A compound probiotic oligosaccharide prepared from algal residue and its method

Abstract

This invention discloses a method for preparing a composite probiotic oligosaccharide based on algal residue. Using algal residue as raw material, the method involves treatment with a lithium bromide molten salt hydrate system, followed by acid conditioning, methanol solubilization, gradient temperature reaction, dialysis desalting, alcohol precipitation, and drying. This process yields a composite probiotic oligosaccharide composed of glucose, fucose, mannose, and rhamnose, with a degree of polymerization of 4-10 and a weight-average molecular weight of 600-2000 Da, significantly enhancing intestinal probiotic function. This invention achieves efficient degradation and precise control of the depolymerization process under mild conditions, eliminating the need for high temperature, high pressure, and enzyme preparations. It overcomes the shortcomings of traditional chemical methods, such as uncontrolled degradation and easy destruction of activity, as well as the high cost and single component of enzymatic hydrolysis. It offers advantages such as high-value utilization of solid waste, reduced waste, and ease of large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of high-value utilization of marine biological resources and preparation of functional food ingredients, and particularly relates to a compound probiotic oligosaccharide and method based on algal residue. Background Technology

[0002] Prebiotic oligosaccharides are carbohydrate compounds composed of 2 to 20 monosaccharide molecules linked by glycosidic bonds. They can resist the decomposition by human digestive enzymes, and after reaching the intestines, they selectively proliferate beneficial bacteria and inhibit the growth of harmful microorganisms. They have prebiotic activities such as regulating intestinal microecology, improving metabolic function, enhancing intestinal immunity, and lubricating the intestines.

[0003] Existing seaweed-derived probiotic oligosaccharides are mostly processed from brown algae, with mainstream processes including chemical degradation (alkali extraction and acid hydrolysis) and enzymatic hydrolysis. Chemical degradation relies on acid-base catalysis to break the glycosidic bonds of polysaccharides, offering advantages such as rapid reaction and low cost. However, it suffers from a core drawback: poor controllability of degradation, easily leading to over-hydrolysis and destruction of active structures, ultimately resulting in a disordered product composition and significant waste and purification challenges. Enzymatic hydrolysis, compared to chemical degradation, offers advantages such as milder conditions, lower pollution, and fewer byproducts, but still has significant technical limitations, making it difficult to efficiently prepare highly active probiotic oligosaccharides.

[0004] Chinese invention patent application CN119753056A discloses a "method for preparing high-purity fucoidan oligosaccharides," which involves enzymatic hydrolysis by adding sodium alginate in batches (three times), followed by ultrafiltration and spray drying to obtain fucoidan oligosaccharides. While this method improves substrate degradation efficiency to some extent, it still suffers from problems such as the product's degree of polymerization being significantly affected by enzyme characteristics, reaction conditions, and batch feeding fluctuations, making precise control difficult. Reliance on expensive enzyme preparations not only increases production costs but also poses a risk of allergens due to enzyme protein residues. Furthermore, the high-temperature process in the subsequent spray drying stage (inlet air temperature often exceeding 150°C) easily induces thermal degradation and Maillard reactions in the oligosaccharides, leading to darker product color, decreased solubility, and loss of bioactivity.

[0005] Algal residue is a large byproduct generated during the deep processing of brown algae, red algae, and other seaweeds, including the extraction of gums and polysaccharides. It still contains a large amount of bound polysaccharides and unused oligosaccharide precursors, possessing extremely high resource potential. Currently, the industry's utilization rate of industrial algal residue is extremely low, with disposal mainly consisting of waste or low-value treatment. This not only wastes high-quality marine biological resources but also easily causes secondary environmental pollution.

[0006] Molten salt hydrates (MSH) are solvent systems formed by mixing inorganic salts and water in a specific ratio, and have attracted attention in the field of cellulose conversion in recent years. This system can efficiently disrupt the dense hydrogen bond network of cellulose under mild conditions through the coordination of salt cations with cellulose hydroxyl groups, thereby dissolving insoluble polysaccharides. Existing research shows that while molten salt hydrates (MSH) can effectively disrupt the crystalline structure of cellulose and achieve its dissolution, key technical bottlenecks remain in practical applications: the acid catalysis is difficult to precisely control, the cellulose depolymerization process often fails to stop at the oligosaccharide stage, and the products are easily further hydrolyzed into glucose or excessively degraded into small molecule byproducts such as 5-hydroxymethylfurfural and levulinic acid. For complex biomass raw materials containing polysaccharide impurities, such as brown algae gum extraction residue, the composition of products after MSH treatment is even more complex, making it difficult to selectively obtain high-value-added functional oligosaccharides. Therefore, how to precisely control the cellulose depolymerization process in the MSH system, stopping it at the oligosaccharide stage, and expanding its application to the high-value conversion of seaweed processing byproducts remains a pressing technical challenge. Summary of the Invention

[0007] The present invention aims to solve the above-mentioned technical problems existing in the prior art by providing a compound probiotic oligosaccharide prepared from algal residue and a method thereof.

[0008] The technical solution of the present invention is: a compound probiotic oligosaccharide prepared based on algal residue, which is polymerized from glucose, fucose, mannose and rhamnose, with a degree of polymerization of 4 to 10 and a weight-average molecular weight of 600 to 2000 Da. The mass percentage of each monosaccharide is 70 to 80% glucose, 18 to 25% fucose, 0.5 to 3% mannose and 0.5 to 3% rhamnose.

[0009] A method for preparing compound probiotic oligosaccharides based on algal residue, comprising the following steps: Step 1. Dissolve lithium bromide in deionized water, mix and dissolve to obtain solution I; Step 2. Add sulfuric acid to solution I and stir until homogeneous to obtain solution II; Step 3. Add methanol to solution II and stir until homogeneous to obtain solution III; Step 4. Mix solution III with algal residue to obtain solution IV; Step 5. Evaporate solution IV to remove methanol, obtaining solution V; Step 6. Heat solution V to obtain solution VI; Step 7. Add deionized water to solution VI to dissolve it, filter to remove insoluble matter, and obtain solution VII; Step 8. Dialyze solution VII sequentially to desalt, precipitate with alcohol, and freeze dry to obtain the compound probiotic oligosaccharide.

[0010] Preferably, the mixing and dissolution temperature in step 1 is 80°C, and the mass percentage concentration of solution I is 50-60%.

[0011] Preferably, the sulfuric acid concentration in solution II of step 2 is 0.25~2.25 mol / L.

[0012] Preferably, the volume ratio of solution II to methanol in step 3 is 1:1 to 5.

[0013] Preferably, the liquid-to-solid ratio of solution III to algal residue in step 4 is 2.0~15.0 mL / g.

[0014] Preferably, step 5 involves placing solution IV at 50°C for 10-12 hours to remove methanol.

[0015] Preferably, step 6 involves heating solution V at 80-130°C for 1-4 hours.

[0016] Preferably, step 7 involves mixing solution VI with deionized water at a volume ratio of 1:1 and stirring to dissolve.

[0017] Preferably, step 8 involves dialysis desalting using a dialysis membrane with a molecular weight cutoff of 300-500 Da, followed by alcohol precipitation with 8 times the volume of anhydrous ethanol, and freeze-drying for 24 hours.

[0018] This invention uses seaweed residue from seaweed industrial processing as raw material and employs an optimized molten salt hydrate system for controlled degradation of polysaccharides. This system achieves efficient degradation and precise control of the depolymerization process under mild conditions, eliminating the need for high temperature, high pressure, and the addition of enzymes. It overcomes the shortcomings of traditional chemical methods, such as uncontrolled degradation and easy destruction of activity, as well as the high cost and single composition of enzymatic hydrolysis. The resulting compound probiotic oligosaccharide exhibits a concentrated degree of polymerization, with a yield of 92.28 ± 0.27% (standard deviation) and a purity of 94.25 ± 0.14% (w / w). The compound probiotic oligosaccharide prepared by this invention is synergistically composed of glucose, fucose, mannose, and rhamnose, exhibiting balanced and stable composition and significantly enhancing intestinal probiotic function. Furthermore, this invention offers advantages such as high-value utilization of solid waste, minimal waste generation, and ease of large-scale production. Attached Figure Description

[0019] Figure 1 The chromatograms are of the monosaccharide mixed standard solution and the composite probiotic oligosaccharide prepared in Example 1 of this invention.

[0020] Figure 2 This is a relative molecular mass distribution diagram of the compound probiotic oligosaccharide prepared in Example 1 of the present invention.

[0021] Figure 3 This is the infrared spectrum of the compound probiotic oligosaccharide prepared in Example 1 of the present invention.

[0022] Figure 4 This is an OD600 curve of four probiotic strains co-cultured with the compound probiotic oligosaccharide prepared in Example 1 of this invention.

[0023] Figure 5 This is a schematic diagram illustrating the effect of the compound probiotic oligosaccharide prepared in Example 1 of the present invention on the formation of short-chain fatty acids. Detailed Implementation Example 1

[0024] The present invention discloses a method for preparing compound probiotic oligosaccharides based on algal residue, comprising the following steps: Step 1. Dissolve lithium bromide (LiBr) in deionized water to prepare a lithium bromide aqueous solution with a mass percentage concentration of 60%. Stir continuously at 80°C until completely dissolved to obtain solution I; Step 2. Take 2.35 mL of solution I, add 1.74 mL of sulfuric acid with a concentration of 4 mol / L, stir well, and obtain solution II with a final sulfuric acid concentration of approximately 1.7 mol / L; Step 3. Mix solution II with methanol at a volume ratio of 1:2.5 (v / v) to obtain solution III; Step 4. Take 10 mL of Solution III and add it dropwise to 1.0 g of degummed brown algae residue ( Macrocystis pyrifera Mix thoroughly in the solution to obtain solution IV; Step 5. Place solution IV in a 50°C oven and heat for 12 hours to remove methanol, obtaining solution V; Step 6. Heat solution V at 120°C for 2 hours to obtain solution VI; Step 7. Take 10 mL of solution VI and add 10 mL of deionized water, mix thoroughly to dissolve the soluble components, and obtain solution VII; Step 8. Transfer solution VII to a dialysis bag with a molecular weight cutoff of 300 Da, and dialyze with deionized water for 24 hours to remove salts; after dialysis, add 8 times the volume of anhydrous ethanol for alcohol precipitation, and let stand overnight; filter and collect the precipitate, and freeze-dry for 24 hours to obtain the compound probiotic oligosaccharide.

[0025] The oligosaccharide prepared in Example 1 was a pale yellow powder. The yield was 92.28 ± 0.27% (standard deviation, calculated based on the total polysaccharide content in the algal residue), and the purity was 93.10% ± 0.56% (w / w).

[0026] I. Analysis of the oligosaccharides prepared in Example 1 The oligosaccharides prepared in Example 1 were analyzed by HPLC, and the results are as follows: Figure 1 As shown, Figure 1Figure (A) is the HPLC chromatogram of the monosaccharide mixed standard solution, and Figure (B) is the HPLC chromatogram of the oligosaccharide prepared in Example 1. The analytical results show that the oligosaccharide prepared in this invention is mainly composed of glucose, fucose, rhamnose, and mannose. Calculated by mass percentage, the proportions of each monosaccharide are: glucose 76.02%, fucose 21.76%, rhamnose 1.67%, and mannose 0.56%, with glucose having the highest content, followed by fucose.

[0027] The mass spectrum obtained by MALDI-TOF-MS analysis is shown in the attached figure. Figure 2 As shown. The results show that the oligosaccharides prepared in Example 1 of this invention have molecular weights mainly distributed between 600-2000 Da, and degrees of polymerization (DP) mainly concentrated between 4 and 10. Among them, the components with degrees of polymerization of 4-10 account for 93.3% of the total peak area, confirming that the obtained product is an oligosaccharide.

[0028] Infrared spectroscopy analysis showed the following infrared spectrum: Figure 3 As shown. The results indicate that the oligosaccharide prepared in Example 1 of this invention possesses the typical characteristics of polysaccharide compounds: at 823 cm⁻¹ -1 The presence of a characteristic absorption peak for β-glycosidic bonds at 3500–3200 cm⁻¹ confirms that the product retains the typical β-glycosidic bond structure; the presence of a characteristic absorption peak for β-glycosidic bonds at 3500–3200 cm⁻¹ further confirms that the product retains the typical β-glycosidic bond structure. -1 The broad absorption peak at 3000–2850 cm⁻¹ is due to O–H stretching vibration, indicating the presence of numerous hydrogen bonds between molecules; -1 The absorption peak at 1164 cm⁻¹ corresponds to the C–H stretching vibration. -1 The nearby absorption peaks are attributed to the C–O–C stretching vibration of the hemiacetal in the pyranose ring skeleton, a characteristic absorption of oligosaccharide compounds; 1100–1010 cm⁻¹ -1 The three absorption peaks in the region indicate that the product contains a pyranose ring structure.

[0029] II. Prebiotic Activity Experiment of the Oligosaccharides Prepared in Example 1 1. Activated Akkermansia, Lactobacillus paracasei, Bacteroides parabens, and Bifidobacterium adolescentis bacterial suspensions (all purchased externally) were inoculated at a 1% inoculum into a basal culture medium with a final concentration of 2 g / L of the oligosaccharide of this invention as the sole carbon source. The medium was anaerobically cultured at 37°C for 48 h, with OD600 values ​​measured every 6 h. A probiotic growth curve was plotted, with glucose and inulin used as controls. Results are as follows: Figure 4 As shown, Figure 4 Figures (A) to (D) in the figure are OD600 value curves of Akkermansia, Lactobacillus paracasei, Bacteroides paracasei, and Bifidobacterium adolescentis, respectively.

[0030] The results showed that the oligosaccharide prepared in Example 1 of this invention had a particularly outstanding growth-promoting effect on Lactobacillus paracasei, with a final biomass 31% higher than that of inulin. Although the growth-promoting effect on Bifidobacterium adolescentis and Bacteroides paracasei was delayed, the final biomass could reach 80% of that of the glucose group and 94% of that of the inulin group. This indicates that the oligosaccharide prepared in this invention is a complex prebiotic that can be utilized differentially by various probiotics, providing a basis for developing functional products that precisely regulate the intestinal flora.

[0031] 2. SPF-grade mice aged 6–8 weeks and weighing 20±2 g were selected, and fresh feces were collected to prepare intestinal flora fermentation broth. The compound probiotic oligosaccharide prepared in Example 1 of this invention was added to the fermentation system at a final concentration of 2 g / L as a carbon source. Anaerobic fermentation was carried out at 37℃ for 48 h, with a blank control and inulin as controls. After fermentation, the content of various short-chain fatty acids in the fermentation broth was determined by gas chromatography, and a bar chart showing the differences in short-chain fatty acid production between the oligosaccharide, inulin, and blank groups was generated. Figure 5 As shown in the figure. The results indicate that, compared with the control group, oligosaccharides significantly increased the content of short-chain fatty acid metabolites in feces, especially acetic acid and butyric acid; compared with the inulin group, although the acetic acid content in the oligosaccharide group was lower, the contents of propionic acid and butyric acid were significantly higher than those in the inulin group. Therefore, the oligosaccharides prepared in this invention have a significant prebiotic effect. Example 2

[0032] The present invention discloses a method for preparing compound probiotic oligosaccharides based on algal residue, comprising the following steps: Step 1. Dissolve lithium bromide (LiBr) in deionized water to prepare a lithium bromide aqueous solution with a mass percentage concentration of 60%. Stir continuously at 80°C until completely dissolved to obtain solution I; Step 2. Take 2.35 mL of solution I, add 0.78 mL of sulfuric acid with a concentration of 4 mol / L, stir well, and obtain solution II with a final sulfuric acid concentration of approximately 1.0 mol / L; Step 3. Mix solution II with methanol at a volume ratio of 1:2.5 (v / v) to obtain solution III; Step 4. Take 10 mL of Solution III and add it dropwise to 1.0 g of degummed brown algae residue ( Kjellmaniella crassifolia Mix thoroughly in the solution to obtain solution IV; Step 5. Place solution IV in a 50°C oven and heat for 12 hours to remove methanol, obtaining solution V; Step 6. Heat solution V at 90°C for 1.5 hours to obtain solution VI; Step 7. Take 10 mL of solution VI and add 10 mL of deionized water, mix thoroughly to dissolve the soluble components, and obtain solution VII; Step 8. Transfer solution VII to a dialysis bag with a molecular weight cutoff of 300 Da, and dialyze with deionized water for 24 hours to remove salts; after dialysis, add 8 times the volume of anhydrous ethanol for alcohol precipitation, and let stand overnight; filter and collect the precipitate, and freeze-dry for 24 hours to obtain the compound probiotic oligosaccharide.

[0033] The oligosaccharide prepared in Example 2 was a pale yellow powder. The yield was 76.30 ± 0.18%, and the purity was 91.71 ± 0.32% (w / w). Following the characterization method of Example 1, the structural characteristics of the obtained product were basically the same as those in Example 1, mainly composed of glucose, fucose, rhamnose, and mannose. Example 3

[0034] The present invention discloses a method for preparing compound probiotic oligosaccharides based on algal residue, comprising the following steps: Step 1. Dissolve lithium bromide (LiBr) in deionized water to prepare a lithium bromide aqueous solution with a mass percentage concentration of 60%. Stir continuously at 80°C until completely dissolved to obtain solution I; Step 2. Take 2.35 mL of solution I, add 0.78 mL of sulfuric acid with a concentration of 4 mol / L, stir well, and obtain solution II with a final sulfuric acid concentration of approximately 1.0 mol / L; Step 3. Mix solution II with methanol at a volume ratio of 1:2.5 (v / v) to obtain solution III; Step 4. Take 10 mL of Solution III and add it dropwise to 1.0 g of the carrageenan-extracted red algae caryophyllum residue ( Gelidium amansii Mix thoroughly in the solution to obtain solution IV; Step 5. Place solution IV in a 50°C oven and heat for 12 hours to remove methanol, obtaining solution V; Step 6. Heat solution V at 110°C for 2.0 hours to obtain solution VI; Step 7. Take 10 mL of solution VI and add 10 mL of deionized water, mix thoroughly to dissolve the soluble components, and obtain solution VII; Step 8. Transfer solution VII to a dialysis bag with a molecular weight cutoff of 300 Da, and dialyze with deionized water for 24 hours to remove salts; after dialysis, add 8 times the volume of anhydrous ethanol for alcohol precipitation, and let stand overnight; filter and collect the precipitate, and freeze-dry for 24 hours to obtain the compound probiotic oligosaccharide.

[0035] The oligosaccharide prepared in Example 3 was a pale yellow powder. The yield was 90.20 ± 0.23%, and the purity was 94.25 ± 0.14% (w / w). Following the characterization method of Example 1, the structural characteristics of the obtained product were basically the same as those in Example 1, mainly composed of glucose, fucose, rhamnose, and mannose.

Claims

1. A compound probiotic oligosaccharide prepared from algal residue, characterized in that... It is composed of glucose, fucose, mannose and rhamnose, with a degree of polymerization of 4 to 10 and a weight-average molecular weight of 600 to 2000 Da. The mass percentage of each monosaccharide is 70 to 80% glucose, 18 to 25% fucose, 0.5 to 3% mannose and 0.5 to 3% rhamnose.

2. A method for preparing compound probiotic oligosaccharides based on algal residue, characterized in that... Perform the following steps in sequence: Step 1. Dissolve lithium bromide in deionized water, mix and dissolve to obtain solution I; Step 2. Add sulfuric acid to solution I and stir until homogeneous to obtain solution II; Step 3. Add methanol to solution II and stir until homogeneous to obtain solution III; Step 4. Mix solution III with algal residue to obtain solution IV; Step 5. Evaporate solution IV to remove methanol, obtaining solution V; Step 6. Heat solution V to obtain solution VI; Step 7. Add deionized water to solution VI to dissolve it, filter to remove insoluble matter, and obtain solution VII; Step 8. Dialyze solution VII sequentially to desalt, precipitate with alcohol, and freeze dry to obtain the compound probiotic oligosaccharide.

3. The method for preparing compound probiotic oligosaccharides based on algal residue according to claim 2, characterized in that... The mixing and dissolution temperature in step 1 is 80°C, and the mass percentage concentration of solution I is 50-60%.

4. The method for preparing compound probiotic oligosaccharides based on algal residue according to claim 3, characterized in that... The sulfuric acid concentration in solution II of step 2 is 0.25~2.25 mol / L.

5. The method for preparing compound probiotic oligosaccharides based on algal residue according to claim 4, characterized in that... The volume ratio of solution II to methanol in step 3 is 1:1~5.

6. The method for preparing compound probiotic oligosaccharides based on algal residue according to claim 5, characterized in that... The liquid-to-solid ratio of solution III to algal residue in step 4 is 2.0~15.0 mL / g.

7. The method for preparing compound probiotic oligosaccharides based on algal residue according to claim 6, characterized in that... Step 5 involves placing solution IV at 50°C for 10-12 hours to remove methanol.

8. The method for preparing compound probiotic oligosaccharides based on algal residue according to claim 7, characterized in that... Step 6 involves heating solution V at 80-130°C for 1-4 hours.

9. The method for preparing compound probiotic oligosaccharides based on algal residue according to claim 8, characterized in that... Step 7 involves mixing solution VI with deionized water at a volume ratio of 1:1 and stirring to dissolve.

10. The method for preparing compound probiotic oligosaccharides based on algal residue according to claim 9, characterized in that... Step 8 involves dialysis desalting using a dialysis membrane with a molecular weight cutoff of 300-500 Da, followed by alcohol precipitation with 8 times the volume of anhydrous ethanol, and freeze-drying for 24 hours.

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