Preparation of galactomannan and application of galactomannan in improvement of diarrhea

Abstract

The invention discloses a preparation method of galactomannan and application of the galactomannan in relieving diarrhea, and belongs to the technical field of biological medicines.The preparation method comprises the steps that guar gum is subjected to composite enzymolysis, centrifugation, enzyme deactivation, impurity removal and ultrafiltration, and the galactomannan with the specific molecular weight (10000-20000 Da) is obtained. Animal experiments prove that the galactomannan can quickly and effectively relieve diarrhea symptoms of mice and weaned piglets, the diarrhea index and the water sample amount of the mice can be remarkably reduced within 2 hours after intervention on the diarrhea mice, meanwhile, the intestinal barrier of the mice can be effectively consolidated, intestinal moisture absorption is promoted, intestinal flora is adjusted, and the diarrhea resistance of the mice and the weaned piglets is improved. The invention provides special and effective novel prebiotics for the treatment of diarrhea, and provides a theoretical basis for the practical application of galactomannan.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the preparation of galactomannan and its application in improving diarrhea. Background Art

[0002] Galactomannan is a natural water-soluble macromolecular polysaccharide, which consists of mannose connected by β-(1,4)-glycosidic bonds as the main chain and galactose connected by α-(1,6)-glycosidic bonds as the side chain. Its main sources include guar gum, locust bean gum, fenugreek gum, and tianqing gum. Galactomannan obtained from different sources and different extraction methods has different molecular weights and ratios of galactose to mannose. Guar gum has the characteristics of high viscosity and good stability. It is often used as a thickener and stabilizer in food, industry and other fields. However, the high molecular weight makes guar gum too viscous, and it is very viscous even at low concentrations, which is not conducive to practical application. Therefore, a composite enzymatic hydrolysis method can be used to degrade the macromolecular guar gum into galactomannan with a smaller molecular weight for further application. As a special long-chain macromolecular polysaccharide, galactomannan can smoothly reach the distal end of the human body and be used by intestinal microorganisms in the distal end of the intestine, thereby regulating intestinal homeostasis. However, if the molecular weight is too small, it cannot form a long-chain structure and is difficult to reach the distal end of the intestine. Therefore, it is necessary to find a suitable molecular weight range that not only meets the characteristics of low viscosity suitable for application, but also ensures its long-chain structure and maintains good beneficial activity.

[0003] Diarrhea is a common disease, with typical symptoms of increased bowel movements and thin stools. When it occurs, it affects the patient's work, study and life, and even endangers the patient's life in severe cases. The intestines of weaned piglets are not fully developed, the immune function is reduced, and the intestinal microbial barrier is easily damaged, resulting in disordered intestinal flora and reduced content of short-chain fatty acids in the intestine, which in turn damages the intestinal mechanical and chemical barriers, and is prone to decreased appetite, digestive disorders, diarrhea, etc., which leads to growth retardation.

[0004] Prebiotics are a type of indigestible substance that cannot be digested and absorbed by the host, but can be selectively used by microorganisms in the intestine, thereby promoting the proliferation of probiotics and improving the intestinal microenvironment. Currently, prebiotics used to relieve diarrhea are mostly small molecules, which achieve the effect of preventing diarrhea through long-term intervention. However, this method of preventing diarrhea by regulating intestinal flora has a long cycle and usually requires more than two weeks of intervention. It has poor short-term treatment effects on acute diarrhea. Therefore, the development of a new prebiotic that can quickly relieve diarrhea has broad application prospects. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing galactomannan, and to apply the method to relieve diarrhea, so as to solve the above-mentioned deficiencies of existing prebiotics. The galactomannan obtained by composite enzymatic hydrolysis of guar gum can quickly and significantly relieve diarrhea symptoms, and play the role of strengthening the intestinal barrier and improving the intestinal microecology.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing galactomannan, comprising the following steps:

[0008] Step 1, after dissolving the guar gum, adding two enzymes and mixing them thoroughly to perform composite enzymolysis;

[0009] Step 2, adjusting the pH of the enzymatic hydrolyzate to 6.0-6.2, and then centrifuging to obtain a supernatant;

[0010] Step 3, inactivating enzymes, removing impurities, decolorizing and filtering the supernatant, ultrafiltration to retain products of 10000Da-20000Da, and drying to obtain galactomannan powder.

[0011] Preferably, the enzymes in step 1 are acid mannanase and cellulase, and the added amounts are 200-2000U of acid mannanase and 300-1000U of cellulase per gram of guar gum, respectively.

[0012] Preferably, in step 1, the concentration of the guar gum solution is 5%-25%, the enzymolysis time is 10-24h, and the enzymolysis temperature is 30-60°C.

[0013] Preferably, in step 2, calcium hydroxide is used to adjust the pH, the centrifugal speed is 4000 rpm, and the centrifugal time is 10-30 min.

[0014] Preferably, the conditions for the enzyme inactivation treatment in step 3 are 90° C. and 30 min.

[0015] Preferably, in step 3, activated carbon and diatomaceous earth are used for decolorization and impurity removal.

[0016] Preferably, in step 3, a spiral ultrafiltration membrane is used, and the molecular weight cut-off range is 10000 Da-20000 Da.

[0017] Preferably, in the galactomannan obtained by the present method, the ratio of mannose to galactose is 1:1.4-1:1.5.

[0018] The present invention also provides the application effect of the galactomannan prepared by the method in alleviating diarrhea in mice and piglets.

[0019] The galactomannan disclosed in the present invention is applied to a diarrhea mouse model induced by senna leaves, and significantly reduces the diarrhea symptoms of mice within 2 hours; increases the ratio of villus length to crypt depth and intestinal wall thickness, reduces intestinal structural damage, significantly increases the expression of intestinal tissue tight junction proteins ZO-1 and Occludin, and mucin MUC2, and consolidates the intestinal barrier; significantly increases the expression of intestinal tissue water channel proteins AQP3 and AQP4, promotes the absorption of water by the intestine, and reduces the water content of feces; regulates the composition of intestinal flora, promotes the proliferation of beneficial bacteria, reduces the abundance of harmful bacteria, promotes the production of short-chain fatty acids, and improves the intestinal microenvironment.

[0020] The galactomannan disclosed in the present invention is applied to weaned piglets, which significantly alleviates the diarrhea symptoms of piglets within 7 days, improves the intestinal microenvironment of piglets, enriches the diversity of intestinal flora, reduces the abundance of harmful bacteria, promotes the proliferation of beneficial bacteria, and increases the content of intestinal short-chain fatty acids.

[0021] The present invention has at least the following beneficial effects:

[0022] 1. The galactomannan preparation method disclosed in the present invention adopts composite enzymatic hydrolysis, and the obtained product has a specific chemical composition (molecular weight 10000-20000Da, ratio of mannose to galactose is 1:1.4-1:1.5);

[0023] 2. The galactomannan provided by the present invention can quickly relieve diarrhea symptoms. Mouse experiments show that the diarrhea symptoms of mice are significantly alleviated within 2 hours after galactomannan intervention;

[0024] 3. The present invention uses HE staining, real-time quantitative polymerase chain reaction (RT-qPCR), 16s rRNA sequencing, high-performance liquid chromatography and other methods to explore the effects of galactomannan on the intestinal tissue structure, intestinal barrier, intestinal flora composition and metabolites of diarrhea mouse models. The results show that galactomannan can comprehensively relieve mouse diarrhea by strengthening the intestinal barrier, reducing intestinal water absorption and regulating intestinal flora.

[0025] 4. The present invention broadens the application field of galactomannan, provides experimental basis for the application of galactomannan in relieving diarrhea, and can be used as a special long-chain prebiotic in food or feed to quickly relieve diarrhea symptoms in humans or animals, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the results of measuring the molecular weight of galactomannan in Example 1;

[0027] Figure 2 This is a diagram showing the results of determining the monosaccharide composition of galactomannan in Example 1;

[0028] Figure 3 The effect diagram of galactomannan in Example 2 on relieving symptoms in mice includes diarrhea scores and the number of watery stools in mice within 2 hours after intervention. The data are expressed as mean ± standard error. ANOVA analysis of variance and LSD multiple comparisons were used. P < 0.05 was considered significant. Groups with significant differences are represented by different letters.

[0029] Figure 4 The HE staining observation diagram of the jejunum and colon structure of different groups of mice in Example 2, the scale bar is 200 μm;

[0030] Figure 5 The values ​​are as follows: the measured values ​​of jejunal villus length (A), crypt depth (B), villus length / crypt depth ratio (C), jejunal wall thickness (D), and colon wall thickness (E) of different groups of mice in Example 2. Ten regions were randomly selected from each slice for measurement. The data are expressed as mean ± standard error. ANOVA analysis of variance and LSD multiple comparisons were used. P < 0.05 was considered significant. Groups with significant differences are represented by different letters.

[0031] Figure 6 The effect of galactomannan on the mRNA expression level of genes in the intestinal tissues of mice in Example 2, ileum: ileum, jejunum: jejunum, colon: colon, ZO-1: zonula occludens 1, Occludin: occludens, MUC2: mucin 2, AQP3: aquaporin 3, AQP4: aquaporin 4, data are expressed as mean ± standard error, ANOVA analysis of variance and LSD multiple comparisons were used, P < 0.05 was considered significant, and groups with significant differences were represented by different letters;

[0032] Figure 7 This is a Venn diagram of OTUs of intestinal flora of mice in different groups in Example 2;

[0033] Figure 8 This is a PCoA analysis of the intestinal flora of different groups of mice in Example 2 based on OTU classification, analyzed using braycurtis distance and ANOSIM test;

[0034] Fig. 9 The relative abundance of intestinal flora composition at the phylum (A), family (B), genus (C) level, and differential bacteria (DG) at the genus level in different groups of mice in Example 2 are presented as mean ± standard error, and ANOVA analysis of variance and LSD multiple comparisons were used. P < 0.05 was considered significant, and groups with significant differences are represented by different letters;

[0035] Fig.10This is the LEfSe analysis diagram of the intestinal flora of different groups of mice in Example 2, comparing each group with the model group, and the LDA threshold is 2.0;

[0036] Fig.11 The contents of total short-chain fatty acids (A), lactic acid (B), formic acid (C), acetic acid (D), and isobutyric acid (E) in the intestinal contents of different groups of mice in Example 2 are expressed as mean ± standard error, and ANOVA analysis of variance and LSD multiple comparisons were used. P < 0.05 was considered significant, and groups with significant differences are represented by different letters;

[0037] Fig.12 The difference analysis of the α diversity index of the intestinal flora of piglets in different groups in Example 3 was performed using the Kruskal-Wallis rank sum test and FDR multiple verification validation. P<0.05 was considered to be significantly different (*), and P<0.01 was considered to be extremely significantly different (**);

[0038] Fig.13 The relative abundance of intestinal flora composition of different groups of piglets at the phylum (A) and family (B) levels in Example 3;

[0039] Fig.14 The difference flora of different groups of piglets in Example 3 compared with the control group (CK group) at the genus level was analyzed using the Wilcoxon rank sum test and FDR multiple verification validation. P<0.05 was significantly different (*), and P<0.01 was extremely significantly different (**);

[0040] Fig.15 The contents of propionic acid (A), isobutyric acid (B), and n-butyric acid (C) in the feces of different groups of piglets in Example 3 are expressed as mean ± standard error. ANOVA analysis of variance and LSD multiple comparisons were used. P < 0.05 was considered significantly different (*), and P < 0.01 was considered extremely significantly different (**). DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1 Preparation and chemical composition analysis of galactomannan

[0043] 1. Preparation of Galactomannan

[0044] Take 50g guar gum and dissolve it in 300mL water, add acid mannanase and cellulase, and hydrolyze at 50℃ for 20 hours. After the enzymolysis is completed, adjust the pH of the enzymolysis solution to 6.0-6.2 with calcium hydroxide, and then centrifuge at 4000rpm for 10min. Take the supernatant after centrifugation, add activated carbon and diatomaceous earth, heat it to 90℃, and keep it warm for 30min to kill the enzyme. After filtering and removing impurities, ultrafiltration is used to retain the product of 10000Da-20000Da, and spray drying is performed to obtain galactomannan powder with a yield of 65%-75%.

[0045] 2. Analysis of chemical composition of galactomannan

[0046] The galactomannan powder was dissolved at a concentration of 10 mg / mL and the molecular weight was determined using a high performance liquid chromatography-differential refractive index detector. Figure 1 As shown, the molecular weight of the product is distributed in the range of 10000-20000Da.

[0047] The monosaccharide composition of galactomannan was determined by PMP derivatization-HPLC, with lactic acid as the internal standard. The results showed that the monosaccharides that make up galactomannan are galactose, mannose, and glucose, and the ratio of mannose to galactose is 1:1.4-1:1.5 ( Figure 2 ).

[0048] Example 2 Evaluation of the effect of galactomannan on improving diarrhea

[0049] 1. Experimental Methods

[0050] 1.1 Preparation of galactomannan with different molecular weights

[0051] The preparation method of 10000Da-20000Da galactomannan (LMGM) is the same as that in Example 1. In addition, under the same conditions, the hydrolysis is carried out for 24 hours, and the product of 2000Da-10000Da is retained by ultrafiltration to obtain small molecular galactomannan (SMGM).

[0052] 1.2 Animal Experiment Grouping and Drug Treatment

[0053] Thirty 6-week-old SPF male BALB / c mice (initial weight 17.0±0.5g) were randomly divided into a normal group, a model group, a small molecule galactomannan group (SMGM), a low-dose galactomannan group (LMGM-L), a high-dose galactomannan group (LMGM-H), and a positive control group (Montmorillonite) after adaptive feeding for 1 week. The mice in the normal group were gavaged with 0.2mL of normal saline, and the mice in the other four groups were gavaged with 0.2mL of senna leaf extract to establish a model of diarrhea mice. After diarrhea was observed in mice, intervention treatment was immediately performed. The mice in the model group were gavaged with 0.2mL of normal saline, the mice in the high-dose galactomannan group were gavaged with 2.5g / kg galactomannan, the mice in the low-dose galactomannan group were gavaged with 0.5g / kg galactomannan, and the mice in the positive control group were gavaged with 1.2g / kg montmorillonite powder.

[0054] 1.3 Diarrhea evaluation

[0055] The fecal morphology of mice after galactomannan intervention was observed and recorded. The fecal grade standard was: grade 0, formed and dry feces; grade 1, formed but moist feces; grade 2, feces tended to be formed; grade 3, unformed feces; grade 4, watery stools. The fecal morphology of each mouse within 2 hours after intervention was counted, and all grades of defecation of the mouse during this period were added up to obtain the diarrhea score of each mouse.

[0056] 1.4 Observation of intestinal tissue morphology

[0057] The jejunal and colon tissues of mice were fixed with 4% paraformaldehyde and then stained with hematoxylin-eosin to observe the intestinal tissue morphology, and to measure the intestinal wall thickness, jejunal villus length and crypt depth.

[0058] 1.5 Real-time quantitative polymerase chain reaction (RT-qPCR)

[0059] The jejunum, ileum and colon tissues of mice were collected and ground with liquid nitrogen to extract total RNA. GADPH was used as a housekeeping gene to measure genes related to intestinal barrier and water absorption.

[0060] 1.6 Analysis of intestinal flora composition and diversity

[0061] The contents of mouse cecum were collected for 16s rRNA sequencing analysis. Total genomic DNA was extracted from all samples, and then the 16s rRNA V3-V4 region was amplified. After sequencing, splicing, quality control, and filtering, OTUs at a 97% similarity level were clustered and further analyzed.

[0062] 1.7 Determination of intestinal short-chain fatty acid content

[0063] The intestinal contents of mice were taken, dissolved in normal saline, and the content of short-chain fatty acids was determined by high performance liquid chromatography.

[0064] 1.8 Statistical analysis

[0065] SPSS25.0 statistical software was used for one-way analysis of variance, and LSD method was used for multiple comparisons. The test results were expressed as mean ± standard error, and P < 0.05 was considered significant, represented by different letters.

[0066] 2. Results and Analysis

[0067] 2.1 Evaluation of diarrhea in mice

[0068] like Figure 3 As shown in the figure, no diarrhea was observed in the mice in the normal group, while the diarrhea index and the number of grade 4 watery loose stools in the mice in the model group increased significantly within 2 hours after intervention. Compared with the model group, the diarrhea index and the number of grade 4 watery loose stools in the mice in the small molecular galactomannan group (SMGM), low-dose galactomannan group (LMGM-L), and high-dose galactomannan group (LMGM-H) were significantly reduced within 2 hours after intervention, but the diarrhea index of the low-dose galactomannan group (LMGM-L) and the high-dose galactomannan group (LMGM-H) was lower, which was significantly different from the small molecular galactomannan group (SMGM), indicating that galactomannan can significantly alleviate the diarrhea symptoms of mice within 2 hours, and the molecular weight of 10000Da-20000 Da is more effective.

[0069] 2.2 Effects of galactomannan on intestinal tissue morphology in mice

[0070] like Figure 4 As shown in the figure, the jejunal and colonic tissue structures of mice were observed, and it was found that the intestinal structure of mice in the normal group was intact without obvious lesions; the jejunal villi of mice in the model group were irregular, the crypt structure was destroyed, and obvious inflammatory cell infiltration was observed in the colon. Compared with the model group, the intestinal structure of mice in the galactomannan group was intact, the villi grew, and the inflammatory cell infiltration was significantly improved. The measurement found that the ratio of jejunal villus length / crypt depth of mice in the galactomannan group increased, and the intestinal wall thickened ( Figure 5 ).

[0071] 2.3 Effect of galactomannan on intestinal barrier gene expression in mice

[0072] like Figure 6As shown in AI, compared with the normal group mice, the expression of tight junction proteins ZO-1 and Occludin in the jejunum, ileum, and colon tissues of the model group mice decreased significantly; after galactomannan intervention, the expression of tight junction proteins in the mice increased significantly and returned to the same level as that of the normal group mice. The expression of mucin MUC2 in the jejunum, ileum, and colon tissues of the model group mice was significantly lower than that of the normal group mice, and galactomannan significantly inhibited the downregulation of MUC2 expression. The above results show that galactomannan can promote the expression of tight junction proteins and mucins, consolidate the intestinal mechanical and chemical barriers, avoid the occurrence of diarrhea, and reduce the damage of diarrhea to the intestine.

[0073] 2.4 Effect of galactomannan on the expression of water absorption genes in the intestine of mice

[0074] like Figure 6 As shown in Figure JO, the expression of water channel proteins AQP3 and AQP4 in the jejunum, ileum, and colon of mice was measured. The expression of water channel proteins in the jejunum, ileum, and colon of mice in the model group was significantly reduced, indicating that the intestinal absorption of water in diarrheal mice was significantly reduced, resulting in increased water content in the intestinal contents and thinner feces. Intervention with galactomannan restored the expression of intestinal water channel proteins in mice to the same level as that of mice in the normal group, indicating that galactomannan can promote intestinal absorption of water by upregulating the expression of water channel proteins, thereby reducing fecal water content and alleviating diarrhea.

[0075] 2.5 Effects of galactomannan on the composition and diversity of intestinal flora in mice

[0076] like Figure 7 As shown, there were 546 OTUs in the five groups, and the normal group, model group, positive control group, high-dose galactomannan group, and low-dose galactomannan group had 236, 114, 70, 106, and 91 unique OTUs, respectively.

[0077] The PCoA results showed that the intestinal flora composition of the model group mice was significantly different from that of the normal group mice, and the clusters of the high-dose galactomannan group and the model group mice were significantly separated ( Figure 8 ).

[0078] At the phylum level, Firmicutes and Bacteroidetes were the two most abundant dominant phyla in the cecal contents of mice. The abundance of Bacteroidetes in the model group was significantly increased, while the abundance of Firmicutes was significantly decreased ( Fig. 9 A).

[0079] At the family level, the abundance of Bacteroidetes and Prevotellaceae in the model group increased, while the abundance of Lachnospiraceae, Oscillospiraceae, and Ruminococcaceae decreased. However, high-dose galactomannan decreased the abundance of Prevotellaceae and increased the abundance of Lachnospiraceae ( Fig. 9 B).

[0080] At the genus level, the abundance of the beneficial bacteria genera Eubacterium and Butyricoccus in the model group mice was significantly reduced, and the abundance of the harmful bacteria genera Prevotella and Parabacteroides was significantly increased. Both high and low doses of galactomannan significantly reversed these changes, promoted the proliferation of beneficial bacteria genera, and reduced the abundance of harmful bacteria genera ( Fig. 9 CG). LEfSe analysis was further performed at the genus level to compare the differential biomarkers between each group and the model group ( Fig.10 ).

[0081] The above results indicate that the intestinal flora of diarrheal mice is destroyed, and galactomannan can improve the imbalance of intestinal flora, promote the proliferation of beneficial bacteria, reduce the abundance of harmful bacteria, and restore the intestinal flora to normal homeostasis.

[0082] 2.6 Effect of galactomannan on the content of short-chain fatty acids in the intestine of mice

[0083] like Fig.11 As shown in the results, the total short-chain fatty acid content of mice in the model group was significantly reduced, while the intervention of galactomannan restored the content of short-chain fatty acids in the intestinal contents of mice. Compared with the model group, high-dose galactomannan significantly increased the content of lactic acid, and low-dose galactomannan significantly increased the content of formic acid and acetic acid.

[0084] Example 3 Evaluation of the effect of galactomannan on improving diarrhea and intestinal flora in piglets

[0085] 1. Experimental Methods

[0086] The preparation method of galactomannan is the same as that in Example 1.

[0087] 1.1 Experimental design and experimental animals

[0088] 48 healthy and disease-free 30-day-old Duroc Changda Sanyuan pig weaned piglets were selected and randomly divided into three groups, including 30 pigs in the control group (CK group), 9 pigs in the high-dose galactomannan oligosaccharide group (HG group, LG group) and 9 pigs in each group. The control group was fed with a basic diet and antibiotics, and the high-dose galactomannan oligosaccharide group and the low-dose galactomannan oligosaccharide group were fed with 0.32% and 0.16% galactomannan oligosaccharide in the basic diet, respectively, and the experimental period was 14 days.

[0089] 1.2 Feeding and management

[0090] The experiment was conducted in a pig farm in Jiulong Street, Fangzi District, Weifang City. The temperature of the pig house was 25℃, the humidity was 75%, and it was kept clean and hygienic. During the experiment, all piglets were allowed to eat and drink freely. The feeding, mental state and diarrhea of ​​the piglets were observed and recorded daily. The immunization and health care procedures were carried out according to the routine management methods of the pig farm.

[0091] 1.3 Analysis of intestinal flora composition and diversity

[0092] Piglet fecal samples were collected on days 7 and 14 of the experiment for 16s rRNA sequencing analysis. Total genomic DNA was extracted from all samples, and then the 16s rRNA V3-V4 region was amplified. After sequencing, splicing, quality control, and filtering, OTUs at a 97% similarity level were clustered and further analyzed.

[0093] 1.4 Determination of intestinal short-chain fatty acid content

[0094] Piglet feces samples were collected on the 7th and 14th days of the experiment, dissolved in saline and then determined by HPLC for the content of short-chain fatty acids.

[0095] 1.5 Statistical analysis

[0096] SPSS 25.0 statistical software was used for one-way analysis of variance, and the LSD method was used for multiple comparisons. The test results are expressed as mean ± standard error, P < 0.05 was considered to be significantly different (*), and P < 0.01 was considered to be extremely significantly different (**).

[0097] 2. Results and Analysis

[0098] 2.1 Evaluation of piglet diarrhea

[0099] Table 1 Ratio of the number of diarrhea piglets in each group of Example 3 on different feeding days to the number of piglets in the group

[0100] Feeding days CK LG HG 2 2 / 30 0 / 9 0 / 9 5 7 / 30 2 / 9 1 / 9 7 4 / 30 1 / 9 0 / 9 10 5 / 30 0 / 9 0 / 9 14 4 / 30 0 / 9 0 / 9

[0101] The diarrhea of ​​piglets in different feeding days was recorded. As shown in Table 1, piglets in the galactomannan group (LG group and HG group) had no diarrhea symptoms on the second day of feeding, while piglets in the control group (CK group) had diarrhea symptoms; piglets with diarrhea accounted for the largest proportion in the control group (CK group) on the fifth and seventh days of feeding, followed by the low-dose galactomannan group (LG group) and the high-dose galactomannan group (HG group); piglets in the galactomannan group (LG group and HG group) had no diarrhea on the tenth and fourteenth days of feeding, while piglets in the control group (CK group) still had diarrhea symptoms. The above results show that galactomannan can effectively alleviate the diarrhea symptoms of weaned piglets.

[0102] 2.2 Effect of galactomannan on the diversity of intestinal flora in piglets

[0103] The intestinal microbial α diversity index of each group was analyzed. Fig.12As shown, compared with the control group, feeding low-dose galactomannan (LG) for 7 consecutive days significantly reduced the Shannon index and significantly increased the Simpson index, while feeding high-dose galactomannan (HG) for 7 consecutive days significantly increased the Sobs index and ACE index; feeding high and low doses of galactomannan (HG, LG) for 14 consecutive days significantly increased the Sobs index, ACE index, Chao index and Shannon index, while feeding low-dose galactomannan for 14 consecutive days significantly reduced the Simpson index. The results showed that feeding high-dose galactomannan for 7 consecutive days can significantly increase the richness of piglet microbiota, and feeding high and low doses of galactomannan for 14 consecutive days can significantly increase the richness and diversity of piglet microbiota.

[0104] 2.3 Effect of galactomannan on the composition of intestinal flora in piglets

[0105] like Fig.13 As shown in the figure, at the phylum level, the dominant phyla in each group were Firmicutes and Bacteroidetes, and the sum of the two accounted for more than 95%. Among them, Firmicutes accounted for a higher proportion, greater than 50%. At the family level, the abundance of Prevotellaceae was the highest in each group. Compared with the control group, the abundance of Prevotellaceae in HG_7, LG_14, and HG_14 groups decreased, and the abundance of Lactobacillaceae in LG_7 increased.

[0106] The differential bacterial communities at the genus level were further analyzed, such as Fig.14 As shown in the figure, compared with the control group, the abundance of Prevotella in the HG_7, LG_14, and HG_14 groups was significantly reduced, the abundance of UCG-005 in the HG_7 group was extremely significantly increased, and the abundance of UCG-002 in the HG_14 and LG_14 groups was extremely significantly increased. The abundance of the strict Clostridium 1 genus in the control group was 0%, and the abundance of this genus increased in each experimental group, among which the abundance of the strict Clostridium 1 genus in the HG_7, LG_14, and HG_14 groups was extremely significantly increased.

[0107] 2.4 Effect of galactomannan on the content of short-chain fatty acids in the intestine of piglets

[0108] like Fig.15 As shown in the data, compared with the control group, feeding high-dose galactomannan (HG) for 14 consecutive days significantly increased the concentration of propionic acid in the feces of piglets, feeding low-dose galactomannan (LG) for 7 consecutive days significantly increased the concentration of isobutyric acid in the feces of piglets, and feeding high and low doses of galactomannan (HG, LG) for 7 consecutive days could slightly increase the concentration of n-butyric acid in the feces of piglets, but there was no significant difference compared with the control group.

[0109] In summary, galactomannan can quickly relieve diarrhea symptoms, significantly reduce the diarrhea score and the number of watery stools in mice within 2 hours after intervention; consolidate the intestinal barrier of mice and repair intestinal structural damage; promote the absorption of water by the intestine, thereby reducing the moisture content of feces; regulate the composition of the intestinal flora of mice, maintain the homeostasis of the intestinal flora, and promote the production of short-chain fatty acids; effectively relieve diarrhea symptoms in weaned piglets, regulate the composition of the intestinal flora of piglets, enrich the diversity of the intestinal flora, and promote the production of short-chain fatty acids.

[0110] The above-described embodiments are only used to describe the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, ordinary technicians in this field may make other different forms of changes or modifications to the technical solutions of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this application shall be included in the scope of protection of this application.

[0111] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0112] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing galactomannan, characterized in that: The following steps are involved: Step 1, after dissolving the guar gum, adding two enzymes and mixing them thoroughly to perform composite enzymolysis; Step 2, adjusting the pH of the enzymatic hydrolyzate to 6.0-6.2, and then centrifuging to obtain a supernatant; Step 3, inactivating enzymes, removing impurities, decolorizing and filtering the supernatant, ultrafiltration to retain products of 10000Da-20000Da, and drying to obtain galactomannan powder.

2. The preparation method according to claim 1, characterized in that: The enzymes described in step 1 are acid mannanase and cellulase, and the added amounts are 200-2000U of acid mannanase and 300-1000U of cellulase per gram of guar gum.

3. The preparation method according to claim 1, characterized in that: In step 1, the concentration of the guar gum solution is 5%-25%, the enzymolysis time is 10-24h, and the enzymolysis temperature is 30-60°C.

4. The preparation method according to claim 1, characterized in that: In step 2, calcium hydroxide is used to adjust the pH, the centrifugal speed is 4000 rpm, and the centrifugal time is 10-30 min.

5. The preparation method according to claim 1, characterized in that: The conditions for the enzyme inactivation treatment in step 3 are 90° C. and 30 min.

6. The preparation method according to claim 1, characterized in that: In step 3, activated carbon and diatomaceous earth are used for decolorization and impurity removal.

7. The preparation method according to claim 1, characterized in that: In step 3, a spiral ultrafiltration membrane is used with a molecular weight cutoff range of 10000 Da to 20000 Da.

8. The galactomannan prepared by the method according to claim 1, characterized in that: The ratio of mannose to galactose is 1:1.4-1:1.

5.

9. Use of the galactomannan prepared by the method according to claim 1 in relieving diarrhea, characterized in that: It can quickly relieve diarrhea symptoms, strengthen the intestinal barrier, promote the intestinal absorption of water, and regulate the intestinal microecology.

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