a magnetic stirrer for 2 h. After 2 h of enzymolysis, a guar gum enzymolysis solution was obtained. The obtained guar gum enzymolysis solution was sterilized by inactivating enzymes in a boiling water bath, and then freeze-dried after filtration to obtain PHGG.

[0053] Example 6

[0054] Guar gum was hydrolyzed by cellulase and mannanase, with a guar gum concentration of 0.1%, a pH of 6, and a temperature of 60°C. First, 1 g of guar gum was dissolved in 1000 mL of phosphate buffer, and then 8000 U / g of cellulase and 100 U / g of mannanase were added simultaneously, and then stirred at 200 rpm for 14 h in a magnetic stirrer. After 14 h of enzymolysis, a guar gum enzymolysis solution was obtained. The obtained guar gum enzymolysis solution was sterilized by inactivating enzymes in a boiling water bath, and then freeze-dried after filtration to obtain PHGG.

[0055] Comparative Example 1

[0056] Guar gum was hydrolyzed by cellulase and mannanase in sections, with a guar gum concentration of 0.5%, a pH of 5, and a temperature of 50°C. First, 5 g of guar gum was dissolved in 1000 mL of phosphate buffer, and then 8000 U / g of cellulose preparation was added, and then the mixture was stirred at 200 rpm for 2 h in a magnetic stirrer. After 2 h of enzymolysis, a guar gum enzymolysis solution 1 was obtained, and the obtained guar gum enzymolysis solution 1 was sterilized by inactivating enzymes in a boiling water bath. After the guar gum enzymolysis solution 1 was cooled, 100 U / g of mannanase was added thereto, and then the mixture was stirred at 200 rpm for 2 h in a magnetic stirrer. After 2 h of enzymolysis, a guar gum enzymolysis solution 2 was obtained, and the obtained guar gum enzymolysis solution 2 was sterilized by inactivating enzymes in a boiling water bath, and then freeze-dried after filtration to obtain PHGG.

[0057] Comparative Example 2

[0058] Guar gum was hydrolyzed by cellulase and xylanase in a coordinated manner. The concentration of guar gum was 0.5%, the pH value was 5, the temperature was 50°C, the addition amount of cellulase was 8000 U / g, and the addition amount of mannanase was 100 U / g.

[0059] The remaining conditions are the same as those in Example 1.

[0060] Results test:

[0061] 1. PHGG molecular weight determination

[0062] 1.1. Determination method

[0063] The HPLC system used a 105-103-101 column and a differential detector. Dextran standards of different molecular weights were used to determine the retention time, and the linear relationship between the retention time and the logarithm of the molecular weight was calibrated.

[0064] 1.2. Measurement results

[0065] Table 1

[0066]

[0067] 2. PHGG viscosity determination

[0068] 2.1. Determination method

[0069] The apparent viscosity of Examples 1-4 was measured using a spindle type rotational viscometer. First, after fixing a specific spindle, the viscometer was automatically zeroed in air, and then the apparent viscosity of each solution was measured. The apparent viscosity of each sample at a rotation speed of 100 rpm was measured using a spindle No. 61 rotor.

[0070] 2.2. Measurement results

[0071] Table 2

[0072] Guar Gum Example 1 Example 2 Example 3 Example 4 Viscosity / cps <![CDATA[494.5±1.25 a ]]> <![CDATA[4.26±0.43 b ]]> <![CDATA[4.14±0.07 b ]]> <![CDATA[4.08±0.00 b ]]> <![CDATA[4.02±0.19 b ]]>

[0073] Note: Values ​​are expressed as mean ± standard deviation (n = 3); the mean values ​​with different letters in the same row are significantly different (p < 0.05).

[0074] The viscosity of guar gum is 494.5 cps. As the synergistic enzymatic hydrolysis time of cellulase and mannanase increases, the viscosity of the example gradually decreases.

[0075] 3. Acid stability test

[0076] 3.1. Determination test

[0077] The reducing sugar conversion rate of Examples 1-4 at different acidic pH values ​​was measured to investigate the acid resistance of the samples. The sample solutions were adjusted to pH = 1, 2, 3, 4, and 5 with 0.1 mol / L hydrochloric acid. After hydrolysis for 1 h, the pH was adjusted back to the original pH value and the reducing sugar content was measured. The reducing sugar conversion rate formula is:

[0078] RRS (%) = (C1-C0) / C

[0079] Among them, C1 is the reducing sugar content after hydrolysis;

[0080] C0 is the reducing sugar content before hydrolysis;

[0081] C is the total sugar content.

[0082] 3.2 Test Results

[0083] The conversion rate of PHGG reducing sugar at acidic pH is as follows Figure 1 As shown. Under acidic conditions with a pH value of 2.0-5.0, RRS% increased slightly and was less than 1.0%. When the pH value reached 1.0, the conversion rate of reducing sugars increased significantly. pH < 2.0 will cause PHGG to degrade, which is manifested by an increase in the conversion rate of reducing sugars. The molecular weight of PHGG will affect its acid stability. PHGG with a large molecular weight has the lowest conversion rate of reducing sugars at pH 1.0, indicating that it has the highest acid stability. It was preliminarily determined that PHGG with a high molecular weight has more stable properties in an acidic environment.

[0084] 4. Evaluation of intestinal flora

[0085] 4.1 Evaluation Method

[0086] 4.1.1 High-throughput sequencing of 16S rRNA gene

[0087] The 24h fermentation precipitate stored at -80℃ was taken out for fecal flora DNA extraction and 16S rRNA high-throughput sequencing. The V3-V4 variable region of the 16S rRNA gene was PCR amplified using the upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). Sequencing was performed using the Illumina HiSeq 2500 platform. Data processing and bioinformatics analysis were performed based on the I-sanger Bioinformatics Cloud Platform (http: / / www.isanger.com / index.html) of Shanghai Meiji Biopharmaceutical Technology Co., Ltd., and the intestinal flora composition at different levels of each group was analyzed using the R language tool; the bacterial groups with significant differences in genus abundance between different groups were determined using linear discriminant analysis effect size (LEfSe) analysis (LDA>2.5, P<0.05).

[0088] 4.1.2 Determination of short-chain fatty acids

[0089] Take 800 μL of supernatant and add 200 μL of 50% (w / v) concentrated sulfuric acid to vortex and mix, then immediately add 1 mL of ether and vortex for 1 min, let stand on ice for 10 min, add anhydrous CaCl2 for dehydration after centrifugation at 12000 r / min for 10 min, and draw the supernatant to pass through the membrane for testing. The mass concentration of each short-chain fatty acid was calculated by external standard method, the detector was a hydrogen flame detector (Flame ionization detector, FID), the carrier gas was N2, the detector temperature was 250°C, the injection volume was 1 μL, the injection port temperature was 240°C, the initial temperature was 80°C (0.5 min), the temperature was raised to 150°C (4°C / min), and then the temperature was raised to 230°C (20°C / min) and maintained for 10 min.

[0090] 4.2 Evaluation Results

[0091] 4.2.1 Effects of PHGG with different molecular weights on in vitro fermentation pH

[0092] The change of pH value reflects to some extent the change of intestinal acidity and alkalinity during the fermentation process, which affects the production of short-chain fatty acids and the structure of microbial flora. Therefore, the present invention monitors the acidity of the culture medium at each sampling point. Figure 2 As shown, the initial pH values ​​of the fermentation broths of the blank group, GG group and embodiment group were between 7 and 8; during the fermentation 0-6h stage, the pH of the blank group decreased and then tended to be flat, maintaining at about 7; as the fermentation time increased, the pH values ​​of the GG group and the embodiment group gradually decreased, and at 24h, the pH value was about 5; during 24-48h, due to the gradual depletion of the substrate, the pH values ​​of the GG group and the embodiment group decreased but basically tended to be flat. During the overall fermentation process, the pH values ​​of the fermentation broths of PHGG with different molecular weights were similar.

[0093] 4.2.2 Effects of PHGG with different molecular weights on in vitro fermentation of SCFAs

[0094] The results are as follows Figure 3As shown, among the short-chain fatty acids, GG and PHGG produced the largest butyrate content. At 24h, the acetic acid production of GG group, Example 1 group, Example 2 group, Example 3 group, and Example 4 group was 0.55μg / mL, 1.03μg / mL, 1.17μg / mL, 1.20μg / mL, and 1.05μg / mL, respectively; at 48h, it was 0.92μg / mL, 1.87μg / mL, 1.97μg / mL, 1.80μg / mL, and 1.56μg / mL, respectively; it was also found that PHGG did not produce acetic acid from 0 to 12h, but acetic acid increased from 12 to 48h and then tended to be flat, and the change trend of acetic acid of PHGG with different molecular weights was similar. The propionic acid production of PHGG increased rapidly in 6-24h. After 24h, the production of propionic acid in Example 2 group increased more than that in Example 1 group, Example 3 group, and Example 4 group; the butyric acid production of PHGG increased rapidly in 12-36h. At 24h, there was no significant difference in the butyric acid content produced by different molecular weights. At 48h, the effect of Example 2 group was better. After 24h of fermentation, the viscosity of GG decreased and butyric acid began to be produced, and the total acid content showed a rapid upward trend. According to the acid production results, PHGG has good prebiotic potential. The above results show that intestinal microorganisms can utilize PHGG, leading to its fermentation and production of a large amount of SCFAs, thereby lowering the pH value.

[0095] 4.2.3 Effects of PHGG with different molecular weights on the diversity of human intestinal flora

[0096] 4.2.3.1 Effects of different molecular weights on the OTU index of intestinal flora

[0097] OTU refers to a species classification unit with a similarity sequence of 97%. The OTU in GG and PHGG prepared in Example 1-4 was analyzed using 16S RNA sequencing technology. Figure 4 As shown, the specific population of intestinal flora in the blank group was 316, and the specific population OTU number of the GG group was 3. However, after enzymatic hydrolysis, the OTU number of intestinal flora of PHGG with different molecular weights increased, among which the OTU number of Example 2 group was the largest, and its specific population number was 10.

[0098] 4.2.3.2 Effects of different molecular weights on intestinal flora diversity

[0099] (1) α-diversity of intestinal flora

[0100] The α-diversity of colonic fermentation intestinal microorganisms was analyzed, which mainly included the Ace index, Chao index, Shannon index and Simpson index. Among them, the Ace index and Chao index are indices used to evaluate the number of OTUs in the community, representing the abundance of the flora; while the Shannon index and Simpson index are indices used to estimate the diversity of microorganisms. The research results are shown in Table 3. Compared with the blank group, the Ace index, Chao index and Shannon index of the GG group and the embodiment group decreased significantly (p < 0.05); but compared with the GG group, the Ace index of the embodiment 2 group increased significantly (p < 0.05); for the Shannon index, the embodiment group increased significantly compared with the GG group (p < 0.05); the Simpson index of the GG group and the embodiment group increased. The above results show that the GG group caused the richness and uniformity of the intestinal flora to decrease, but the embodiment group can increase the richness and uniformity of the intestinal flora, and the effect of the embodiment 2 group is more significant.

[0101] Table 3

[0102]

[0103] Note: Values ​​are expressed as mean ± standard deviation (n = 3); means with different letters in a column are significantly different (p < 0.05).

[0104] (2) β-diversity of intestinal flora

[0105] β-diversity is a comparison of the composition of microbial communities between different samples. The present invention further performs PCoA principal component analysis on fecal samples at the OTU level based on Bray-Curtis distance. In the PCoA graph, the closer the distance between two samples, the more similar the sample community composition and β-diversity. Figure 5 It can be seen that in the PCoA diagram, the blank group, GG group and embodiment group are distributed in different quadrants, indicating that there are significant differences in the intestinal flora OTU levels of the above three groups; after GG is enzymatically hydrolyzed, PHGG of different molecular weights are close, and there are some overlapping areas, indicating that the β diversity of intestinal microbial OTU levels of PHGG of different molecular weights is similar, and GG and PHGG can change the intestinal β diversity. This is consistent with the results of Venn diagram analysis and α diversity.

[0106] 4.2.3.3 Effects of PHGG on the intestinal flora species level and differential species analysis

[0107] The present invention analyzes the effects of the GG group and the example group on the intestinal microbial structure of healthy people at the genus level. Figure 6 and Figure 7As shown. According to the species analysis at the genus level, there were 15 species of microorganisms with significant differences at the genus level between the GG group and the example group (p < 0.05), and the top five in abundance were Escherichia-Shigella, Clostridium_sensu_stricto_1, Prevotella, Citrobacter, and Dialister. The abundance of Clostridium_sensu_stricto_1 in the GG group and the example group increased significantly (p < 0.05); compared with the blank group, the abundance of Escherichia-Shigella in the GG group increased significantly (p < 0.05), but the example group could significantly reduce the abundance of Escherichia-Shigella (p < 0.05). At the same time, the study showed that Parabacteroides in the example 2 group increased significantly (p < 0.05).

[0108] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing PHGG of a specific molecular weight, characterized in that: The following steps are involved: (1) guar gum, cellulase and mannanase are mixed in a phosphate buffer, and then stirred for enzymolysis to obtain a guar gum enzymolysis solution; (2) sterilizing the obtained guar gum enzymatic hydrolysis solution, filtering it, and then freeze-drying it to obtain PHGG; In step (2), the molecular weight of the PHGG is 5-500 kDa.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of guar gum is 0.001-0.01 g / mL; the amount of cellulase added is 8000 U / g; and the amount of mannanase added is 100 U / g.

3. The preparation method according to claim 2, characterized in that: In step (1), the pH value of the phosphate buffer is 4-6; and the stirring speed is 200 rpm.

4. The preparation method according to claim 3, characterized in that: In step (1), the enzymatic hydrolysis temperature is 40-60°C; and the enzymatic hydrolysis time is 2-14h.

5. The preparation method according to claim 4, characterized in that: In step (1), the concentration of the guar gum is 0.005 g / mL; in step (2), the molecular weight of the PHGG is 8.9-35 kDa.

6. The preparation method according to claim 5, characterized in that: In step (1), the enzymatic hydrolysis time is 6-14 h; in step (2), the molecular weight of PHGG is 8.9-25 kDa.

7. The preparation method according to claim 6, characterized in that: In step (1), the enzymatic hydrolysis time is 6-10 h; in step (2), the molecular weight of the PHGG is 15-25 kDa.

8. The preparation method according to claim 7, characterized in that: In step (1), the enzymatic hydrolysis time is 6 h; in step (2), the molecular weight of PHGG is 25 kDa.

9. PHGG prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the PHGG according to claim 9 in regulating human intestinal flora.

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