Bacillus and application thereof in extraction of enteromorpha dietary fibers

Abstract

The invention discloses a bacillus and application of the bacillus in extraction of enteromorpha dietary fibers, and particularly relates to a bacillus KICET-3 strain, and the strain is separated from naturally fermented enteromorpha. The extraction process of the enteromorpha dietary fiber comprises the following steps: 1, pretreating enteromorpha; and 2, fermenting and extracting the enteromorpha dietary fiber. According to the method, the extraction rate of the dietary fibers is effectively increased from the enteromorpha, the requirements of industrial production for high-yield functional components are met, meanwhile, the functional characteristics of the enteromorpha dietary fibers are improved, the purposes of high efficiency, environmental protection, low cost and sustainability are achieved, and meanwhile high-value utilization of the enteromorpha in green tide disasters is achieved.

Description

Technical Field

[0001] This invention belongs to the field of microbial screening and application technology, and in particular relates to a Bacillus species and its application in the extraction of dietary fiber from seaweed. Background Technology

[0002] The green tide problem caused by benthic algae blooms is becoming increasingly serious worldwide, affecting many regions in Asia, Europe, and the United States. In Brittany, France, the decaying algae release sulfur gas that pollutes the environment and even kills horses. In the United States, green tides cause fish to die from lack of oxygen, impacting the fishing and tourism industries.

[0003] Ulva prolifera is a dominant algae species in the Qingdao sea area during green tides, exhibiting rapid proliferation characteristics. Its resource utilization is a core direction for alleviating the pressure of green tides. Ulva prolifera contains nutrients such as carbohydrates (43-51%), and dietary fiber (DF) is a key functional component, which is divided into soluble (SDF) and insoluble (IDF). SDF has functions such as preventing diabetes and alleviating cardiovascular diseases, but the current extraction rate is generally low.

[0004] Existing dietary fiber extraction methods include physical methods (high energy consumption, easily damaging the structure), chemical methods (easily introducing impurities and polluting the environment), and biological methods (enzymatic methods are costly, and while microbial fermentation has advantages, the extraction efficiency and functional enhancement effects of existing strains need improvement). For example, the yield of SDF extracted from Ulva prolifera is about 21.8%, and the obtained DF has room for improvement in water retention, oil retention, cholesterol adsorption, and antioxidant properties. There is a lack of efficient and sustainable dietary fiber extraction technology for Ulva prolifera.

[0005] In conclusion, how to effectively increase the extraction rate of dietary fiber from *Ulva prolifera* to meet the demand for high-yield functional components in industrial production, while improving the functional properties of dietary fiber in *Ulva prolifera* to achieve high efficiency, environmental protection, low cost and sustainability, and realize the high-value utilization of *Ulva prolifera* during green tide disasters, has become a problem that technicians in the field of microbiology urgently need to solve. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is to provide an efficient, environmentally friendly, low-cost and sustainable extraction method that effectively improves the extraction rate of dietary fiber and improves the functional properties of dietary fiber in seaweed, as well as the application of Bacillus subtilis in the extraction of dietary fiber from seaweed.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a Bacillus strain, classified and named as Bacillus sp. KICET-3, which is isolated from naturally fermented seaweed. The specific isolation process includes the following steps:

[0008] I. Source of the strain:

[0009] (1) Fresh seaweed harvested from the Qingdao sea area was placed in a sterilized iron barrel and fermented naturally for 30 days;

[0010] (2) Collect samples from the middle part of the fermented seaweed and store them at 4℃ for later use;

[0011] II. Strain screening:

[0012] (3) Add the sample to a test tube containing sterile water at a ratio of 1:9 (w / v) and mix with a shaker for 30 min.

[0013] (4) Serial dilution of sterile water to 10 ﹣2 -10 ﹣8 Spread the diluted solution onto cellulose Congo red medium, pick out colonies that produce a clear zone, and repeat the streak culture on potato dextrose agar (PDA) medium until a pure single strain is obtained.

[0014] III. Strain Identification:

[0015] (5) The pure single strain obtained in step (4) was subjected to DNA extraction, PCR amplification and sequencing. Homologous sequences were searched using the BLAST tool of NCBI. A phylogenetic tree was constructed using the neighbor connection algorithm of MEGA11.0 software and identified as Bacillus sp. KICET-3.

[0016] IV. Strain Expansion Culture and Preservation:

[0017] (6) Inoculate the pure single strain obtained in step (4) onto LB liquid medium and culture with shaking at 37°C and 150 rpm to control the strain concentration to 10. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0018] (9) Take the bacterial solution and 40% glycerol (by mass) of sterilization treatment and mix them at a ratio of 1:1 (v / v), and store them in a refrigerator at -80℃.

[0019] The above-mentioned Bacillus strains are used in the extraction of dietary fiber from seaweed.

[0020] The above-mentioned application, the extraction process of dietary fiber from *Ulva prolifera* includes the following steps:

[0021] I. Pretreatment of Ulva prolifera:

[0022] S1. Wash the fresh seaweed, dry it at 65-80℃, pulverize it to 100 mesh, and sterilize it with high pressure steam at 121℃, 0.1MPa, and 20min to obtain the seaweed raw material for later use.

[0023] II. Fermentation and extraction of dietary fiber from seaweed:

[0024] S2. Preparation before fermentation: Inoculate the (Bacillus sp.) KICET-3 strain preserved in step (9) into the seed culture medium from the glycerol tube, and culture with shaking at 35-38℃ and 130-155rpm until the strain concentration reaches 10. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0025] S3. Fermentation process: Mix the raw material of Ulva prolifera with distilled water at a liquid-to-solid ratio of 30-33, add seed liquid with a concentration of 2-5% (v / w), adjust the pH to 6-7.5, and culture with shaking at 35-38℃ and 130-155rpm for 28-36h to obtain the fermented Ulva prolifera mixture.

[0026] S4: Dietary fiber extraction: SDF and IDF were extracted based on the AOAC enzyme gravimetric method. Thermostable α-amylase was added to the fermented Ulva lactuca mixture, and the pH was adjusted to 5.5-6.5. The mixture was stirred for 45 min in a 65℃ water bath and then cooled to room temperature.

[0027] S5: Add papain and adjust the pH to 6-6.5. Stir for 120 min in a 60℃ water bath. Boil for 10 min to inactivate the enzyme and filter to separate the filtrate from the residue.

[0028] S6: Add a certain amount of 95% ethanol to the filtrate and mix overnight. Collect the precipitate and freeze-dry to obtain fermented soluble dietary fiber (XSDF). Wash the filter residue twice with 95% ethanol and acetone alternately and freeze-dry to obtain fermented insoluble dietary fiber (XIDF).

[0029] In the above application, in S1, the drying temperature of the fresh seaweed after washing is 70°C.

[0030] In the above application, in S2, the shaking culture conditions are 37°C and 150 rpm.

[0031] In the above application, in S3, the liquid-to-solid ratio of the *Ulva prolifera* raw material to distilled water is 32.061, the concentration of the added seed liquid is 3.282% (v / w), the pH is adjusted to 6.938, and the mixture is cultured at 37°C and 150 rpm for 30.294 h with shaking.

[0032] In the above application, in step S6, the volume ratio of added ethanol to filtrate is 1:4.

[0033] The advantages of this invention regarding Bacillus sp. and its application in extracting dietary fiber from Ulva prolifera are as follows: It solves the problem of low SDF yield in existing Ulva prolifera dietary fiber extraction methods. Existing enzymatic methods yield approximately 21.8% SDF, while this invention, through fermentation technology using Bacillus sp. KICET-3 strain combined with response surface methodology-optimized process parameters (fermentation time 30.294 h, pH 6.938, inoculum size 3.282%, liquid-to-solid ratio 32.061), increases the SDF yield to 31%, a 9.2 percentage point improvement over enzymatic methods. Furthermore, the strain can be screened in-situ from decaying Ulva prolifera, eliminating the need for complex culture conditions and significantly reducing raw material and production costs, thus laying the foundation for large-scale production of Ulva prolifera dietary fiber. This invention improves the functional properties of dietary fiber from *Ulva prolifera*. Compared to enzymatic methods, the fermentation method of this invention increases the water-holding capacity (WHC), oil-holding capacity (OHC), and swelling capacity (SC) of SDF by 20.31%, 19.08%, and 36.84%, respectively; while the corresponding indicators of IDF increase by 44.16%, 11.12%, and 43.90%, respectively. The loose and porous microstructure better maintains food texture and reduces oil loss, making it suitable for food processing applications such as baking and meal replacements. This invention overcomes the limitations of existing extraction technologies, such as high energy consumption of physical methods, environmental pollution of chemical methods, and high costs of enzymatic methods. It achieves efficient, environmentally friendly, low-cost, and sustainable extraction of dietary fiber from *Ulva prolifera*, while also enabling the high-value utilization of *Ulva prolifera* affected by green tides. Attached Figure Description

[0034] Figure 1 This is a phylogenetic tree of the strains isolated in this invention;

[0035] Figure 2 This is a schematic diagram illustrating the mechanism of dietary fiber extraction from seaweed using Bacillus sp. KICET-3 strain fermentation in this invention.

[0036] Figure 3 SEM images of the soluble dietary fiber (XSDF) and insoluble dietary fiber (XIDF) obtained in Example 2;

[0037] Figure 4 The graphs show the performance test results of water-holding capacity (WHC), oil-holding capacity (OHC), and swelling capacity (SC) of soluble and insoluble dietary fiber prepared by fermentation and traditional enzymatic methods, respectively, in Example 2.

[0038] Figure 5 The graphs show the performance test results of total phenolic content, total flavonoid content, reducing power, and DPPH scavenging power of soluble and insoluble dietary fiber prepared by fermentation and traditional enzymatic methods, respectively, in Example 2.

[0039] Figure 6 The graphs show the adsorption performance of soluble and insoluble dietary fiber prepared by fermentation and traditional enzymatic methods, respectively, in Example 2.

[0040] Figure 7 SEM comparison images of (XSDF) and (XIDF) prepared in Example 2 with (MSDF) and (MIDF) prepared by enzymatic method;

[0041] Figure 8 This is a diagram for sustainability analysis. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] In this invention, unless otherwise stated, directional terms such as "upper" and "lower" generally refer to the upper and lower positions of the device in its actual use or operating state, specifically the drawing directions in the accompanying drawings; while "inner" and "outer" refer to the outline of the device. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". The terms first, second, third, etc., are used merely as illustrative purposes and do not impose numerical requirements or establish an order. The term "multiple" means "two or more".

[0044] Strain verification:

[0045] Bacillus sp. KICET-3 is a known strain proposed by the Korea Institute of Ceramic Engineering and Technology (KICET). Specific information is as follows:

[0046] Whole-genome sequencing data of Bacillus strain KICET-3 have been obtained via GenBank accession number CP137345, 16S rRNA accession number OR807426, BioProject accession number PRJNA1031784, and BioSample accession number SAMN37973075. Its SRA numbers are SRR2664315 and SRR2664316. This strain is deposited at the Korean Center for Microbial Culture (KCCM) under accession number KCCM 13299P.

[0047] This strain was isolated by a South Korean team from the traditional fermented food "doenjang" (Korean soybean paste) and is not *Ulva prolifera*; it is documented in both the South Korean patent KR-20240116408-A and the 2025 genome paper (Microbiol Resour Announc).

[0048] like Figure 1As shown, 16S rRNA gene sequence alignment revealed 100% homology between the target strain and Bacillus sp. KICET-3 (Firmwallis). Further phylogenetic analysis showed that the two clustered into the same branch with 85% confidence, both belonging to a high-confidence (100%) large branch of Bacillus sonorensis-related strains. Based on the comprehensive molecular-level evidence of phylogenetic relationships, the target strain can be identified as belonging to the genus Bacillus and is closely related to Bacillus sp. KICET-3, exhibiting extremely high species similarity.

[0049] To further verify the precise attribution of the strain, this invention supplements the whole-genome-level verification: based on the tandem sequence alignment of 31 housekeeping genes, including dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, and rplD, the target strain showed 99.9% homology with Bacillus sp. KICET-3; the average nucleotide identity (ANI) of the whole genome was 99.7%, and the digital DNA-DNA hybridization (dDDH) value was 94.8%, which are far higher than the thresholds defined at the strain level (ANI≥99%, dDDH≥90%).

[0050] The morphological verification results highly matched the classification characteristics: after culturing on LB medium, the strain formed yellow, well-defined, and thick colonies with uniform morphology and no abnormal discoloration. Microscopic observation revealed that the cells were Gram-positive rod-shaped cells with regular morphology and capable of producing oval spores. The location and morphology of the spores conformed to the typical characteristics of the genus Bacillus. These morphological characteristics were highly consistent with the morphological descriptions of Bacillus sp. KICET-3 and known strains of the genus Bacillus, further corroborating the reliability of the molecular identification results. The Bacillus sp. KICET-3 strain screened from Ulva prolifera in this invention has the same whole genome sequence and gene information as the Bacillus sp. KICET-3 isolated from soybean paste.

[0051] Key equipment and instruments:

[0052] 1. Cultivation equipment: constant temperature shaking incubator (temperature control accuracy ±1℃, rotation speed range 50-300rpm), ultra-clean workbench (Class 100 cleanliness), -80℃ ultra-low temperature freezer, freeze dryer (vacuum degree ≤10Pa, cold trap temperature ≤-50℃).

[0053] 2. Testing equipment: UV-Vis spectrophotometer (wavelength range 200-1000nm, accuracy ±0.5nm), scanning electron microscope (model SU8600, magnification 3000x), X-ray diffractometer (operating current 40mA, voltage 40kV, 2θ angle 5-80° scanning), Fourier transform infrared spectrophotometer (wavenumber range 400-4000cm⁻¹), thermogravimetric analyzer (heating rate 15℃ / min, temperature range 30-500℃), high-speed centrifuge (maximum speed 10000r / min).

[0054] 3. Other equipment: high-pressure steam sterilizer, pulverizer (fineness up to 100 mesh), pH meter (accuracy ±0.01), electronic analytical balance (accuracy 0.1mg).

[0055] A Bacillus strain, classified as Bacillus sp. KICET-3, was isolated from naturally fermented seaweed. The isolation process included the following steps:

[0056] I. Source of the strain:

[0057] (1) Fresh seaweed harvested from the Qingdao sea area was placed in a sterilized iron barrel and fermented naturally for 30 days;

[0058] (2) Collect samples from the middle part of the fermented seaweed and store them at 4℃ for later use;

[0059] II. Strain screening:

[0060] (3) Add the sample to a test tube containing sterile water at a ratio of 1:9 (w / v) and mix with a shaker for 30 min.

[0061] (4) Serial dilution of sterile water to 10 ﹣2 -10 ﹣8 Spread the diluted solution onto cellulose Congo red medium, pick out colonies that produce a clear zone, and repeat the streak culture on potato dextrose agar (PDA) medium until a pure single strain is obtained.

[0062] III. Strain Identification:

[0063] (5) The pure single strain obtained in step (4) was subjected to DNA extraction, PCR amplification and sequencing. Homologous sequences were searched using the BLAST tool of NCBI. A phylogenetic tree was constructed using the neighbor connection algorithm of MEGA11.0 software and identified as Bacillus sp. KICET-3.

[0064] IV. Strain Expansion Culture and Preservation:

[0065] (6) Inoculate the pure single strain obtained in step (4) onto LB liquid medium and culture with shaking at 37°C and 150 rpm to control the strain concentration to 10. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0066] (9) Take the bacterial solution and 40% glycerol (by mass) of sterilization treatment and mix them at a ratio of 1:1 (v / v), and store them in a refrigerator at -80℃.

[0067] Carboxymethyl cellulase activity assay: The strain was inoculated into liquid fermentation enzyme-producing medium (CMC-Na 10g, (NH4)2SO4 4g, KH2PO4 2g, MgSO4·7H2O 0.5g, peptone 10g, beef extract 5g, and distilled water to a final volume of 1000mL), and cultured at 28℃ with shaking at 150r / min; centrifuged at 3000r / min for 10min, and the supernatant was taken as crude enzyme solution. Take 0.1 mL of crude enzyme solution and add 1.9 mL of 1% CMC-Na solution. Hydrolyze at 50℃ for 20 min, add 1.5 mL of DNS solution and boil in a water bath for 10 min. Cool and bring the volume to 25 mL. Measure the absorbance (A) at 520 nm and compare with the glucose standard curve to obtain the glucose content (m1). Take another 0.1 mL of supernatant and add 1.9 mL of sterile water. Measure the absorbance of the glucose content (m2) using the same method. Calculate the enzyme activity using the formula: Enzyme activity (U) = (m1-m2)×250 / (20×0.1×180). Ensure that the enzyme activity of the strain meets the standard (peak value 54.86±1.1U, enzyme activity 74.3±1.3U at the optimal pH 6.0).

[0068] The application of this Bacillus in the extraction of dietary fiber from Ulva prolifera: The extraction process of dietary fiber from Ulva prolifera includes the following steps:

[0069] I. Pretreatment of Ulva prolifera:

[0070] S1. Wash the fresh seaweed, dry it at 65-80℃, pulverize it to 100 mesh, and sterilize it with high pressure steam at 121℃, 0.1MPa, and 20min to obtain the seaweed raw material for later use.

[0071] II. Fermentation and extraction of dietary fiber from seaweed:

[0072] S2. Preparation before fermentation: Inoculate the (Bacillus sp.) KICET-3 strain preserved in step (9) of claim 1 into seed culture medium (LB liquid medium) from the glycerol tube, and culture with shaking at 35-38℃ and 130-155 rpm until the strain concentration reaches 10. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0073] S3. Fermentation process: Mix the raw material of Ulva prolifera with distilled water at a liquid-to-solid ratio of 30-33, add seed liquid with a concentration of 2-5% (v / w), adjust the pH to 6-7.5, and culture with shaking at 35-38℃ and 130-155rpm for 28-36h to obtain the fermented Ulva prolifera mixture.

[0074] S4: Dietary fiber extraction: SDF and IDF were extracted based on the AOAC enzyme gravimetric method. Thermostable α-amylase was added to the fermented Ulva lactuca mixture, and the pH was adjusted to 5.5-6.5. The mixture was stirred for 45 min in a 65℃ water bath and then cooled to room temperature.

[0075] S5: Add papain and adjust the pH to 6-6.5. Stir for 120 min in a 60℃ water bath. Boil for 10 min to inactivate the enzyme and filter to separate the filtrate from the residue.

[0076] S6: Add a certain amount of 95% ethanol to the filtrate and mix overnight. Collect the precipitate and freeze-dry to obtain fermented soluble dietary fiber (XSDF). Wash the filter residue twice with 95% ethanol and acetone alternately and freeze-dry to obtain fermented insoluble dietary fiber (XIDF).

[0077] like Figure 2 As shown, the core mechanism of Bacillus sp. KICET-3 fermentation of seaweed for dietary fiber extraction is that this strain achieves efficient degradation of the seaweed cell wall, targeted release and structural modification of dietary fiber through the synergistic effect of secreting a complex functional enzyme system, regulating the metabolic microenvironment, and physically deconstructing the tissue. Ultimately, this increases the proportion of soluble dietary fiber and its functional properties. Specifically, this can be summarized in three points:

[0078] 1. Enzymatic hydrolysis core function: The strain secretes a variety of enzymes such as cellulase, alginate lyase, and protease, which precisely cut the glycosidic bonds of the polysaccharide of Ulva prolifera and hydrolyze the impurities that encapsulate the polysaccharide, thus deconstructing the cell wall at the molecular level and clearing obstacles for the dissolution of dietary fiber.

[0079] 2. Fermentation-assisted enhancement: The proliferation of microorganisms produces gas, which destroys the physical structure of Ulva prolifera, increases the contact area between enzymes and substrates, and at the same time, the metabolic acid production lowers the pH of the system, promotes the dissolution and deesterification of polysaccharides, and can also modify the structure of dietary fiber, reduce its molecular weight, retain active groups, and optimize water holding capacity.

[0080] 3. Enhanced efficiency through coupling: In-situ coupling of enzymatic hydrolysis and fermentation allows the enzymatic hydrolysis products to provide a carbon source for the strain, forming a positive feedback loop. The entire process is conducted under mild conditions, avoiding the damage to the structure and activity of dietary fiber caused by chemical methods, while simultaneously achieving the initial enrichment and purification of dietary fiber.

[0081] The present application will be specifically described below through specific embodiments. The following embodiments are only some embodiments of the present application and are not intended to limit the present application.

[0082] Example 1:

[0083] A Bacillus strain, classified as Bacillus sp. KICET-3, was isolated from naturally fermented seaweed. The isolation process included the following steps:

[0084] I. Source of the strain:

[0085] (1) Fresh seaweed harvested from the Qingdao sea area was placed in a sterilized iron barrel and fermented naturally for 30 days;

[0086] (2) Collect samples from the middle part of the fermented seaweed and store them at 4℃ for later use;

[0087] II. Strain screening:

[0088] (3) Add the sample to a test tube containing sterile water at a ratio of 1:9 (w / v) and mix with a shaker for 30 min.

[0089] (4) Serial dilution of sterile water to 10 ﹣2 -10 ﹣8 Spread the diluted solution onto cellulose Congo red medium, pick out colonies that produce a clear zone, and repeat the streak culture on potato dextrose agar (PDA) medium until a pure single strain is obtained.

[0090] III. Strain Identification:

[0091] (5) The pure single strain obtained in step (4) was subjected to DNA extraction, PCR amplification and sequencing. Homologous sequences were searched using the BLAST tool of NCBI. A phylogenetic tree was constructed using the neighbor connection algorithm of MEGA11.0 software and identified as Bacillus sp. KICET-3.

[0092] IV. Strain Expansion Culture and Preservation:

[0093] (6) Inoculate the pure single strain obtained in step (4) onto LB liquid medium and culture with shaking at 37°C and 150 rpm to control the strain concentration to 10. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0094] (9) Take the bacterial solution and 40% glycerol (by mass) of sterilization treatment and mix them at a ratio of 1:1 (v / v), and store them in a refrigerator at -80℃.

[0095] The application of this Bacillus in the extraction of dietary fiber from Ulva prolifera involves the following steps:

[0096] I. Pretreatment of Ulva prolifera:

[0097] S1. Wash the fresh seaweed, dry it at 65℃, pulverize it to 100 mesh, and sterilize it with high pressure steam at 121℃, 0.1MPa, and 20min to obtain the seaweed raw material for later use.

[0098] II. Fermentation and extraction of dietary fiber from seaweed:

[0099] S2. Preparation before fermentation: The (Bacillus sp.) KICET-3 strain preserved in step (9) was inoculated into seed culture medium (LB liquid medium) from a glycerol tube and cultured with shaking at 35°C and 130 rpm until the strain concentration reached 10. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0100] S3. Fermentation process: Mix the raw material of Ulva prolifera with distilled water at a liquid-to-solid ratio of 30, add seed liquid with a concentration of 2% (v / w), adjust the pH to 6, and culture with shaking at 35℃ and 130rpm for 28h to obtain the fermented Ulva prolifera mixture.

[0101] S4: Dietary fiber extraction: SDF and IDF were extracted based on the AOAC enzyme gravimetric method. Thermostable α-amylase was added to the fermented Ulva lactuca mixture, and the pH was adjusted to 5.5. The mixture was stirred for 45 min in a 65℃ water bath and then cooled to room temperature.

[0102] S5: Add papain and adjust the pH to 6. Stir for 120 min in a 60°C water bath. Boil for 10 min to inactivate the enzyme and filter to separate the filtrate from the residue.

[0103] S6: Add a certain amount of 95% ethanol to the filtrate and mix overnight. The volume ratio of the added ethanol to the filtrate is 1:4. Collect the precipitate and freeze-dry it to obtain fermented soluble dietary fiber (XSDF). Wash the filter residue twice with 95% ethanol and acetone alternately, and freeze-dry it to obtain fermented insoluble dietary fiber (XIDF).

[0104] Example 2:

[0105] The isolation and extraction process of the Bacillus sp. KICET-3 strain is the same as in Example 1, and will not be repeated here.

[0106] The application of this Bacillus in the extraction of dietary fiber from Ulva prolifera involves the following steps:

[0107] I. Pretreatment of Ulva prolifera:

[0108] S1. Wash the fresh seaweed, dry it at 70℃, pulverize it to 100 mesh, and sterilize it with high pressure steam at 121℃, 0.1MPa, and 20min to obtain the seaweed raw material for later use.

[0109] II. Fermentation and extraction of dietary fiber from seaweed:

[0110] S2. Preparation before fermentation: Inoculate the (Bacillus sp.) KICET-3 strain preserved in step (9) into seed culture medium (LB liquid medium) from the glycerol tube, and culture with shaking at 37°C and 150 rpm until the strain concentration reaches 10. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0111] S3. Fermentation process: The raw material of Ulva prolifera was mixed with distilled water at a liquid-to-solid ratio of 32.061, and seed liquid with a concentration of 3.282% (v / w) was added. The pH was adjusted to 6.938, and the mixture was cultured with shaking at 37℃ and 150 rpm for 30.294 h to obtain the fermented Ulva prolifera mixture.

[0112] S4: Dietary fiber extraction: SDF and IDF were extracted based on the AOAC enzyme gravimetric method. Thermostable α-amylase was added to the fermented Ulva lactuca mixture, and the pH was adjusted to 6. The mixture was stirred for 45 min in a 65℃ water bath and then cooled to room temperature.

[0113] S5: Add papain and adjust the pH to 6.2. Stir for 120 min in a 60℃ water bath. Boil for 10 min to inactivate the enzyme and filter to separate the filtrate from the residue.

[0114] S6: Add a certain amount of 95% ethanol to the filtrate and mix overnight. The volume ratio of the added ethanol to the filtrate is 1:4. Collect the precipitate and freeze-dry it to obtain fermented soluble dietary fiber (XSDF). Wash the filter residue twice with 95% ethanol and acetone alternately, and freeze-dry it to obtain fermented insoluble dietary fiber (XIDF).

[0115] Example 3:

[0116] The isolation and extraction process of the Bacillus sp. KICET-3 strain is the same as in Example 1, and will not be repeated here.

[0117] The application of this Bacillus in the extraction of dietary fiber from Ulva prolifera involves the following steps:

[0118] I. Pretreatment of Ulva prolifera:

[0119] S1. Wash the fresh seaweed, dry it at 80℃, pulverize it to 100 mesh, and sterilize it with high pressure steam at 121℃, 0.1MPa, and 20min to obtain the seaweed raw material for later use.

[0120] II. Fermentation and extraction of dietary fiber from seaweed:

[0121] S2. Preparation before fermentation: Inoculate the (Bacillus sp.) KICET-3 strain preserved in step (9) into seed culture medium (LB liquid medium) from the glycerol tube, and culture with shaking at 38°C and 155 rpm until the strain concentration reaches 10. 8 -10 9 CFU / mL was used to obtain the seed culture;

[0122] S3. Fermentation process: Mix the raw material of Ulva prolifera with distilled water at a liquid-to-solid ratio of 3:3, add seed liquid with a concentration of 5% (v / w), adjust the pH to 7.5, and culture at 38℃ and 155rpm for 36 hours with shaking to obtain the fermented Ulva prolifera mixture.

[0123] S4: Dietary fiber extraction: SDF and IDF were extracted based on the AOAC enzyme gravimetric method. Thermostable α-amylase was added to the fermented Ulva lactuca mixture, and the pH was adjusted to 6.5. The mixture was stirred for 45 min in a 65℃ water bath and then cooled to room temperature.

[0124] S5: Add papain and adjust the pH to 6.5. Stir for 120 min in a 60℃ water bath. Boil for 10 min to inactivate the enzyme and filter to separate the filtrate from the residue.

[0125] S6: Add a certain amount of 95% ethanol to the filtrate and mix overnight. The volume ratio of the added ethanol to the filtrate is 1:4. Collect the precipitate and freeze-dry it to obtain fermented soluble dietary fiber (XSDF). Wash the filter residue twice with 95% ethanol and acetone alternately, and freeze-dry it to obtain fermented insoluble dietary fiber (XIDF).

[0126] Comparative example (enzymatic extraction)

[0127] The raw material of *Ulva prolifera* was mixed with distilled water to achieve a solid-liquid ratio of 20% (w / v). Cellulase and heat-stable α-amylase (pH=6) were added, and the mixture was stirred in a water bath at 65°C for 45 min. Subsequently, the mixture was cooled to room temperature, papain (pH=6.25) was added, and the mixture was stirred in a water bath at 60°C for 120 min. Finally, the mixture was boiled for 10 min to inactivate the enzymes. The filtrate and residue were separated by filtration. The filtrate was mixed with 95% ethanol (1:4 v / v) and incubated overnight. The precipitate was collected and freeze-dried to obtain MSDF. The residue was washed twice alternately with 95% ethanol and acetone and freeze-dried to obtain MIDF.

[0128] The performance test results of the soluble dietary fiber (XSDF) and insoluble dietary fiber (XIDF) obtained in Example 2 of this invention are as follows:

[0129] 1. Regarding the extraction rate.

[0130] In existing technologies, the yield of soluble dietary fiber (SDF) from *Ulva prolifera* using enzymatic extraction is only about 21.8%, and it requires additional high-priced enzyme preparations, making it difficult to meet the industrial production demand for high-value functional components. This invention utilizes fermentation technology with the *Bacillus sp. KICET-3* strain, combined with response surface methodology-optimized process parameters: fermentation time 30.294 h, pH 6.938, inoculum size 3.282%, and liquid-to-solid ratio 32.061, increasing the SDF yield to 31%, a 9.2 percentage point improvement over enzymatic methods. Furthermore, the strain can be screened in-situ from decaying *Ulva prolifera*, eliminating the need for complex culture conditions and significantly reducing raw material and production costs, laying the foundation for the large-scale production of dietary fiber from *Ulva prolifera*.

[0131] 2. Regarding physical and chemical properties: [Water holding capacity (WHC), Oil holding capacity (OHC), Swelling capacity (SC)].

[0132] Water Holding Capacity (WHC)

[0133] Take 0.3g (M0) of sample into a dried centrifuge tube and record the weight (M1). Then add 15mL of distilled water, let stand at room temperature for 24h, centrifuge at 4000rpm for 15min, remove the supernatant and residual water, and record the mass of the sample and centrifuge tube (M2). The WHC is calculated as follows:

[0134] ;

[0135] Where M0, M1, and M2 are the masses of the sample, centrifuge tube, and sample before and after water absorption, respectively, in grams.

[0136] Oil-holding capacity (OHC)

[0137] Take 0.3g (M0) of the sample into a dried centrifuge tube and record the weight (M1). Then add 15mL of peanut oil, let it stand at room temperature for 24 hours, centrifuge at 4000rpm for 15 minutes, remove the upper layer of oil and any remaining oil droplets, and weigh the sample and centrifuge tube (M2). The OHC calculation method is as follows:

[0138] ;

[0139] Where M0, M1, and M2 are the masses of the sample, centrifuge tube, and sample before and after oil absorption, respectively, in grams.

[0140] Swelling capacity (SC)

[0141] Dry the measuring cylinder in an oven. Take 0.3 g (M0) of sample into the measuring cylinder and record the volume V0. Add 10 mL of distilled water and let it stand at room temperature for 24 h, recording the volume V1. The calculation method for SC is as follows:

[0142] ;

[0143] Where V0 and V1 are the sample volumes before and after expansion, in mL; and M0 is the sample mass, in g.

[0144] like Figure 3 , 4 As shown, XSDF and XIDF are the soluble and insoluble dietary fiber obtained by fermentation in Example 2 of this invention, respectively, while MSDF and MIDF are the soluble and insoluble dietary fiber obtained by enzymatic preparation, respectively. The water-holding capacity (WHC), oil-holding capacity (OHC), and swelling capacity (SC) of fermented SDF are increased by 20.31%, 19.08%, and 36.84%, respectively, compared to the enzymatic method; the corresponding indicators of IDF are increased by 44.16%, 11.12%, and 43.90%, respectively. Figure 3 The loose and porous microstructure can better maintain the texture of food and reduce oil loss, making it suitable for food processing scenarios such as baking and meal replacement.

[0145] 3. Regarding physiological activity [Total phenol content (TPC), Total flavonoid content (TFC)]:

[0146] Total flavonoid content (TFC)

[0147] Pipette 0.00, 0.25, 0.50, 1.00, 2.00, 3.00, 4.00, and 6.00 mL of rutin standard solution, equivalent to 0.00, 0.050, 0.10, 0.20, 0.40, 0.60, 0.80, and 1.20 mg of anhydrous rutin, respectively, into 25 mL stoppered colorimetric tubes. Make up to 5.0 mL with ethanol solution, add 1 mL of sodium nitrite solution, shake well, and let stand for 6 min. Add 1.5 mL of aluminum nitrate solution, shake well, and let stand for 6 min. Add 4 mL of sodium hydroxide solution, and dilute to the mark with ethanol solution, shake well, and let stand for 15 min. Using a 1 cm cuvette, adjust the zero point with a reagent blank, and measure the absorbance at a wavelength of 510 nm. Plot a standard curve corresponding to the absorbance values.

[0148] Pipette 2.0 mL of sample solution (adjust the sample volume appropriately depending on the sample value, and try to control the sample absorbance (ABS) between 0.2 and 0.7) into a 25 mL stoppered colorimetric tube. Make up to 5.0 mL with ethanol solution, add 1 mL of sodium nitrite solution, shake well, and let stand for 6 min. Add 1.5 mL of aluminum nitrate solution, shake well, and let stand for 6 min. Add 4 mL of sodium hydroxide solution, and dilute to the mark with ethanol solution, shake well, and let stand for 15 min. Using a 1 cm cuvette, use the corresponding sample solution without aluminum nitrate solution as a blank for calibration, and measure the absorbance at a wavelength of 510 nm. Calculate the mass of rutin in the sample solution according to the standard curve.

[0149] The total flavonoid content was determined by the aluminum nitrate colorimetric method, and the rutin mass of the sample solution was calculated by comparing it with the rutin standard curve. The total flavonoid content (calculated as rutin) ω (μg / g) in the sample was calculated according to the following formula (1):

[0150] ;

[0151] Where m1 is the total flavonoid mass of the sample solution calculated from the working curve, in milligrams (mg);

[0152] V—The extraction volume of the sample, in milliliters (mL);

[0153] V1—The volume of sample taken during the determination, in milliliters (mL);

[0154] M—Mass of the sample, in grams (g).

[0155] Total phenol content (TPC)

[0156] Accurately weigh 0.5g of sample, add 10mL of 80% methanol solution, mix well, and sonicate at room temperature in the dark for 20min with power set to 200W and water bath temperature set to 50℃. Cool to room temperature. Then centrifuge at 4000rpm for 10min, take the supernatant and dilute to 25mL for later use.

[0157] The total phenol content was determined by the Folin-Ciocalteu colorimetric method, and the content of total polyphenol compounds was quantitatively determined by comparing with the gallic acid standard curve.

[0158] 1. Pipette 0.25 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, and 3.5 mL of gallic acid standard stock solution into 50 mL volumetric flasks, respectively, and dilute to the mark with water to obtain gallic acid standard solutions with mass concentrations of 0.025 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, 0.30 mg / mL, and 0.35 mg / mL, respectively.

[0159] 2. Take 0.2 mL of gallic acid standard solution of each concentration and place it into a 10 mL colorimetric tube. Add 0.5 mL of Folin-Ciocalteu reagent to each tube, shake well, and let stand for 3 min. Add 1.5 mL of 10.0% sodium carbonate solution to each tube, and dilute to the mark with water. Let stand at room temperature in the dark for 20 min.

[0160] 3. Using 75% ethanol as a blank control, the absorbance was measured sequentially at a wavelength of 765 nm using a spectrophotometer. The standard curve and linear equation were plotted with the mass concentration (mg / mL) of gallic acid solution from 0.025 mg / mL to 0.35 mg / mL in Method 1 as the x-axis and the absorbance as the y-axis.

[0161] 4. Pipette 0.2 mL of the sample solution and the blank sample solution into 10 mL colorimetric tubes, add 0.5 mL of Folin-Ciocalteu reagent, shake well, and let stand for 3 min. Then add 1.5 mL of 10.0% sodium carbonate solution, mix well, and dilute to the mark with water. Let stand at room temperature in the dark.

[0162] like Figure 5 As shown, where, Figure 5 (a) represents the total phenol content; Figure 5 (b) represents the total flavonoid content; Figure 5 (c) represents reducing power; Figure 5 (d) DPPH scavenging ability. During fermentation, microbial enzymatic hydrolysis releases more phenols and flavonoids, increasing the total phenol content of XSDF / XIDF by 125.48% and 57.42% respectively, and the total flavonoid content by 61.79% and 105.99% respectively compared to the enzymatic method. The DPPH free radical scavenging ability is close to 100% at concentrations of 4 mg / mL (XSDF) and 10 mg / mL (XIDF), and the total reducing power is significantly higher than that of the enzymatic product. It can be used as a natural antioxidant in the fields of health products and functional beverages.

[0163] 4. Regarding metabolic regulation performance (adsorption capacity).

[0164] (1) Glucose adsorption capacity (GAC)

[0165] Mix 0.1 g of SDF and IDF with 10 mL (80 mmol / L) of glucose solution. Incubate in a 37°C air bath for 4 h, then centrifuge the mixture at 4000 rpm for 20 min. Collect the supernatant and determine its glucose concentration using the DNS colorimetric method.

[0166] ;

[0167] Wherein, C1 is the glucose concentration (mg / mL) of the supernatant of the blank group, C2 is the glucose concentration (mg / mL) of the supernatant of the sample group, W0 is the mass (g) of SDF, and V0 is the volume (mL) of the supernatant.

[0168] (2) Determination of glucose delayed adsorption index (GDRI)

[0169] Mix 0.2 g of SDF with 10 mL of glucose solution (100 mmol / L) and place the mixture in a dialysis bag (8000-14000 MWCO). Then, place the bag in a beaker containing 200 mL of deionized water. Incubate the beaker in a 37°C water bath for 10, 20, 30, 40, 50, and 60 min. After each incubation, collect 2 mL of the dialysate and determine the glucose content using the DNS method. Calculate the GDRI using the following formula:

[0170] ;

[0171] Wherein, G1 is the glucose content (mg) of the sample group and G2 is the glucose content (mg) of the blank group.

[0172] (3) Cholesterol adsorption capacity (CBC)

[0173] Fresh egg yolks were placed in a 500mL beaker, and 9 times their volume of distilled water was added and thoroughly stirred into an emulsion. 0.2g of SDF and 0.15g of IDF samples were placed in a 100mL Erlenmeyer flask, and 25mL of diluted egg yolk solution was added and stirred until homogeneous. The pH of the mixture was adjusted to 2.0 (simulating the stomach environment) and 7.0 (simulating the intestinal environment). The mixture was placed in a constant temperature shaking incubator and incubated at 37℃ for 2 hours. Then, it was centrifuged at 5000 rpm for 20 minutes, and the supernatant was collected. 0.2mL of the supernatant was diluted with 10 times its volume of distilled water, and the cholesterol concentration was determined using the o-phthalaldehyde method. The calculation method is as follows:

[0174] ;

[0175] Where C0 is the cholesterol content of the diluted egg yolk, C1 is the cholesterol content after adding SDF, and M0 is the amount of sample used.

[0176] (4) Sodium cholate adsorption capacity (SCAC)

[0177] In vitro adsorption experiments were conducted to simulate sodium cholate adsorption on dietary fiber. 0.1 g of each sample was mixed with 10 mL of NaCl solution (0.15 M, pH 7.0) and 0.01 g of sodium cholate in a 100 mL Erlenmeyer flask. The mixture was placed in a constant-temperature air bath shaker at 37°C and 150 rpm. Aliquots (1 mL) were collected from the mixture at 0, 30, 60, 90, 120, 150, 180, and 210 minutes during incubation. The sodium cholate concentration was obtained using the furfural method, referring to a standard curve. The calculation method is as follows:

[0178] ;

[0179] Where C0 is the initial concentration of sodium cholate, C1 is the concentration of sodium cholate after adsorption, and m0 is the amount of sample used.

[0180] like Figure 6 As shown, where, Figure 6 (a) represents the cholesterol adsorption capacity at pH=2; Figure 6 (b) represents the cholesterol adsorption capacity at pH=7; Figure 6 (c) represents the glucose delayed adsorption index; Figure 6 (d) represents the adsorption capacity of sodium cholate; Figure 6 (e) represents the glucose adsorption capacity at pH=2; Figure 6 (f) Glucose adsorption capacity at pH=7. In a simulated gastrointestinal environment, XSDF's adsorption capacity for cholesterol (36.595 mg / g at pH 2.0, 33.52 mg / g at pH 7.0), sodium cholate (33.085 mg / g), and glucose (119.46 mg / g in the intestinal environment) are all significantly superior to those of enzymatic products (MSDF / MIDF). Furthermore, XSDF has the highest glucose dialysis delay index (GDRI) of 71.68%, effectively delaying glucose absorption and providing a core raw material for the development of functional foods that assist in regulating blood sugar and blood lipids. Therefore, the soluble dietary fiber (XSDF) and insoluble dietary fiber (XIDF) prepared from *Ulva prolifera* by fermentation in this invention have greater potential for metabolic regulation compared to enzymatic methods (MSDF and MIDF).

[0181] like Figure 7 As shown, where, Figure 7 (a) and (b) are electron microscope images of the soluble dietary fiber (XSDF) and insoluble dietary fiber (XIDF) prepared in this invention, respectively. Figure 7(c) and (d) are electron micrographs of soluble dietary fiber (MSDF) and insoluble dietary fiber (MIDF) prepared by enzymatic method, respectively. After dispersion, the samples were uniformly attached to the surface of conductive carbon ribbon; to improve conductivity, the samples were subjected to ion sputtering pretreatment and gold plating modification in sequence. The morphology of the samples was characterized and analyzed using a Hitachi (Japan) field emission scanning electron microscope (model: SU8600), and observation and image acquisition were completed at 3000x magnification. The adsorption mechanism is as follows:

[0182] The reason why fermented DF has an enhanced ability to adsorb glucose, cholesterol, and sodium cholate is as follows: Figure 7 As shown, the Bacillus sp. KICET-3 strain used for fermentation can secrete cellulase. Cellulase specifically hydrolyzes the β-1,4-glycosidic bonds in dietary fiber, breaking down the originally dense cellulose macromolecular chains and generating more short-chain oligosaccharides and branched polysaccharide fragments, resulting in a more porous structure. Furthermore, DF contains natural groups such as uronic acid, aldehyde, and phenolic groups, and the porous structure exposes more of these groups. For glucose molecules, this change increases the hydrogen bonding sites between DF and glucose molecules and enhances their electrostatic interaction, ultimately improving glucose adsorption capacity. For hydrophobic cholesterol molecules, the porous structure significantly expands their contact area with cholesterol, thereby enhancing the hydrophobic interaction and van der Waals forces between them. Sodium cholate molecules are amphiphilic; their polar head can form hydrogen bonds with exposed active groups, generating electrostatic interactions, while their hydrophobic tail hydrophobically binds to the porous fiber structure, a dual effect that jointly enhances adsorption efficiency. Therefore, during fermentation, enzymatic degradation enables the structural reconstruction of dietary fiber, increasing its specific surface area and exposing active sites, while optimizing its interaction with target molecules of different properties, thereby systematically enhancing the adsorption performance of dietary fiber for glucose, cholesterol, and sodium cholate.

[0183] 5. Sustainability Analysis

[0184] With the continued widening of raw material supply gaps and the gradual improvement of public awareness of environmental protection, sustainable product development has become a core concern for various industries. In the context of contemporary social development, the research and development and implementation of sustainable products have crucial practical value and strategic significance. At its root, this trend is directly related to the increasingly severe global ecological and environmental problems, and stems from the more stringent comprehensive requirements placed on enterprises by consumers, investors, and government regulatory departments regarding their economic output, social value creation capabilities, and environmental impact management levels.

[0185] To more effectively assess the sustainability of fermentation and enzymatic extraction methods for seaweed dietary fiber, we adopted the Overall Sustainability Footprint (OSF) approach. Based on the principles of ecological sustainability, economic sustainability, and social sustainability, we compared and analyzed the comprehensive sustainability levels of the two extraction technologies in terms of raw material utilization and the process itself from multiple dimensions.

[0186] We selected eight specific factors for evaluation, including Green Friendly (GF), SDF Extraction Rate (SE), Waste Utilization (WU), Ease of Preparation (SP), Safety (SF), Cost (CT), Technological Scalability (TP), and Public Awareness (PC). Performance for each factor was assessed on a three-tiered basis, scored according to a rating scale of Low (L, i=1), Medium (M, i=2), and High (H, i=3). Notably, within the "Cost" factor, the negative impact of cost on material sustainability corresponds to ranking values ​​i=3, i=2, and i=1 for L, M, and H, respectively. The Overall Sustainability Footprint (OSF) was calculated based on the reported sustainability impacts using the following formula.

[0187] ;

[0188] Where “j” represents sustainability and “i” represents the corresponding ranking score.

[0189] We compared the previously studied methods of enzymatic extraction of dietary fiber from pear pomace, ultrasound-assisted extraction of dietary fiber from mulberry, three-phase separation extraction of dietary fiber from pear pomace, and Bifidobacterium fermentation extraction of dietary fiber from millet bran with Bacillus fermentation and enzymatic extraction of dietary fiber from Ulva prolifera.

[0190] like Figure 8 As shown, this invention demonstrates outstanding green sustainability, balancing ecological and economic benefits. Existing chemical extraction methods easily introduce impurities and pollute the environment, physical methods are energy-intensive, and traditional enzymatic methods are costly and have low waste utilization rates. This patent offers multiple sustainability advantages: Eco-friendly: Using *Ulva prolifera*, a species affected by green tides, as raw material, it achieves "disaster resource utilization," reducing the manpower and material resources required for *Ulva prolifera* harvesting and disposal; the fermentation process is free of chemical reagent pollution, and the product has no harmful residues, meeting green production standards. Sustainable process: The overall sustainability footprint (OSF) reaches 83.3%, higher than existing processes such as enzymatic extraction of *Ulva prolifera* (DF) (75%) and ultrasound-assisted extraction of mulberry (DF) (70.83%); the operation process is simple (no complex precision equipment required), the technology is highly scalable, and suitable for implementation by small and medium-sized enterprises.

[0191] High economic value: Compared with existing technologies, this patent reduces raw material costs while increasing the added value of product functions, which can promote the transformation of seaweed from "waste" to high-value functional food raw material, forming a virtuous cycle of "ecological governance - resource utilization - industrial upgrading".

[0192] In summary, this invention is the first to apply Bacillus sp. KICET-3 strain to the extraction of dietary fiber from Ulva prolifera. Utilizing the complex enzyme system of cellulase and hemicellulase secreted by the strain, an integrated "extraction-modification" process for dietary fiber is achieved: enzymatic hydrolysis breaks the β-1,4-glycosidic bonds of cellulose, transforming the fiber structure from a dense, blocky form to a loose, porous one. This not only improves extraction efficiency but also enhances adsorption performance by exposing active groups such as hydroxyl and carboxyl groups. This "microbial enzymatic hydrolysis-driven structural reconstruction" technical approach provides a valuable research paradigm for the high-value utilization of other algae (such as Ulva prolifera and kelp) or agricultural waste (such as soybean residue and rice bran), promoting the upgrading of dietary fiber extraction technology towards "high efficiency, greenness, and functional orientation." It has significant economic benefits and is suitable for industrial-scale application.

[0193] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should be protected by the present invention.

Claims

1. A Bacillus strain, classified as Bacillus sp. KICET-3, characterized in that... The strain was isolated from naturally fermented seaweed, and the specific isolation process included the following steps: I. Source of the strain: (1) Fresh seaweed harvested from the Qingdao sea area was placed in a sterilized iron barrel and fermented naturally for 30 days; (2) Collect samples from the middle part of the fermented seaweed and store them at 4℃ for later use; II. Strain screening: (3) Add the sample to a test tube containing sterile water at a ratio of 1:9 (w / v) and mix with a shaker for 30 min. (4) Serial dilution of sterile water to 10 -2 -10 -8 Spread the diluted solution onto cellulose Congo red medium, pick out colonies that produce a clear zone, and repeat the streak culture on potato dextrose agar (PDA) medium until a pure single strain is obtained. III. Strain Identification: (5) The pure single strain obtained in step (4) was subjected to DNA extraction, PCR amplification and sequencing. Homologous sequences were searched using the BLAST tool of NCBI. A phylogenetic tree was constructed using the neighbor connection algorithm of MEGA11.0 software and identified as Bacillus sp. KICET-3. IV. Strain Expansion Culture and Preservation: (6) Inoculate the pure single strain obtained in step (4) onto LB liquid medium and culture with shaking at 37°C and 150 rpm to control the strain concentration to 10. 8 -10 9 CFU / mL was used to obtain the seed culture; (9) Take the bacterial solution and 40% glycerol (by mass) of sterilization treatment and mix them at a ratio of 1:1 (v / v), and store them in a refrigerator at -80℃.

2. The application of Bacillus as described in claim 1 in the extraction of dietary fiber from Ulva prolifera.

3. The application according to claim 2, characterized in that, The extraction process of dietary fiber from *Ulva prolifera* includes the following steps: I. Pretreatment of Ulva prolifera: S1. Wash the fresh seaweed, dry it at 65-80℃, pulverize it to 100 mesh, and sterilize it with high pressure steam at 121℃, 0.1MPa, and 20min to obtain the seaweed raw material for later use. II. Fermentation and extraction of dietary fiber from seaweed: S2. Preparation before fermentation: Inoculate the (Bacillus sp.) KICET-3 strain preserved in step (9) of claim 1 into the seed culture medium from the glycerol tube, and culture with shaking at 35-38℃ and 130-155rpm until the strain concentration reaches 10. 8 -10 9 CFU / mL was used to obtain the seed culture; S3. Fermentation process: Mix the raw material of Ulva prolifera with distilled water at a liquid-to-solid ratio of 30-33, add seed liquid with a concentration of 2-5% (v / w), adjust the pH to 6-7.5, and culture with shaking at 35-38℃ and 130-155rpm for 28-36h to obtain the fermented Ulva prolifera mixture. S4: Dietary fiber extraction: SDF and IDF were extracted based on the AOAC enzyme gravimetric method. Thermostable α-amylase was added to the fermented Ulva lactuca mixture, and the pH was adjusted to 5.5-6.

5. The mixture was stirred for 45 min in a 65℃ water bath and then cooled to room temperature. S5: Add papain and adjust the pH to 6-6.

5. Stir for 120 min in a 60℃ water bath. Boil for 10 min to inactivate the enzyme and filter to separate the filtrate from the residue. S6: Add a certain amount of 95% ethanol to the filtrate and mix overnight. Collect the precipitate and freeze-dry to obtain fermented soluble dietary fiber (XSDF). Wash the filter residue twice with 95% ethanol and acetone alternately and freeze-dry to obtain fermented insoluble dietary fiber (XIDF).

4. The application according to claim 3, characterized in that: In step S1, the drying temperature of the fresh seaweed after washing is 70°C.

5. The application according to claim 3, characterized in that: In S2, the shaking culture conditions are 37°C and 150 rpm.

6. The application according to claim 3, characterized in that: In step S3, the liquid-to-solid ratio of the *Ulva prolifera* raw material to distilled water was 32.061, the concentration of the added seed liquid was 3.282% (v / w), the pH was adjusted to 6.938, and the mixture was cultured at 37°C and 150 rpm for 30.294 h with shaking.

7. The application according to claim 3, characterized in that: In step S6, the volume ratio of added ethanol to filtrate is 1:4.

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