Modified fucoidin, preparation method thereof and application of modified fucoidin in regulating intestinal flora structure

Abstract

The invention belongs to the technical field of food microbial fermentation, and particularly relates to modified fucoidin, a preparation method thereof and application of the modified fucoidin in regulating an intestinal flora structure. The method comprises the following steps: inoculating a fermentation culture medium containing fucoidin with an activated bifidobacterium longum seed solution, carrying out anaerobic fermentation, taking a supernatant, desalting, and drying to prepare the modified fucoidin. The main chain structure and monosaccharide composition of the modified fucose fructose are unchanged, and the content of sulfuric acid groups of the modified fucose fructose is reduced by 25%-35% compared with that before modification. When the modified fucoidin is used for regulating the intestinal flora, the molecular weight of the modified fucoidin is reduced to 15% or below compared with that before modification, the proportion of the intestinal beneficial flora can be remarkably increased, the intestinal utilization rate of the modified fucoidin by the intestinal flora is increased, and a basis is provided for subsequent research and development of food for improving the intestinal beneficial flora.

Description

Technical Field

[0001] The present invention belongs to the technical field of food microbial fermentation, and specifically relates to a modified fucoidan and a preparation method thereof, and an application thereof in regulating the structure of intestinal flora. Background Art

[0002] Fucoidan (FUC) is a natural polysaccharide found in brown algae and marine invertebrates. It is mainly composed of L-fucose, sulfate groups and a small amount of other monosaccharides. It has multiple biological activities such as anticoagulation, anti-tumor and immunomodulation. However, the molecular weight of fucoidan is large and it is not easily absorbed, which seriously limits its application. For example, fucoidan is difficult to be digested and absorbed by the human gastrointestinal tract. Its function and regulation of body health depend to a large extent on the utilization of intestinal flora. At the same time, studies have found that because fucoidan is rich in sulfate groups in its structure, it will cause the environmental pH to decrease when used by microorganisms, which will have a certain effect on microbial proliferation.

[0003] As a new type of "functional organ", intestinal flora is vital to human health. It can regulate the immune system, inhibit the adhesion of pathogens on the intestinal surface, maintain the epithelial barrier, decompose indigestible carbohydrates and produce important metabolites. Among them, Bifidobacterium longum is a kind of probiotic, belonging to the genus Bifidobacterium. Because the surface-related proteins, extracellular polysaccharides or metabolites of Bifidobacterium longum interact with the host, it regulates the health of the host through direct or indirect pathways, so it has the effect of regulating intestinal health and can effectively prevent brain and intestinal diseases and autoimmune diseases.

[0004] Therefore, developing a method that can effectively reduce the molecular weight of fucoidan in vitro and reduce the content of sulfate groups in fucoidan is a favorable way to increase the utilization of fucoidan by intestinal flora and further improve its physiological efficacy. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing modified fucoidan in view of the deficiencies in the prior art.

[0006] The technical problem that the present invention needs to solve is to provide modified fucoidan prepared by the above preparation method.

[0007] The final technical problem to be solved by the present invention is to provide the application of the modified fucoidan mentioned above, and an intestinal flora regulator containing the modified fucoidan.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing modified fucoidan comprises the following steps: inoculating activated Bifidobacterium longum seed liquid into a fermentation medium containing fucoidan for anaerobic fermentation, taking the supernatant, desalting and drying to obtain the modified fucoidan.

[0010] Wherein, the Bifidobacterium longum powder is commercially available Bifidobacterium longum powder, and the fucoidan is fucoidan extracted from sea cucumber or brown algae or a commercially available product thereof.

[0011] Wherein, the inoculation amount of the Bifidobacterium longum seed solution is 0.01% to 5% v / v.

[0012] Preferably, the inoculum amount is 0.05% v / v.

[0013] The fermentation medium containing fucoidan has the following formula: 8.2-12.5 g / L of peptone, 0.5-5.0 g / L of beef extract powder, 0.5-6.0 g / L of fucoidan, 3.1-5.6 g / L of yeast extract powder, 15.2-25.7 g / L of glucose, 0.5-1.5 mL / L of Tween-80, 0.8-3.7 g / L of dipotassium hydrogen phosphate, 2.7-6.9 g / L of sodium acetate, 9.8-18.2 g / L of agar, 0.5-3.7 g / L of triammonium citrate, 0.1-0.4 g / L of magnesium sulfate, and 0.01-0.15 g / L of manganese sulfate.

[0014] Preferably, the concentration of fucoidan is 0.5 g / L or 5.0 g / L, and a more preferred concentration is 5.0 g / L.

[0015] The anaerobic fermentation is carried out under the following conditions: anaerobic culture at 37° C. for 4 to 48 hours.

[0016] Among them, after the preparation method of the modified fucoidan, the number and growth morphology of the long bifidobacterium were significantly improved. The length-to-width ratio of the bacteria was significantly increased, and the specific surface area was also increased, which was particularly significant in the fermentation group containing 5g / L fucoidan (i.e., 5FUC fermentation group).

[0017] The modified fucoidan prepared by the modified fucoidan preparation method is also within the scope of protection of the present invention.

[0018] Specifically, the modified fucoidan is desulfurized fucoidan. During the fermentation by Bifidobacterium longum, Bifidobacterium longum will degrade the sulfate groups of fucoidan, so that the sulfate group content of the modified fucoidan is reduced by 25% to 35% compared with that before modification.

[0019] Specifically, after the modified fucoidan is fermented and modified by Bifidobacterium longum, its molecular weight is about 70% of that before modification.

[0020] The application of the modified fucoidan in regulating the structure of intestinal flora is also within the scope of protection of the present invention.

[0021] Among them, the molecular weight of the modified fucoidan significantly decreased when regulating the intestinal flora structure, and compared with the pre-modification, it was reduced to less than 15%. This shows that the fucoidan after mixed intestinal flora fermentation is partially utilized by intestinal microorganisms. Compared with the molecular weight change after simple fermentation with Bifidobacterium longum, the molecular weight change is significant, indicating that the intestinal utilization rate of the fucoidan after mixed fermentation is higher.

[0022] Among them, the intestinal flora includes any one or a combination of several of the phylum Bacteroidota, phylum Firmicutes and phylum Proteobacteria.

[0023] Specifically, in healthy intestinal flora, the Bacteroidetes are the main responders of fucoidan fermentation.

[0024] An intestinal flora regulator is also within the scope of protection of the present invention.

[0025] Specifically, the intestinal flora regulator contains the modified fucoidan.

[0026] Beneficial effects:

[0027] (1) The present invention uses Bifidobacterium longum to ferment fucoidan to prepare a modified fucoidan. The preparation method partially desulfurizes fucoidan, which is not well absorbed and utilized by the intestine, through fermentation with Bifidobacterium longum, and effectively reduces the molecular weight of fucoidan without changing its main chain structure.

[0028] (2) The modified fucoidan prepared by the present invention can significantly increase the proportion of beneficial intestinal flora in regulating intestinal flora and improve the intestinal utilization rate of modified fucoidan by intestinal flora.

[0029] (3) The raw material of the fucoidan of the present invention is easy to obtain and easy to prepare, and has a significant effect on promoting intestinal health. It has broad prospects for industrial application and provides a basis for subsequent research and development of foods that increase beneficial intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0031] Figure 1 The effect of inoculum size on the molecular weight and sulfate group content of fucoidan fermented by Bifidobacterium longum;

[0032] Figure 2To study the effect of culture time on the molecular weight and sulfate group content of fucoidan fermented by Bifidobacterium longum;

[0033] Figure 3 The effect of different substrate concentrations on the molecular weight of fucoidan fermented by Bifidobacterium longum;

[0034] Figure 4 It is a quantitative analysis diagram of sulfate content before and after fermentation of fucoidan with different concentrations;

[0035] Figure 5 The infrared spectra of fucoidan with different concentrations before and after fermentation;

[0036] Figure 6 The monosaccharide composition profiles of different concentrations of fucoidan after fermentation;

[0037] Figure 7 The comparison of the morphology of Bifidobacterium longum before and after fermentation at magnifications of 8000× and 20000×. Among them, A and D are blank groups, representing that the fermentation system does not contain FUC; B and E are 0.5FUC fermentation groups, representing that the fermentation system contains 0.5g / L FUC; C and F are 5FUC fermentation groups, representing that the fermentation system contains 5g / L FUC;

[0038] Figure 8 This is the HPSEC molecular weight distribution diagram of modified fucoidan after fermentation by mouse intestinal flora. DETAILED DESCRIPTION

[0039] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0040] In the following examples, the Bifidobacterium longum powder is commercially available Bifidobacterium longum powder, and the fucoidan is fucoidan extracted from sea cucumber or brown algae or a commercially available product thereof.

[0041] In the following examples, the molecular weight of the fucoidan was detected by high performance gel chromatography. The system used consisted of an Agilent 1260 high performance liquid chromatograph and a differential refractive index detector, the chromatographic column model was Shodex OHpak SB-806HQ (8mm×300mm), 0.2M Na2SO4 was used as the mobile phase, the flow rate during elution was 0.6mL / min, and the column temperature was maintained at 35°C. All samples were processed through a 0.22μm microporous filter membrane, and the injection volume during detection was 30μL.

[0042] In the following examples, the sulfate group content of the fucoidan is determined by acid hydrolysis-ion chromatography, and the operation is as follows: weigh 5 mg of the sample to be tested, add 2M trifluoroacetic acid (1 mL) to seal the tube, shake it vigorously to dissolve it, and hydrolyze it at 110°C for 10 hours. Then blow dry in an oven at 80°C to remove the residual trifluoroacetic acid, add distilled water to dissolve the dried product and make it constant to 25 mL. The hydrolyzed sample is analyzed on an ion chromatograph equipped with a DS5 conductivity detector and an IonPac AS14 column (4mm×250mm), and the suppressor model is AERS500. The mobile phase is a mixture of 3.6mM Na2CO3 and 4.5mM NaHCO3, the flow rate is 0.5mL / min, the injection volume is 25μL, and anhydrous sodium sulfate is used as the standard. After gradient dilution, a sulfate concentration standard curve is established, and the peak area value of the sample curve is substituted into the standard curve to obtain the sulfate group content in the polysaccharide sample by calculation.

[0043] In the following examples, the monosaccharide composition analysis of the fermented fucoidan was determined by 1-phenyl-3-methyl-5-pyrazolone (PMP)-high performance liquid chromatography (HPLC). The specific operation is as follows: 100 μL of the fermentation broth was mixed with 0.6M NaOH at 1:1 (v / v), and 100 μL of the supernatant was transferred to a new test tube after oscillation. After adding the same volume of 0.5MPMP-methanol solution to the solution, the tube was sealed and oscillated evenly, and the reaction was carried out in a metal bath at 70°C for 100 min. After the derivatization reactant was cooled to room temperature, 0.3M HCl solution was added to the reactant to make the pH value of the aqueous solution neutral, and then the mixture was transferred to an oven and dried at 50°C. 1 mL of distilled water was added to dissolve the sample, and the excess PMP in the solution was removed by adding 1 mL of chloroform for continuous extraction 3 times. The upper aqueous phase was drawn and treated with a 0.22 μm microporous filter membrane, and then waited for HPLC detection and analysis. Standard monosaccharide samples including glucose, galactose, fucose, etc. were also derivatized according to the above steps. The test was carried out on a Thermo UltiMate 3000 chromatograph using an XDB-C18 column (4.6mm×150mm, 5μm) with a column temperature stabilized at 30°C. The mobile phase consisted of a pH 6.7 phosphate buffer solution (0.1M) and acetonitrile in a ratio of 83:17 (v / v), the sample injection volume was 20μL, the flow rate was 1mL / min, the elution time was 40min, and the UV detector detected the sample at a wavelength of 245nm.

[0044] Example 1: Optimization of anaerobic fermentation of fucoidan using Bifidobacterium longum

[0045] (1) Bacterial Activation

[0046] 0.1 g of Bifidobacterium longum powder was inoculated into 10 mL of MRS liquid culture medium and cultured anaerobically at constant temperature and humidity (37° C., 75% RH) for 24 h to obtain activated Bifidobacterium longum seed liquid.

[0047] (2) Effect of inoculation amount on the fermentation of fucoidan by Bifidobacterium longum

[0048] The activated Bifidobacterium longum seed liquid in step (1) was inoculated into a fermentation medium containing 5 g / L fucoidan according to different inoculation amounts (0.01%, 0.05%, 1%, 3%, 5% v / v), and placed in an incubator at 37°C for anaerobic culture for 24 h. After the fermentation was completed, samples were taken and the molecular weight and sulfate group content of the modified fucoidan after fermentation were determined by high performance liquid chromatography and Fourier transform infrared spectrometer, respectively.

[0049] Among them, the fermentation medium is an MRS medium added with fucoidan, and its formula is: peptone 10.0g / L, beef extract powder 5.0g / L, fucoidan 5.0g / L, yeast extract powder 4.0g / L, glucose 20.0g / L, Tween-801.0mL / L, dipotassium hydrogen phosphate 2.0g / L, sodium acetate 5.0g / L, agar 15.0g / L, triammonium citrate 2.0g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, pH 6.2±0.2.

[0050] The results are as follows Figure 1 As shown in the figure, it can be seen that after 24 hours of fermentation, there are differences in the molecular weight of modified fucoidan in different inoculation groups. The molecular weight of modified fucoidan in the inoculation group of 0.01% v / v is greater than that in other groups, and the molecular weight of modified fucoidan in the inoculation groups of 0.05%, 1%, 3%, and 5% v / v is not much different. Therefore, under this condition, the inoculation amount of 0.05% v / v can meet the needs.

[0051] (3) Effect of culture time on the fermentation of fucoidan by Bifidobacterium longum

[0052] The long bifidobacterium seed liquid activated in step (1) was inoculated into a fermentation medium containing 5 g / L fucoidan at an inoculation rate of 0.05% v / v, and placed in a 37°C incubator for anaerobically cultured for 4 to 36 hours. The fermentation broths of different culture times (4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, and 36 hours) were sampled, and the molecular weight of the modified fucoidan after fermentation was measured by high performance liquid chromatography, the sulfate group content was measured by acid hydrolysis-ion chromatography, and the characteristic peak changes of the sulfate group were analyzed by infrared spectroscopy.

[0053] The results are as follows Figure 2As shown, after 4 hours of fermentation by Bifidobacterium longum, the molecular weight of fucoidan can be reduced from the original 1663.09 kDa to 1311.663 kDa, and the sulfate group content is reduced from the original 25.68% to 18.65%. With the extension of fermentation time, the molecular weight of modified fucoidan is slightly reduced in the range of 4 to 20 hours; in the range of 20 to 36 hours, the molecular weight of modified fucoidan has no obvious change. At 24 hours of fermentation, the molecular weight of modified fucoidan is reduced to 1180.619 kDa, which is 29.01% lower than the original molecular weight of 1663.09 kDa. By analyzing the structure of modified fucoidan, it is found that there is no change in the main chain structure. After 4 hours of fermentation, the sulfate group content does not change significantly with the extension of fermentation time. At 24 hours of fermentation, the content of sulfate group is 18.19%, which is 29.17% lower than the original content of 25.68%.

[0054] (4) Effects of different substrate (fucoidan) concentrations on the fermentation of fucoidan by Bifidobacterium longum

[0055] After Bifidobacterium longum was activated at 37°C for 24 hours, 0.05% v / v of the activated bacterial solution was taken and inoculated into fermentation medium containing 0g / L (blank group), 0.5g / L (0.5FUC fermentation group), and 5g / L (5FUC fermentation group) of fucoidan, respectively, and fermented and cultured at 37°C for 24 hours. After the fermentation was completed, it was inactivated and centrifuged, and the supernatant was retained. After dialysis, the fermentation solution was freeze-dried to obtain a dry solid sample. Using unfermented fucoidan as a control, the molecular weight, sulfate group content, and monosaccharide composition of the modified fucoidan after fermentation were determined.

[0056] The results of the change of substrate concentration on the molecular weight of modified fucoidan are as follows: Figure 3 As shown in the figure, after 24h of fermentation with Bifidobacterium longum, the effect of different substrate concentrations on the reduction of fucoidan molecular weight was not significantly different. The results of the change of substrate concentration on the change of sulfate group content are shown in the figure. Figure 4 As shown in the figure, the reduction of sulfate content by different substrate concentrations was not significantly different, but sulfate degradation was significant after fermentation of modified fucoidan. This result is consistent with the results of infrared spectroscopy analysis ( Figure 5 ), Bifidobacterium longum will degrade the sulfate groups of fucoidan during fermentation. By comparing the peak time and peak area of ​​the chromatograms of the modified fucoidan samples and the standard after two fermentations, it was found that the monosaccharide composition of fucoidan did not change before and after Bifidobacterium longum fermentation ( Figure 6 ).

[0057] Example 2: Changes in bacterial morphology in the fucoidan fermentation system of Bifidobacterium longum

[0058] Scanning electron microscopy was used to observe the effect of fucoidan on the morphology of Bifidobacterium longum during the fermentation process. The specific operation was as follows: After Bifidobacterium longum was activated at 37°C for 24 hours, 0.05% v / v of the activated bacterial solution was taken and inoculated into MRS medium containing 0g / L (blank group), 0.5g / L (0.5FUC fermentation group), and 5g / L (5FUC fermentation group) fucoidan, respectively. After fermentation and cultivation at 37°C for 24 hours, 8mL of the fermentation solution was transferred to a 5mL centrifuge tube, and the centrifuge was set to a speed of 1200r / min for 5min, and the precipitate was collected to collect the bacteria. Then the bacteria were rinsed with 0.1M PBS buffer, and the operation was repeated 3 times. Subsequently, 2.5% glutaraldehyde-0.1M PBS solution was added to the bacteria for fixation, and the solution was left to stand overnight at 4°C. The fixed cells were dehydrated with different concentrations of ethanol (30%, 50%, 75%, 100%), and then supercritically dried with carbon dioxide at 95 bar. The dried samples were sprayed with gold and the cell morphology was observed by SEM at 8000 and 20000 magnifications.

[0059] The results are as follows Figure 7 As shown in the figure, Bifidobacterium longum showed a typical rod-shaped morphology in all treatment groups. However, with the increase of fucoidan concentration, the length-to-width ratio of the bacteria increased significantly, and the specific surface area also increased, which was particularly significant in the fermentation group containing 5g / L fucoidan (i.e., 5FUC fermentation group). Bifidobacterium longum can adhere to intestinal epithelial cells and prevent the growth of harmful microorganisms by producing antibacterial substances such as organic acids and bacteriocins. The increase in the specific surface area of ​​Bifidobacterium longum is more conducive to its biological activity.

[0060] Example 3: Utilization of modified fucoidan by in vitro cultured intestinal flora

[0061] The modified fucoidan prepared by fermentation for 4 h in step (3) of Example 1 was used as the experimental group, and the original fucoidan was used as the control group. They were added to MRS fermentation medium at a concentration of 5 g / L and fermented at 37° C. for 48 h. The samples were centrifuged and the molecular weight of the fermentation product was determined by liquid phase method.

[0062] The mouse intestinal flora was inoculated into the MRS culture medium of the above experimental group and control group at a ratio of 10% v / v, respectively as the mouse-experimental group and mouse-control group, and fermented at 37°C for 48 hours; the samples were centrifuged, and the molecular weight of the fermentation product was determined by liquid phase method. Among them, the mouse intestinal flora used in the experiment was prepared by dispersing fresh feces of normal mice in physiological saline and mixing them evenly.

[0063] Liquid phase results are shown in Figure 8, the X value corresponding to the peak value of the vertical axis response value is the peak time, and the relative molecular weight = 10^(-0.6192X+15.547). After calculation, it was found that after the intestinal flora used modified fucoidan for fermentation (i.e., the mouse-experimental group), its molecular weight was significantly reduced. Compared with the fermentation using only original fucoidan (control group), the molecular weight of fucoidan in the mouse-experimental group was reduced from the original 1663.09kDa to 239.18Da; compared with the fermentation using only Bifidobacterium longum (i.e., the experimental group), its molecular weight was also significantly reduced, indicating that after the intestinal flora used modified fucoidan for fermentation, part of the fucoidan was utilized by intestinal microorganisms; compared with the intestinal flora only using original fucoidan for fermentation (i.e., the mouse-control group), its molecular weight was reduced to a greater extent. The above results show that after the intestinal flora uses modified fucoidan for fermentation, the intestinal utilization rate of modified fucoidan is higher.

[0064] Example 4 Application of modified fucoidan in regulating intestinal flora structure

[0065] The fermentation product obtained in Example 3 was analyzed by high-throughput sequencing to study the effect of modified fucoidan on the composition of intestinal microbial flora.

[0066] The relative abundance results of intestinal microbial communities at the phylum level are shown in Table 1. After 48 h of fermentation, the dominant bacterial communities in each group were Bacteroidota, Firmicutes, and Proteobacteria, but they showed differences in relative abundance.

[0067] Table 1 Effects of fucoidan on the phylum-level composition of intestinal microbiota

[0068]

[0069] Compared with the control group, the experimental group showed that due to the fermentation modification of fucoidan, the number of Bacteroidetes in the fucoidan-containing intestinal flora increased most significantly (p=0.024), and the number of Proteobacteria was also greatly reduced (p=0.029). This shows that in healthy intestinal flora, Bacteroidetes is the main responder to fucoidan fermentation, and its increase in abundance is related to the accumulation of propionic acid in the fucoidan-containing intestinal flora. The genus Bacteroidetes has functional diversity and high genomic homogeneity. In the intestinal ecosystem, when the polysaccharide utilization sites in the genome are activated, some Bacteroidetes can promote the secretion of their own polysaccharide degrading enzymes, thereby cutting off specific glycosidic bonds and degrading a variety of polysaccharides. In the present invention, due to the proliferation of Bacteroidetes, the fucoidan hydrolase increases, so that the molecular weight of the modified fucoidan is reduced during the fermentation process.

[0070] The present invention provides a modified fucoidan and its preparation method and application ideas and methods for regulating the structure of intestinal flora. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing modified fucoidan, characterized in that: The activated Bifidobacterium longum seed liquid is inoculated into a fermentation medium containing fucoidan for anaerobic fermentation, and the supernatant is taken, desalted and dried to obtain the modified fucoidan.

2. The preparation method according to claim 1, characterized in that: The inoculation amount of the Bifidobacterium longum seed solution is 0.01% to 5% v / v.

3. The preparation method according to claim 1, characterized in that: The fermentation medium containing fucoidan has the following formula: 8.2-12.5 g / L of peptone, 0.5-5.0 g / L of beef extract powder, 0.5-6.0 g / L of fucoidan, 3.1-5.6 g / L of yeast extract powder, 15.2-25.7 g / L of glucose, 0.5-1.5 mL / L of Tween-80, 0.8-3.7 g / L of dipotassium hydrogen phosphate, 2.7-6.9 g / L of sodium acetate, 9.8-18.2 g / L of agar, 0.5-3.7 g / L of triammonium citrate, 0.1-0.4 g / L of magnesium sulfate, and 0.01-0.15 g / L of manganese sulfate.

4. The preparation method according to claim 1, characterized in that: The anaerobic fermentation conditions are as follows: anaerobic culture at 37° C. for 4 to 48 hours.

5. The modified fucoidan prepared by the preparation method according to any one of claims 1 to 4.

6. The modified fucoidan according to claim 5, characterized in that: The modified fucoidan is desulfurized fucoidan, and the sulfate group content of the modified fucoidan is reduced by 25% to 35% compared with that before modification.

7. Use of the modified fucoidan according to claim 5 in regulating the structure of intestinal flora.

8. The use according to claim 7, characterized in that: When the modified fucoidan regulates the intestinal flora structure, its molecular weight is reduced to less than 15% compared with that before modification.

9. The use according to claim 7, characterized in that: The intestinal flora includes any one or a combination of several of the phyla Bacteroidota, Firmicutes and Proteobacteria.

10. An intestinal flora regulator, characterized in that The intestinal flora regulator comprises the modified fucoidan described in claim 5.

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