Method for producing alginate oligosaccharide by fermenting bacillus coagulans by using kelp as main carbon source

Abstract

The invention relates to the field of microbial fermentation, and provides a method for producing alginate oligosaccharide by fermenting bacillus coagulans by using kelp as a main carbon source. A specific bacillus coagulans strain is screened, rich algin in kelp is used as a main carbon source for fermentation, and an efficient fermentation process is carried out. In the fermentation process, the bacillus coagulans can effectively degrade algin and synthesize alginate oligosaccharide with biological activity. The fermentation method provided by the invention not only improves the yield of alginate oligosaccharide, but also optimizes the fermentation conditions, so that the whole process is more environment-friendly and economic. In addition, the prepared alginate oligosaccharide has wide application potential in the fields of animal husbandry, food, medicine and the like. The invention provides an innovative alginate oligosaccharide production technology which has an important application value.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, in particular to a method for producing brown algae oligosaccharides by fermenting Bacillus coagulans using kelp as a main carbon source. Background Art

[0002] Fucoidan is a degradation product of alginate and a low molecular weight polymer. Compared with polysaccharides, fucoidan has a smaller molecular weight and is easier to absorb and utilize. Fucoidan has the characteristics of high water solubility, strong stability, safety and non-toxicity. It has broad prospects in the fields of food, feed, agriculture, water treatment and medical care.

[0003] Brown algae oligosaccharides can be obtained through different preparation methods, such as chemical degradation, physical degradation, or enzymatic hydrolysis. Physical and chemical degradation methods have high production efficiency, but they have problems such as high energy consumption, high impurities, and environmental pollution. Enzymatic hydrolysis has relatively mild reaction conditions, simple operation, and is more environmentally friendly, but the composition of the enzymatic hydrolysis product is relatively complex, and the obtained brown algae oligosaccharides contain some impurities and incomplete enzymatic hydrolysis polysaccharides, which reduces the purity of the brown algae oligosaccharides. Therefore, it is of great significance to develop an environmentally friendly and efficient method for preparing brown algae oligosaccharides.

[0004] Bacillus coagulans is a specialized spore-forming probiotic and the only spore-forming lactic acid bacterium on the "List of Edible Mushrooms." It not only possesses the beneficial effects of lactic acid bacteria, such as maintaining intestinal microecological balance, stimulating immunity, improving overall health, and promoting digestion and absorption, but also, due to its spore-forming nature, possesses strong environmental resistance, including resistance to high temperatures, pressures, acids, alkalis, and desiccation. This makes it a sought-after probiotic in the market, particularly for its use in the food industry.

[0005] Kelp is a nutrient-rich brown algae with medicinal properties. It is rich in a variety of active ingredients beneficial to the human body, such as mannitol, alginate, iodine, cellulose, and vitamins. These active ingredients are also important raw materials for industries such as medicine, healthcare, chemicals, and agricultural fertilizers. Kelp is a very important marine plant resource in my country. my country leads the world in both production and scale of kelp cultivation, making it a major player in kelp resources and comprehensive utilization. Besides being used directly for food, kelp is a major raw material in the brown algae industry. However, for many years, my country's kelp development has focused on the same three main ingredients: iodine, alginate, and mannitol. The industry has generally been low-quality, with low added value, low efficiency, and high energy consumption. Mature production processes for deep processing and efficient, comprehensive utilization of kelp resources are relatively lacking, resulting in weak market competitiveness for both products and enterprises. Furthermore, the comprehensive utilization rate of kelp is only approximately 30%, with over 50% of its active ingredients becoming waste. Furthermore, large amounts of waste rich in organic matter and nutrients are discharged into water bodies during industrial kelp production, causing a series of environmental pollution problems. In recent years, with the rapid development of bioengineering technology, microbial fermentation technology has been widely used in various industries. The fermentation of kelp using Bacillus coagulans has the advantages of simple operation, mild reaction conditions, high specificity, few by-products and high yield. It is an effective way to achieve high-value development and utilization and deep processing of kelp. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for producing brown algae oligosaccharides by fermentation with Bacillus coagulans using kelp as a main carbon source.

[0007] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides Bacillus coagulans ( Bacillus coagulans ) SDHY-2402, strain SDHY-2402 was classified and named Bacillus coagulans Bacillus coagulan It has been deposited in the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, with the deposit number CGMCC No.32910 and the deposit date December 5, 2024.

[0008] In a second aspect, the present invention provides the use of Bacillus coagulans SDHY-2402 in the fermentation production of brown algae oligosaccharides.

[0009] In a third aspect, the present invention provides a method for producing brown algae oligosaccharides by fermenting Bacillus coagulans using kelp as the main carbon source, comprising preparing a fermentation medium with kelp powder as the main carbon source; inoculating a bacterial liquid of Bacillus coagulans SDHY-2402 with a preservation number of CGMCC No. 32910 into the fermentation medium for fermentation; and determining the content of brown algae oligosaccharides in the fermentation liquid after the fermentation is completed.

[0010] The kelp powder is obtained by washing, drying, and crushing the kelp.

[0011] Furthermore, the fermentation medium includes the following components in parts by weight: 5-10 parts of glucose, 30-40 parts of kelp powder, 20-30 parts of egg white, 2-8 parts of beef extract, 5-10 parts of fermented soybean meal, 3-6 parts of sodium acetate, 1-3 parts of dipotassium hydrogen phosphate, 0.5-1.0 parts of magnesium sulfate, 0.1-0.3 parts of manganese sulfate, 0.2-0.5 parts of calcium chloride, 0.1-0.3 parts of ferrous sulfate and 200-250 parts of water.

[0012] Furthermore, the fermentation conditions are: 45°C~47°C, pH 5.5~6.0, dissolved oxygen concentration 20%~40%, and rotation speed 300~400 rpm.

[0013] Furthermore, determining the content of brown algae oligosaccharide in the fermentation broth includes steps such as centrifugation, filtration, and nanofiltration.

[0014] In a fourth aspect, the present invention provides a method for producing brown algae oligosaccharides by fermentation with Bacillus coagulans, comprising the following steps: S1. Pre-treating the kelp: washing, drying, and crushing the kelp to obtain kelp powder; mixing the kelp powder with water and disrupting the cell walls using ultrasound; then chemically treating the kelp powder with dilute acid and dilute alkali, enzymatically hydrolyzing the resulting product, and using the enzymatic hydrolysis product as a carbon source for fermentation production of brown algae oligosaccharides; S2. A fermentation medium is prepared using the enzymatic hydrolysis product of step S1, and fermentation is performed using Bacillus coagulans SDHY-2402 with a deposit number of CGMCC No. 32910 as the fermentation strain; after the fermentation is completed, the content of brown algal oligosaccharides in the fermentation liquid is determined.

[0015] Furthermore, step S1 includes: (1) Mix 30-50 g of kelp powder with 750-1250 mL of water and use ultrasound to break the cell walls. The ultrasound conditions are: probe diameter 10 mm, power 600 W (effective power 450 W), working cycle 3 s on, 2 s off, each working cycle 3 min, 5 working cycles, each working cycle separated by 3 min of ice bath, for a total of 27 min. (2) The kelp powder of step (1) is ultrasonically treated and then centrifuged to collect the precipitate, which is hydrolyzed with dilute acid at 80-100°C, and the hydrolyzate is chemically treated with dilute alkali at 80-100°C; the dilute acid is selected from sulfuric acid or hydrochloric acid; and the dilute alkali is selected from sodium hydroxide or potassium hydroxide; (3) The product obtained by chemical treatment in step (2) is contacted with a complex enzyme and enzymatically hydrolyzed at 40-60°C; the complex enzyme is selected from two or more of lipase, alginate, and pectinase.

[0016] Furthermore, steps (2) and (3) comprise: mixing 50 g of the precipitate with 750-1000 mL of sulfuric acid, and hydrolyzing the mixture at 80-100° C. for 120-240 min (preferably 120 min); after the hydrolysis is completed, adding 1000-1500 mL of sodium hydroxide thereto, and treating the mixture at 80-100° C. for 240-360 min (preferably 240 min); adding a complex enzyme to the treated product so that the final concentrations of cellulase, alginate enzyme, and pectinase in the enzymatic hydrolysis system are 40-60 U / mL, 50-80 U / mL, and 20-30 U / mL, respectively; performing enzymatic hydrolysis at 40-60° C. for 480-600 min (preferably 480 min), and directly using the obtained enzymatic hydrolysis product for fermentation, or removing insoluble residue from the obtained enzymatic hydrolysis product to obtain a clarified liquid for fermentation; Wherein, the concentration of sulfuric acid is 0.1-0.5M, and the concentration of sodium hydroxide is 0.5-1M.

[0017] In a fifth aspect, the present invention provides brown algal oligosaccharides produced according to the method.

[0018] In the present invention, through the directional cutting of the composite enzyme system, the degree of polymerization (DP) of the product brown algae oligosaccharide is concentrated between 2-6, accounting for >85%, of which the DP4 (tetrasaccharide) content reaches 32.5±1.8%; the half-peak width of the molecular weight distribution (Mw / Mn) is reduced to 1.18, and the monodispersity is improved by 40%; the ratio of mannuronic acid (M) to guluronic acid (G) (M / G ratio) is achieved to 3:1 through process control.

[0019] In a sixth aspect, the present invention provides the use of the brown algae oligosaccharide in preparing products; The product can be selected from any one of cosmetics, medicines, foods or food additives, feeds or feed additives, and the like.

[0020] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (1) Environmental protection: Using kelp as the main carbon source reduces the use of chemical reagents and reduces environmental pollution.

[0021] (2) Low cost: Kelp is a cheap natural resource, which reduces production costs.

[0022] (3) High efficiency: By optimizing the fermentation conditions, the yield and purity of brown algae oligosaccharides are improved.

[0023] (IV) Wide range of applications: The prepared brown algae oligosaccharides can be used in multiple fields and have broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a colony morphology diagram of Bacillus coagulans SDHY-2402 of the present invention.

[0025] Figure 2 This is a Gram staining microscopic examination image of Bacillus coagulans SDHY-2402 of the present invention.

[0026] Figure 3 This is a diagram of the physiological and biochemical reactions of Bacillus coagulans SDHY-2402 of the present invention.

[0027] Figure 4 This is a calcium dissolution reaction diagram of Bacillus coagulans SDHY-2402 of the present invention.

[0028] Figure 5 This is a diagram of amylase, lipase and protease produced by Bacillus coagulans SDHY-2402 of the present invention.

[0029] Figure 6 This is a growth curve diagram of Bacillus coagulans SDHY-2402 of the present invention.

[0030] Figure 7 The results are the sensitivity test results of Bacillus coagulans SDHY-2402 of the present invention to different antibiotics. DETAILED DESCRIPTION

[0031] The present invention aims to provide a Bacillus coagulans fermentation method using kelp as a main carbon source, which can efficiently prepare brown algal oligosaccharides and has the advantages of being environmentally friendly and low-cost.

[0032] The present invention adopts the following technical solutions: 1. Screening and cultivation of Bacillus coagulans: Bacillus coagulans strains that can use kelp as a carbon source were screened from the marine environment and cultured.

[0033] 2. Preparation of fermentation medium: Pre-treat the kelp, remove impurities and then crush it to use as the main carbon source of the fermentation medium.

[0034] 3. Optimization of the fermentation process: Optimize the fermentation process by adjusting the fermentation conditions (such as temperature, pH value, inoculation amount, etc.) to increase the yield and purity of brown algae oligosaccharides.

[0035] 4. Extraction and purification of brown algae oligosaccharides: After fermentation, brown algae oligosaccharides are extracted and purified by physical and chemical methods.

[0036] 5. Application of brown algae oligosaccharides: The prepared brown algae oligosaccharides can be used in food, medicine, cosmetics and other fields.

[0037] The details are as follows: The present invention provides Bacillus coagulans ( Bacillus coagulans ), which is Bacillus coagulans SDHY-2402, and its deposit number is CGMCC No.32910.

[0038] The present invention also provides a Bacillus coagulans fermentation method using kelp as the main carbon source, comprising the following steps: screening and culturing a Bacillus coagulans strain that can use kelp as a carbon source; preparing a fermentation medium with kelp powder as the main carbon source; inoculating the cultured Bacillus coagulans liquid into the fermentation medium for fermentation; and determining the brown algae oligosaccharide content of the fermentation liquid after the fermentation is completed.

[0039] The present invention provides a fermentation and culturing method for Bacillus coagulans, comprising the steps of: inoculating a seed liquid of Bacillus coagulans into a Bacillus coagulans fermentation medium, and culturing the culture medium under the conditions of 45° C. to 47° C., a pH value of 5.5 to 6.0, a dissolved oxygen concentration of 20% to 40%, and a rotation speed of 300 to 400 rpm.

[0040] The present invention also provides a Bacillus coagulans fermentation medium, which comprises, by weight, 5 to 10 parts of glucose, 30 to 40 parts of kelp powder, 20 to 30 parts of egg white, 2 to 8 parts of beef extract, 5 to 10 parts of fermented soybean meal, 3 to 6 parts of sodium acetate, 1 to 3 parts of dipotassium hydrogen phosphate, 0.5 to 1.0 parts of magnesium sulfate, 0.1 to 0.3 parts of manganese sulfate, 0.2 to 0.5 parts of calcium chloride, 0.1 to 0.3 parts of ferrous sulfate, and 200 to 250 parts of water.

[0041] It should be noted that the fermentation and culture method of Bacillus coagulans in the specific embodiment of the present invention is applicable to the shake flask fermentation stage, the small-scale test stage, the pilot stage, the large-tank trial production stage and the industrial production stage. The fermentation scale includes but is not limited to a 50L fermenter, a 100L fermenter, a 500L fermenter, a 3-ton fermenter, a 5-ton fermenter, etc. Those skilled in the art can rely on their own professional knowledge to prepare the seed liquid step by step according to the fermentation scale.

[0042] Furthermore, the extraction and detection of brown algae oligosaccharides includes steps such as centrifugation, filtration, and nanofiltration.

[0043] The present invention also provides brown algae oligosaccharide produced by the method.

[0044] The present invention also provides a product, comprising the Bacillus coagulans SDHY-2402 or the brown algae oligosaccharide produced according to the method; the product is any one selected from cosmetics, medicines, foods or food additives, feeds or feed additives.

[0045] The present invention also provides the use of the Bacillus coagulans SDHY-2402 or the brown algae oligosaccharide produced according to the method in preparing a product; the product is any one selected from cosmetics, medicines, food or food additives, feed or feed additives.

[0046] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0047] Example 1 Screening and cultivation of Bacillus coagulans 1. Isolation and culture of strains 1. Modified bromocresol purple glucose broth: 10 g egg white, 3 g beef extract powder, 10 g glucose, 5 g sodium chloride, 0.04 g bromocresol purple, 0.2 g sodium azide, 1 L water, adjust pH to 6.8-7.0, and sterilize at 115°C for 15 min.

[0048] 2. Modified bromocresol purple glucose broth agar: 10g egg white, 3g beef extract powder, 10g glucose, 5g sodium chloride, 15g agar powder, 0.04g bromocresol purple, 0.2g sodium azide, 1L water, adjust pH to 6.8-7.0, and sterilize at 115℃ for 15min.

[0049] 3. The formula of the liquid culture medium with kelp as the sole carbon source is as follows: 40g / L kelp powder, 30g / L protein, 8g / L beef extract, 5.0g / L sodium acetate, 2.0g / L dipotassium hydrogen phosphate, 1.0g / L magnesium sulfate, 0.5g / L calcium carbonate and 0.2g / L manganese sulfate. The pH of the seed culture medium is adjusted to between 6.3 and 6.5.

[0050] 4. Soil sample collection: Soil samples were collected from a mango field in Hainan. 1 g of the sample was placed in a 100 mL sterile conical flask. 9 mL of sterile physiological saline was added, and an appropriate amount of sterilized glass grinding beads was added. The mixture was shaken and mixed to obtain a soil mixed suspension.

[0051] 5. Enrichment of target bacteria: Use modified bromocresol purple glucose broth as the enrichment medium for enrichment culture. After the soil mixed suspension is filtered through a 100 μm filter membrane, it is inoculated into modified bromocresol purple glucose broth and cultured anaerobically at 55°C for 24-48 hours to obtain the enrichment culture solution.

[0052] 6. Isolation and purification of bacterial strains: The enriched bacterial liquid was graded diluted with sterile saline. After graded dilution, the diluted liquid was spread on a bromocresol purple glucose broth agar medium and cultured anaerobically in an incubator at 55°C for 24-48 hours. After colonies grew, round colonies with light yellow to yellow smooth edges were selected and purified. After repeated streaking and culture, pure colonies were obtained and numbered for storage.

[0053] 7. UV mutagenesis and screening of strains The screened coagulant spore bacilli ( Bacillus coagulans ) were inoculated into a liquid medium containing kelp powder as the sole carbon source and incubated at 47°C in a shaking incubator for 24–36 hours. The bacterial suspension was then graded diluted with saline and plated onto modified bromocresol purple glucose broth agar plates. Strain selection was performed using ultraviolet mutagenesis. Liquid suspensions were prepared from strains with the best antibacterial properties and diluted to a concentration of 1.0 × 10 8 ~5.0×10 8 CFU / mL, 20-30 mL was placed in a 10 cm sterile dish, and induced by 15 W ultraviolet light for 60-150 s. The lethality was controlled at 50%-80%. The induced bacterial liquid was spread on a modified bromocresol purple glucose broth agar plate and cultured at 47 ° C in the dark for 24-36 h. The induced bacteria were selected for cultivation, and finally the strain capable of synthesizing brown algal oligosaccharides was obtained by thin plate chromatography and named SDHY-2402.

[0054] Example 2 Identification of strain SDHY-2402 1. Morphological identification For strain SDHY-2402 in the logarithmic growth phase with stable colony size, the single colony status was described, including colony size, color, transparency, colony surface, and colony edge. The colonies ranged in size from 2 to 4 mm and were yellow, opaque, circular, with a smooth surface, a raised center, and a regular colony edge.

[0055] Next, the SDHY-2402 strain, which was in its logarithmic growth phase, was stained and its bacterial morphology was observed using an optical microscope. The isolated and screened strain, SDHY-2402, was Gram-positive, with rod-shaped cells no larger than 1 μm in diameter, and spores that did not swell.

[0056] 2.16S rRNA sequence homology analysis The selected pure colonies were inoculated into 50 mL of bromocresol purple glucose broth and cultured in a shaking incubator at 47°C for 24 h. The genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit according to the instructions. The 16S rRNA primers of the strain, including 27F: AGAGTTTGATCMTGGCTCAG and 1492R: GGTTACCTTGTTACGACTT, were used to amplify the 16S rDNA fragment. The PCR product was sent to Shanghai Sangon Biotechnology Service Co., Ltd. for sequencing, and then a BLAST comparison was performed (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Based on the comparison results, the sequence was identified as Bacillus coagulans ( Bacillus coagulans ) strains. 16S rDNA gene sequence homology A>99% was used as the identification standard.

[0057] 3. Physiological and biochemical identification Refer to the Bergey's Manual of Bacterial Identification and the Manual of Identification of Common Bacterial Systems.

[0058] The biochemical identification of SDHY-2402 was carried out according to the instructions of the HBI biochemical identification strips. The SDHY-2402 strain was analyzed and tested by two methods: bacterial suspension inoculation and puncture inoculation. The strain's various biochemical reactions were tested, including VP, citrate, gelatin, 7% sodium chloride, pH 5.7, nitrate reduction, starch hydrolysis, propionate, D-xylose, L-arabinose, and D-mannitol. The results are shown in Table 1. SDHY-2402 meets the biochemical characteristics of Bacillus coagulans.

[0059] Table 1 HBI biochemical identification

[0060] The lactic acid production capacity of SDHY-2402 is tested through a calcium dissolving reaction. Calcium carbonate generates soluble calcium lactate under the action of lactic acid. The solid MRS culture medium is sterilized at 121°C. Calcium carbonate is sterilized separately after adding water. After the temperature of the MRS solid culture medium is cooled to 45-55°C, calcium carbonate is added to the solid culture medium at a dosage of 0.5%. After thorough mixing, the plate is quickly poured to avoid precipitation of calcium carbonate. The SDHY-2402 strain is streaked and inoculated into the MRS solid culture medium supplemented with calcium carbonate, cultured at 47°C for 48 hours, and the size of the calcium melting circle is observed. The results are as follows: Figure 4 As shown in the results, SDHY-2402 has a good lactic acid production ability, which is consistent with the lactic acid production characteristics of Bacillus coagulans.

[0061] 4. Gram staining of strains Place a drop of sterilized distilled water on the slide and pick a single colony that grows faster after mutagenesis (see colony morphology for details). Figure 1) is dissolved in water, scraped, and dried on an alcohol lamp for fixation. Add crystal violet staining solution, stain for 2 minutes, rinse with water, and air dry; add iodine solution for mordant staining for 2 minutes, rinse with water, and air dry; add alkaline fuchsin ethanol solution for 50 seconds, rinse with water, and air dry; observe under an ordinary optical microscope. If the bacteria are purple, it is positive; if they are red, it is negative. The results are shown in the table. Figure 2 .

[0062] Example 3 Stress resistance test of Bacillus coagulans SDHY-2402 1. Heat resistance test The Bacillus coagulans SDHY-2402 bacterial solution was placed in a water bath for 20 minutes and treated at 60°C, 70°C, 80°C, and 90°C, respectively, with 3 replicates for each treatment. After the treatment, the viable bacterial count was determined by the pouring method. The determination results are shown in Table 2.

[0063] Table 2 Heat resistance test results

[0064] 2. Acid resistance test Put 10 8 CFU / ml Bacillus coagulans SDHY-2402 was inoculated into MRS medium with pH values ​​of 2.0, 3.0, 4.0, and 5.0, and the number of viable bacteria was determined by the plate pouring method after 4 h.

[0065] In a weakly acidic environment (pH 3.0, 4.0, and 5.0), Bacillus coagulans can grow normally. At pH 2.0, growth of Bacillus coagulans is slightly inhibited, but the viable count is still maintained at 7.04 lg (cfu / ml). This indicates that this strain has a strong tolerance to acid and can withstand the effects of gastric acid. The test results are shown in Table 3.

[0066] Table 3 Acid resistance test results

[0067] 3. Bile salt tolerance test Bile salts were added to MRS liquid medium at concentrations of 0.20%, 0.40%, 0.60%, 0.80%, and 1.0%, respectively. The medium was sterilized at 121°C for 15 minutes. A 2% inoculation of activated Bacillus coagulans SDHY-2402 culture was incubated in a 47°C incubator. Growth was observed using three replicates per sample. "+++" indicates good growth, "++" indicates moderate growth, "+" indicates slight growth, and "-" indicates no growth. The results are shown in Table 4.

[0068] Table 4 Bile salt tolerance test results

[0069] Example 4 Growth Curve Determination of Bacillus coagulans SDHY-2402 The growth curve represents the dynamic changes of bacteria in the whole process of growth and reproduction in a new and suitable environment until aging and death. Bacillus coagulans SDHY-2402 was inoculated into MRS liquid culture medium at a 1% (v / v) inoculation rate and cultured at 42℃ for 24 hours. MRS culture medium without bacterial solution was used as a blank control, and the plates were plated and counted every 2 hours. The experiment was repeated three times, and the results were averaged. The data were recorded and the growth curve was drawn. Figure 3 As shown, from 2 to 14 hours, Bacillus coagulans SDHY-2402 was in the logarithmic growth phase with a high reproduction rate. From 16 to 24 hours, the number of Bacillus coagulans SDHY-2402 tended to be stable.

[0070] Example 5 Sensitivity analysis of Bacillus coagulans SDHY-2402 to different antibiotics Antibiotic susceptibility was determined using antimicrobial susceptibility strips. Several common antibiotics from each class were selected as representatives to comprehensively evaluate the antimicrobial susceptibility of SDHY-2402. A 1% suspension of Bacillus coagulans SDHY-2402 was then added to liquid YPD solid medium and mixed until the YPD medium solidified and dried for 3–5 minutes. A tablet was placed in the center of a plate and gently compacted. Each plate was incubated in a 47°C incubator for 36 hours, and the size of the antimicrobial zone was observed. Three replicates were performed for each tablet. Zones of inhibition with a diameter less than 15 mm were considered resistant, those between 16 and 20 mm were considered moderately sensitive, and those greater than 20 mm were considered sensitive. Analysis of the inhibition diameters showed that Bacillus coagulans SDHY-2402 was sensitive to 16 antibiotics, including penicillin, tetracycline, and erythromycin (Table 5), indicating that B. coagulans SDHY-2402 was not resistant.

[0071] Table 5 Antibiotic susceptibility of Bacillus coagulans SDHY-2402

[0072] This result shows that Bacillus coagulans SDHY-2402 does not have good drug resistance, and is therefore safe and reliable to use as a feed probiotic.

[0073] Example 6 Enzyme Production Characteristics of Bacillus coagulans Activated Bacillus coagulans was inoculated into MRS liquid medium and cultured at 47°C, 200 rpm, for 24 hours to prepare a test culture. The culture was then inoculated onto screening medium containing amylase, protease, and lipase and cultured at 42°C for 48 hours. The size of the enzyme-producing zone was then measured. For the amylase screening medium, dilute iodine solution was added to discolor the starch, and the diameter of the enzyme-producing zone was measured. The results are shown in Table 6.

[0074] Table 6 Evaluation of enzyme production effect of Bacillus coagulans SDHY-2402

[0075] From the results in Table 6, it can be seen that the Bacillus coagulans provided by the present invention has excellent enzyme production effect.

[0076] Amylase, lipase and protease produced by Bacillus coagulans SDHY-2402 Figure 5 .

[0077] The growth curve of Bacillus coagulans SDHY-2402 is shown in Figure 6 .

[0078] The results of the sensitivity test of Bacillus coagulans SDHY-2402 to different antibiotics are shown in Figure 7 .

[0079] Example 7 Fermentation Test of Bacillus coagulans SDHY-2402 This embodiment provides a fermentation and cultivation method for Bacillus coagulans, wherein the fermentation scale is a 3-ton fermentation tank.

[0080] 1. Kelp Pretreatment In order to improve the utilization rate of kelp and promote the fermentation of brown algae oligosaccharides by Bacillus coagulans, the following pretreatment methods can be used: Kelp was pretreated by washing, drying, and pulverizing the kelp to obtain kelp powder. The kelp powder was then mixed with water and ultrasonically disrupted. 50 g of kelp powder was then mixed with 1250 mL of water and ultrasonically disrupted. Ultrasonication conditions were: a 10 mm probe diameter, 600 W power (450 W effective power), a 3-second on / 2-second off duty cycle, and a 3-minute cycle. Five cycles were repeated, each cycle separated by a 3-minute ice bath, for a total of 27 minutes. After ultrasonic treatment, the kelp powder was centrifuged and the precipitate collected.

[0081] 50 g of precipitate was mixed with 1000 mL of 0.5 M sulfuric acid and hydrolyzed at 100°C for 120 min. After the hydrolysis, 1500 mL of 1 M sodium hydroxide was added and the mixture was treated at 100°C for 240 min. A composite enzyme was added to the treated product so that the final concentrations of cellulase, alginate, and pectinase in the enzymatic hydrolysis system were 60 U / mL, 80 U / mL, and 30 U / mL, respectively. The product was hydrolyzed at 60°C for 480 min. The resulting enzymatic hydrolysis product was either used directly for fermentation or the clear liquid was obtained after removing the insoluble residue from the enzymatic hydrolysis product for fermentation.

[0082] 2. Preparation of seed solution 1. Seed activation Take Bacillus coagulans stored in a -80°C glycerol tube. After the glycerol tube of Bacillus coagulans is completely thawed, draw 1.0 mL of the bacterial liquid and inoculate it into 50 mL of MRS liquid culture medium. Place it in a shake flask at 45°C and 250 rpm for 15 hours. Then pick the bacterial liquid and streak it on an MRS culture medium plate. Place it in anaerobically at 45°C for 24 hours to obtain a single colony of Bacillus coagulans.

[0083] 2. Preparation of primary seed solution Use a flame-sterilized inoculation loop to pick up a single colony of Bacillus coagulans growing on an MRS medium plate, then inoculate it into 50 mL of MRS liquid medium. Place the culture in a shake flask at 45°C and 250 rpm for 12-14 hours and set aside.

[0084] 3. Preparation of Secondary Seed Solution Transfer the first-level seed liquid to 1.2 L of MRS liquid culture medium at an inoculum volume ratio of 2%, place it in a shaking flask at 45°C and 250 r / min for 14 to 16 hours, and set aside.

[0085] 4. Preparation of Seed Tank Culture Medium A 100L fermentation tank was selected as the seed tank. Seed culture medium was added to 60% of the tank volume, and 1‰ (volume ratio V / V) of defoaming agent was added. The pH was adjusted to 6.3 with sodium hydroxide, and then sterilized at 121°C for 20 minutes. After sterilization, cold water was passed into the jacket to rapidly cool the tank, and sterile air was introduced into the tank to maintain a pressure of 0.05 MPa. The seed culture medium consisted of 10g / L glucose, 40g / L kelp powder, 30g / L protein, 8g / L beef extract, 5.0g / L sodium acetate, 2.0g / L dipotassium hydrogen phosphate, 1.0g / L magnesium sulfate, 0.5g / L calcium carbonate, 0.2g / L iron sulfate, and 0.2g / L manganese sulfate, and the pH of the seed culture medium was adjusted to 6.3.

[0086] When the tank temperature of the seed tank is cooled to 47°C, the secondary seed liquid is inoculated at a volume ratio of 2%, the initial ventilation ratio is 0.5vvm, the tank pressure is maintained at 0.05MPa, the temperature is controlled at 47°C, and the fermentation is started at the lowest stirring speed. The dissolved oxygen is maintained at not less than 20% during the entire fermentation process. The culture is carried out until the conidiation rate reaches more than 80%, and then the culture is placed in a water bath at 85°C for 15 minutes to obtain the seed tank culture solution.

[0087] 3. Fermentation production in 3-ton fermentation tanks 1. Fermentation medium The fermentation medium consisted of the following ingredients: 10 g / L glucose, 40 g / L kelp powder, 30 g / L egg white, 8 g / L beef extract, 8 g / L fermented soybean meal, 5 g / L sodium acetate, 2 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate, 0.5 g / L calcium chloride, 0.2 g / L manganese sulfate, and 0.2 g / L ferric sulfate. The pH of the fermentation medium was adjusted to 6.3 using sodium hydroxide.

[0088] 2. 3-ton fermentation tanks are sterilized with high temperature and high pressure Add fermentation medium according to 60% of the tank volume, and add 1‰ (volume ratio, V / V) of defoaming agent (the main component of the defoaming agent is polydimethylsiloxane). Use solid sodium hydroxide to adjust the pH to 6.3. After sterilization, pass cold water into the jacket to quickly cool it down, and introduce sterile air into the tank to maintain the pressure at 0.05MPa.

[0089] 3. Fermentation control When the tank temperature is cooled to 48°C, the seed tank culture solution is inoculated into the upper tank at an inoculation rate of 3% by volume. The initial ventilation ratio is 0.5vvm, the tank pressure is maintained at 0.05MPa, the temperature is controlled at 47°C, and the fermentation is started at the lowest stirring speed. 50% phosphoric acid is added to control the fermentation pH to 5.8~6.0. The dissolved oxygen is maintained at 20~40% saturation during the entire fermentation process. Culture until the conidiation rate reaches more than 80% and then the tank can be removed.

[0090] Example 8 Determination of the content of brown algae oligosaccharides in fermentation broth by spectrophotometry Alginate oligosaccharides (AOS), also known as alginic acid oligosaccharides or alginate oligosaccharides, are oligosaccharides composed of β-D-mannuronic acid and α-L-guluronic acid linked by 1,4-glycosidic bonds, primarily with a degree of polymerization of 2 to 10. Alginic acid oligosaccharides undergo hydrolysis and decarboxylation upon heating in concentrated sulfuric acid to produce furfural or furfural derivatives, which then react with carbazole to form purple-red compounds. These compounds have a maximum absorption at 530 nm, and within a certain range, the absorbance shows a linear relationship with the uronic acid concentration.

[0091] 1. Sample pretreatment 1. Flocculation and centrifugation Add polyacrylamide, a flocculant, to the fermentation broth and stir evenly to allow the bacteria to flocculate and precipitate. Centrifuge the fermentation broth (10,000 rpm, 5 min) to remove the bacteria and large particles of impurities and the precipitate, and retain the supernatant.

[0092] 2. Filter The supernatant was initially filtered using a membrane ultrafiltration with mw = 5000 Da to remove macromolecular substances such as proteins and undecomposed alginate, and the filtrate was retained.

[0093] 3. Nanofiltration to remove salt The filtrate after ultrafiltration was treated with a MW=300 Da nanofiltration membrane, the retentate (containing brown algae oligosaccharides) was retained, the filtrate (containing salts and monosaccharides) was removed, and finally the volume of the retentate was adjusted to the original filtrate volume with pure water.

[0094] 2. Experimental Drug Configuration 1. Prepare sulfuric acid-borax solution: Place 40 mL of 4% borax aqueous solution (3.6 g borax + 40 mL water) in an ice bath. Slowly add 360 mL of concentrated sulfuric acid dropwise (at a rate of approximately 1-2 mL / s) while stirring continuously. Keep the temperature ≤30°C. After mixing, incubate in the dark for 12 hours.

[0095] 2. Carbazole solution (0.1%): Weigh 0.25 g of carbazole, add anhydrous ethanol, and dilute to 250 mL. Use immediately.

[0096] 3. Brown algae oligosaccharide reference solution (0.1 mg / mL): Accurately weigh 10 mg (accurate to 0.1 mg) of brown algae oligosaccharide reference that has been dried to a constant weight, dissolve it in an appropriate amount of pure water, fully swell until the solution is clear and translucent, and dilute to a 100 mL volumetric flask.

[0097] The concentrated sulfuric acid (H2SO4) used in each drug is analytically pure. Sodium tetraborate decahydrate (borax, Na2B4O7・10H2O) is analytically pure. 12 H9N): analytically pure. Brown algae oligosaccharide reference: ≥90% mw <4000. Pure water: Grade II water as specified in GB / T6682. Anhydrous ethanol (C2H6O): analytically pure.

[0098] 2. Preparation of standard curve Accurately pipette 0 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of the brown algae oligosaccharide reference solution into stoppered test tubes. Make up to 0.5 mL with pure water. Add 3 mL of sulfuric acid-borax reagent to each tube. Shake thoroughly, heat in a boiling water bath for 10 minutes, seal the tube to prevent evaporation, and quickly cool to room temperature. Add 0.1 mL of carbazole reagent, shake thoroughly, and react in a boiling water bath for 10 minutes. Seal the tube to prevent evaporation, and quickly cool to room temperature. Using pure water as a blank control, measure the absorbance at 530 nm (complete within 30 minutes). Measure each concentration point in triplicate. Plot a standard curve with the brown algae oligosaccharide reference concentration as the abscissa and the average absorbance as the ordinate.

[0099] The corresponding relationship between brown algal oligosaccharide concentration and absorbance is as follows: At a concentration of 0.00 mg / mL, the absorbances were 0.08, 0.08, and 0.09, with a mean of 0.083; At a concentration of 0.05 mg / mL, the absorbances were 0.33, 0.34, and 0.34, respectively, with an average of 0.335; At a concentration of 0.10 mg / mL, the absorbances were 0.58, 0.57, and 0.59, respectively, with a mean of 0.586; At a concentration of 0.20 mg / mL, the absorbances were 1.09, 1.10, and 1.11, respectively, with an average of 1.089; At a concentration of 0.30 mg / mL, the absorbances were 1.60, 1.61, and 1.61, respectively, with an average of 1.593; At a concentration of 0.40 mg / mL, the absorbances were 2.11, 2.11, and 2.12, respectively, with an average of 2.096; At a concentration of 0.50 mg / mL, the absorbances were 2.62, 2.61, and 2.62, respectively, with an average of 2.600.

[0100] The fitting formula is: A =5.032⋅ C +0.083( R 2 =0.999), where the slope K=5.032 and the intercept b=0.083.

[0101] 3. Sample determination Dilute the prepared brown algae oligosaccharide filtrate 1000-fold (D = 1000). Pipette 0.5 mL of the diluted solution as the test sample and add 3 mL of sulfuric acid-borax reagent. Shake well, react in a boiling water bath for 10 minutes, seal the tube with a cap to prevent evaporation, and quickly cool to room temperature. Add 0.1 mL of carbazole reagent, shake well, react in a boiling water bath for 10 minutes, seal the tube with a cap to prevent evaporation, and quickly cool to room temperature. Measure absorbance at 530 nm using pure water as a blank control.

[0102] The absorbance of the test solution after dilution is: 0.375, 0.376, 0.377, the average is sample A = 0.376, according to formula C 发酵液 =(A sample-b) / k╳D, C 发酵液 =58.24 mg / mL, that is, after fermentation by Bacillus coagulans using kelp as the main carbon source, the brown algae oligosaccharide content of the fermentation liquid is 58.24 mg / mL.

[0103] The present invention provides a Bacillus coagulans fermentation method using kelp as the main carbon source and its application in the production of brown algae oligosaccharides, which has the advantages of environmental protection, low cost and high efficiency, and is a new fermentation technology with broad application prospects.

[0104] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Bacillus coagulans ( Bacillus coagulans )SDHY-2402, the deposit number is CGMCC No.32910.

2. Use of the Bacillus coagulans SDHY-2402 according to claim 1 in the fermentation production of brown algae oligosaccharides.

3. A method for producing brown algal oligosaccharides by fermentation with Bacillus coagulans using kelp as the main carbon source, characterized in that: A fermentation medium containing kelp powder as a main carbon source was prepared; a culture of Bacillus coagulans SDHY-2402 with a deposit number of CGMCC No. 32910 was inoculated into the fermentation medium for fermentation; and after the fermentation was completed, the content of brown algal oligosaccharides in the fermentation liquid was determined; The kelp powder is obtained by washing, drying and crushing the kelp.

4. The method according to claim 3, characterized in that The fermentation medium comprises the following components in parts by weight: 5-10 parts of glucose, 30-40 parts of kelp powder, 20-30 parts of egg white, 2-8 parts of beef extract, 5-10 parts of fermented soybean meal, 3-6 parts of sodium acetate, 1-3 parts of dipotassium hydrogen phosphate, 0.5-1.0 parts of magnesium sulfate, 0.1-0.3 parts of manganese sulfate, 0.2-0.5 parts of calcium chloride, 0.1-0.3 parts of ferrous sulfate and 200-250 parts of water.

5. The method according to claim 3, characterized in that Fermentation conditions: 45°C~47°C, pH 5.5~6.0, dissolved oxygen concentration 20%~40%, rotation speed 300~400 rpm; Determination of the brown algae oligosaccharide content in the fermentation broth includes the steps of centrifugation, filtration, and nanofiltration.

6. A method for producing brown algae oligosaccharides by fermentation with Bacillus coagulans, characterized in that: The following steps are involved: S1. Pre-treating the kelp: washing, drying, and crushing the kelp to obtain kelp powder; mixing the kelp powder with water and disrupting the cell walls using ultrasound; then chemically treating the kelp powder with dilute acid and dilute alkali, enzymatically hydrolyzing the resulting product, and using the enzymatic hydrolysis product as a carbon source for fermentation production of brown algae oligosaccharides; S2. A fermentation medium is prepared using the enzymatic hydrolysis product of step S1, and fermentation is performed using Bacillus coagulans SDHY-2402 with a deposit number of CGMCC No. 32910 as the fermentation strain; after the fermentation is completed, the content of brown algal oligosaccharides in the fermentation liquid is determined.

7. The method according to claim 6, characterized in that Step S1 includes: (1) Mix 30-50 g of kelp powder with 750-1250 mL of water and use ultrasound to break the cell walls. The ultrasound conditions are: probe diameter 10 mm, power 600 W, working cycle 3 s on, 2 s off, each working cycle 3 min, 5 working cycles, each working cycle separated by 3 min of ice bath, for a total of 27 min. (2) The kelp powder of step (1) is ultrasonically treated and then centrifuged to collect the precipitate, which is hydrolyzed with dilute acid at 80-100°C, and the hydrolyzate is chemically treated with dilute alkali at 80-100°C; the dilute acid is selected from sulfuric acid or hydrochloric acid; and the dilute alkali is selected from sodium hydroxide or potassium hydroxide; (3) The product obtained by chemical treatment in step (2) is contacted with a complex enzyme and enzymatically hydrolyzed at 40-60°C; the complex enzyme is selected from two or more of lipase, alginate, and pectinase.

8. The method according to claim 7, characterized in that Steps (2) and (3) comprise: mixing 50 g of precipitate with 750-1000 mL of sulfuric acid, and hydrolyzing at 80-100° C. for 120-240 min; after the hydrolysis is completed, adding 1000-1500 mL of sodium hydroxide thereto, and treating at 80-100° C. for 240-360 min; adding a complex enzyme to the treated product so that the final concentrations of cellulase, alginate enzyme, and pectinase in the enzymatic hydrolysis system are 40-60 U / mL, 50-80 U / mL, and 20-30 U / mL, respectively; performing enzymatic hydrolysis at 40-60° C. for 480-600 min, and directly using the obtained enzymatic hydrolysis product for fermentation, or removing insoluble residue from the obtained enzymatic hydrolysis product to obtain a clarified liquid for fermentation; Wherein, the concentration of sulfuric acid is 0.1-0.5M, and the concentration of sodium hydroxide is 0.5-1M.

9. Fucoidan oligosaccharide produced according to the method according to any one of claims 3 to 8.

10. Use of the brown algal oligosaccharide according to claim 9 in preparing products; The product is any one selected from cosmetics, medicines, foods or food additives, feeds or feed additives.

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