Laver lysing extract as well as preparation method and application thereof

Abstract

The invention relates to a laver lysing extract as well as a preparation method and application thereof, and belongs to the technical field of biology. According to the method, a strain is obtained from a separating medium in the porphyra yezoensis drying processing process, it is tested that the strain can secrete lysozyme capable of rapidly dissolving porphyra cell walls, the lysozyme comprises amylase and protease, and intracellular phycobiliprotein, low-molecular sulfuric acid galactan and the like can be released after cell dissolution; the purpose that laver cells are lysed and intracellular components are efficiently released is achieved, and the strain is identified as bacillus tequilensis T7. The laver lysate extract prepared by the invention has the effects of resisting oxidation, preventing ultraviolet radiation and the like, and can be applied to the fields of cosmetics, functional foods, beverages and the like.

Description

Technical Field

[0001] This invention relates to a laver cell lysate extract, its preparation method and application, belonging to the field of biotechnology. Background Technology

[0002] Seaweed (Algae) is a general term for marine algae. The most common seaweeds include brown algae (kelp, wakame, Sargassum, etc.), red algae (porphyria, agar, Gracilaria, carrageenan, Euphorbia, etc.), and green algae (Ulva, Ulva, etc.). Red algae mostly grow in the ocean, are widely distributed, and have many species, estimated at approximately 558 genera and over 3700 species, further divided into two subclasses: Bangioideae and Florideae. Red algae have significant economic value, serving not only as food but also as raw materials for medicine, textiles, and food industries. Porphyria is rich in protein, polysaccharides and gums, dietary fiber, vitamins, and abundant inorganic elements. Porphyria polysaccharides (containing 20-40%) exist in the form of porphyrin, possessing good biological activity and significant development value. However, due to their large molecular weight, high viscosity, and insolubility in water, their development and application are limited. Therefore, reducing the molecular weight and viscosity of laver polysaccharides has become the key to the development and utilization of laver polysaccharides.

[0003] Biological fermentation refers to the use of microorganisms to produce metabolic enzymes under aerobic or anaerobic conditions, which are then used to degrade macromolecular compounds. This method is widely used in the food, biological, and chemical industries. Its advantages include that the fermentation process is generally carried out at ambient temperature and pressure, making it safe, efficient, and requiring relatively simple conditions. Currently, the research and utilization of fermented products and extracts in fields such as nutrition and health care, clinical medicine, beauty and skincare, weight loss, health maintenance, and sub-health management are receiving considerable attention. Based on these characteristics, microbial fermentation technology is increasingly attracting attention. Microbial fermentation lysation is an effective means of obtaining lysates, which is of great significance for improving the availability and product characteristics of laver. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a lysing extract of the red algae *Porphyra yezoensis*, its preparation method, and its applications. This invention involves screening a collection of lysing microorganisms from the separation liquid during the drying process of *Porphyra yezoensis*, and obtaining a lysing bacterium through isolation and purification. This strain was identified as *Bacillus tequilensis* T7. The fermentation broth of this strain was used to incubate *Porphyra yezoensis* for 3-5 hours to induce lysing. The cell walls of the *Porphyra yezoensis* dissolved, the viscosity of the algal liquid decreased, and the cytoplasm separated from the insoluble matter. The cytoplasm obtained was red in color and contained a large amount of phycobiliproteins and specific *Porphyra yezoensis* oligosaccharides. This lysing extract can be further processed into *Porphyra yezoensis* lysing extract stock solution, *Porphyra yezoensis* lysing extract paste, and *Porphyra yezoensis* lysing extract powder. This *Porphyra yezoensis* lysing extract has anti-radiation and antioxidant effects and can be applied in cosmetics, functional foods, etc.

[0005] The technical solution of the present invention is as follows:

[0006] A strain of Bacillus tequilensis T7 was deposited at the China Center for Type Culture Collection (CCTCC) on November 14, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242561.

[0007] The application of Bacillus tekirulatus T7 in dissolving the cell walls of laver.

[0008] According to a preferred embodiment of the present invention, the laver includes Porphyra tenuifolia and Porphyra yezoensis.

[0009] A method for preparing a laver cell lysate extract involves mixing the supernatant from Bacillus tekirae T7 fermentation with laver liquid, incubating for cell lysation, separating the solid and liquid phases, and obtaining the supernatant, which is the laver cell lysate extract.

[0010] According to a preferred embodiment of the present invention, the laver includes Porphyra tenuifolia and Porphyra yezoensis.

[0011] According to a preferred embodiment of the present invention, the preparation method of the fermentation supernatant of Bacillus tekirae T7 is as follows: Bacillus tekirae T7 is activated and cultured on a solid culture medium, then inoculated into a liquid culture medium, seed culture is performed to obtain a seed liquid, the seed liquid is inoculated into a fermentation culture medium, fermentation culture is performed to obtain a fermentation broth, and after separation and sterilization, the supernatant is retained to obtain the fermentation supernatant of Bacillus tekirae T7.

[0012] More preferably, the solid culture medium is LB solid culture medium, and the liquid culture medium is LB liquid culture medium.

[0013] More preferably, the fermentation medium comprises: 5 g / L tryptone, 2 g / L casein, 5 g / L yeast extract, 0.5 g / L soluble starch, 10 g / L sodium chloride, and pH adjusted to 7.2.

[0014] More preferably, the volume ratio of the inoculated seed culture to the fermentation medium is 0.5 to 1:100.

[0015] Further preferred, the OD of the obtained fermentation broth after 10-fold dilution 600 =0.7~0.8.

[0016] More preferably, the temperatures for the activation culture, seed culture, and fermentation culture are all 35–40°C.

[0017] More preferably, the separation and sterilization are carried out by centrifugation at a speed of 4000-5000 rpm for 20-30 minutes.

[0018] According to a preferred embodiment of the present invention, the method for preparing the laver liquid is as follows: dried laver is pulverized and placed in water, with the mass ratio of laver to water being 1:20-30. After mixing and sterilization, the laver liquid is obtained.

[0019] According to a preferred embodiment of the present invention, the mass ratio of the Bacillus tekirae T7 fermentation supernatant to the laver liquid is 2-7:100.

[0020] According to a preferred embodiment of the present invention, the temperature for heat preservation and lysis is 40–50°C, and the time is 3–5 hours.

[0021] According to a preferred embodiment of the present invention, lysis is terminated when the solid content in the reaction system is >3%.

[0022] According to a preferred embodiment of the present invention, the solid-liquid separation is performed by centrifugation at a speed of 4000-5000 rpm for 20-30 minutes.

[0023] A laver cell lysate extract prepared according to the above method.

[0024] The above-mentioned laver lysate extract can be in the form of stock solution, paste, or powder. Laver lysate extract, after being treated with preservatives, becomes laver lysate extract stock solution; laver lysate extract, after being concentrated into a paste and treated with preservatives, becomes laver lysate extract paste; laver lysate extract, after being spray-dried and treated with preservatives, becomes laver lysate extract powder.

[0025] The above-mentioned laver lysate extract is used in the preparation of products with antioxidant or anti-ultraviolet radiation properties.

[0026] According to a preferred embodiment of the present invention, the products include: cosmetics, food, beverages, and health products.

[0027] Beneficial effects:

[0028] 1. This invention obtains a strain of bacteria from the separation liquid during the drying process of Porphyra yezoensis. The test results show that this strain can secrete lysing enzymes that rapidly dissolve the cell walls of Porphyra yezoensis, including amylase and protease. After lysis, it can release intracellular phycobiliproteins and low-molecular-weight sulfated galactan, etc., to achieve the purpose of lysing Porphyra yezoensis cells and efficiently releasing intracellular components. This strain has been identified as Bacillus tequilensis T7.

[0029] 2. This invention provides a method for preparing laver extract using cell lysis technology. This invention utilizes the fermentation broth of *Bacillus terrestris* T7, a laver lysing bacterium, to liquefy laver and achieve cell lysis, which can greatly release intracellular active substances from laver and improve the extraction rate.

[0030] 3. The laver extract prepared by this invention has antioxidant and UV protection effects and can be applied to cosmetics, functional foods, beverages and other fields. Attached Figure Description

[0031] Figure 1 This is a high-performance liquid chromatogram of a sample solution derived from laver polysaccharides; in the figure, Lac represents lactose and Gal represents galactose.

[0032] Figure 2 This is a high-performance liquid chromatogram of a seaweed polysaccharide solution.

[0033] Figure 3 The curve shows the DPPH free radical scavenging rate of the lysate extract of Porphyra yezoensis.

[0034] Figure 4 Bar chart showing the radiation resistance of different concentrations of *Porphyra yezoensis* lysate extracts against UVB radiation in *Escherichia coli*.

[0035] Figure 5 Bar chart showing the radiation resistance of different concentrations of *Porphyra yezoensis* lysate extract to *Brewery yeast* against UVB radiation. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the materials and reagents involved in the embodiments are all commercially available products. Unless otherwise specified, the experimental operations involved in the embodiments are all conventional experimental methods in the art. Unless otherwise specified, the percentages involved in the embodiments are all mass percentages.

[0037] Culture media involved in the examples:

[0038] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, water as solvent, pH adjusted to 7.4.

[0039] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, solvent water, adjust pH to 7.2.

[0040] Seawater LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, agar powder 15 g / L, seawater as solvent, pH adjusted to 7.2.

[0041] Fermentation medium: 5 g / L tryptone, 2 g / L casein, 5 g / L yeast extract, 0.5 g / L soluble starch, 10 g / L sodium chloride, water as solvent, pH adjusted to 7.2.

[0042] Example 1: Isolation and Screening of Lysogenic Microorganisms from Porphyra

[0043] The laver-lysing microorganisms are obtained by separating, purifying, and identifying the microorganisms from the separation liquid during the drying and processing of Porphyra yezoensis Ueda. The specific operation process is as follows: At the laver farming and processing site in Ganyu District, Lianyungang City, Jiangsu Province, China, laver is cut along the rope with ordinary scissors; this process is called "cutting laver." Next is the washing stage, to obtain clean, sand-free laver. This is achieved by using the force of water and the agitation of a spiral machine to wash layer by layer, with sand filtered through an hourglass at the bottom to ensure the laver is clean and sand-free. After washing, the laver is dehydrated, and the washed laver and solution are separated. The separation liquid from the laver processing is collected, concentrated at 80℃ for 30 minutes, and then purified on seawater LB solid medium to obtain strain T7, which was identified as Bacillus tequilensis. The colonies of this strain are white, smooth, opaque, with irregular edges, a slightly raised center, and a diameter of approximately 3 mm.

[0044] Bacillus tekirae T7 was inoculated on LB solid medium containing 1% starch and incubated at 40°C for 24 hours. The amylase clear zone was measured to be >3.0 cm, indicating that Bacillus tekirae T7 can produce amylase and secrete it extracellularly.

[0045] Bacillus tekirae T7 was seeded on LB solid medium containing 1% casein and incubated at 40°C for 24 hours. The protein clear zone was measured to be >3.7 cm, indicating that Bacillus tekirae T7 can produce protease and secrete it extracellularly.

[0046] Bacillus tequilensis T7 was deposited on November 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242561.

[0047] Example 2: Preparation of Bacillus tekiria T7 fermentation broth

[0048] The *Bacillus tekirii* T7 strain obtained in Example 1 was inoculated onto LB solid medium and incubated at 40°C for 24 hours to activate it. The activated strain was then inoculated into 100 mL of LB liquid medium and incubated at 40°C for 24 hours to prepare a seed culture. This seed culture was then inoculated into 100 mL of fermentation medium at a volume ratio of 0.5:100. After inoculation, the culture was carried out at 36°C for 24 hours. The fermentation broth was then diluted 10-fold to obtain the OD... 600 Fermentation was stopped when the temperature reached 0.7-0.8℃, and the temperature was lowered to 10℃. Then, the fermentation broth was centrifuged at 4000 rpm for 30 minutes to separate and sterilize it. The supernatant was retained to obtain the microbial fermentation supernatant with cytolytic activity against laver.

[0049] Example 3: Preparation of Porphyra lysate extract

[0050] Dried laver was pulverized into powder and mixed with water at a mass ratio of 1:25. The mixture was sterilized at 90°C for 10 minutes, cooled to 45°C, and then added to the microbial fermentation supernatant prepared in Example 2 at a mass ratio of 5%. The mixture was stirred and kept warm for 3 hours. When the viscosity of the algal solution decreased significantly and the solution turned red, and the solid content was >3%, the reaction was terminated by high-temperature treatment at 100°C for 10 minutes. The solution was then rapidly cooled to 10°C and centrifuged at 4000 rpm for 30 minutes to separate the solids and liquids. The supernatant was obtained as the laver cell lysate extract with an extraction rate >20%.

[0051] The extraction rate (%) is calculated as follows: (dry weight of laver lysate extract / dry weight of laver) × 100%

[0052] Laver cell lysate extract stock solution: The above supernatant is subjected to preservation treatment and packaging to obtain laver cell lysate extract stock solution.

[0053] Laver cell lysate extract: The above supernatant was concentrated 10 times into a paste, and then subjected to preservation treatment and packaging to obtain laver cell lysate extract.

[0054] Porphyra lysate extract powder: The above supernatant is spray-dried to achieve a moisture content of <8%, and then packaged to obtain porphyra lysate extract powder.

[0055] Example 4: Characteristic polysaccharide extraction from laver lysate extract

[0056] Add 3 times the volume of 95% ethanol to the laver lysate extract obtained in Example 3 above, and precipitate overnight at 4°C; centrifuge at 4000 rpm for 30 min, and collect the precipitate; add an equal volume of ultrapure water to the precipitate to reconstitute, centrifuge at 4000 rpm for 30 min, and collect the supernatant; freeze-dry the supernatant to obtain laver polysaccharide.

[0057] Example 5: Determination of characteristic polysaccharides in laver lysate extract

[0058] (1) Total sugar determination (phenol-sulfuric acid method): The polysaccharide extracted from the seaweed in Example 4 was prepared into a sample solution of 0.1 mg / mL. 0.5 mL of the sample solution was mixed with an equal volume of 6% phenol solution, and then 2.5 mL of concentrated sulfuric acid was added. After mixing thoroughly and cooling to room temperature, the OD value was measured at 490 nm.

[0059] (2) Protein determination (Coomassie Brilliant Blue G-250 method): The polysaccharide extracted from the seaweed in Example 4 was prepared into a sample solution of 10 mg / mL. 0.1 mL of the sample solution was mixed with 5 mL of Coomassie Brilliant Blue G-250, and allowed to stand at room temperature for 2 min. The OD value was then measured at 595 nm.

[0060] (3) Determination of uronic acid (m-hydroxybiphenyl method): The polysaccharide extracted from the seaweed in Example 4 was prepared into a sample solution of 1 mg / mL. 0.5 mL of the sample solution was mixed with 3 mL of sodium tetraborate sulfuric acid solution of 4.78 mg / mL, boiled in a water bath for 5 min, cooled to room temperature in a cold water bath, and then 50 μL of m-hydroxybiphenyl solution of 1.5 mg / mL was added. The mixture was thoroughly mixed and the OD value was measured at 520 nm.

[0061] (4) Determination of sulfate groups (barium sulfate turbidimetric method): Take 10 mg of the polysaccharide extracted from the seaweed in Example 4, prepare a solution of 5 mg / mL with 1M HCl solution, acid hydrolyze at 100℃ for 4 h, cool to room temperature, take 0.2 mL of sample hydrolysate and mix it with 3.8 mL of 3% trichloroacetic acid solution, then add 1 mL of 1% barium chloride-gelatin solution, and use 1 mL of 0.5% gelatin solution as the sample background control. Mix thoroughly and measure the OD value at 360 nm.

[0062] Based on the above measurements, the total sugar content of the laver polysaccharide extracted in Example 4 was 60%, the protein content was 2.6%, the uronic acid content was 5%, and the sulfate group content was 20%, all of which are mass percentages.

[0063] Example 6: Identification of the characteristic polysaccharide and monosaccharide composition of *Porphyra yezoensis* lysate extract

[0064] Specifically, the monosaccharide composition of the laver polysaccharides extracted in Example 4 above was analyzed, and the monosaccharide composition of the laver polysaccharides was determined by the PMP-pre-column derivatization method:

[0065] (1) Sample acid hydrolysis: The extracted laver polysaccharide was prepared into a sample solution of 4 mg / mL. The sample solution was mixed with an equal volume of 4M trifluoroacetic acid solution and acid hydrolyzed in a water bath at 95℃ for 6 h. After cooling to room temperature, the trifluoroacetic acid was removed by rotary evaporation. Then, 1 mL of methanol was added and rotary evaporation was repeated 3 times. The pH was adjusted to neutral with 0.3M NaOH solution and 0.3M HCl solution, and the volume was adjusted to 3 mL with distilled water.

[0066] (2) Derivatization: 450 μL of the sample solution after acid hydrolysis in step (1) and 1 mg / mL lactose and galactose standard solutions were mixed with 50 μL of 1 mg / mL internal standard lactose solution. 200 μL of the mixture containing the internal standard was mixed with 420 μL of 50% PMP-methanol solution and 420 μL of 0.3 M NaOH solution. The mixture was heated in a water bath at 80 °C in the dark for 1 h. After the reaction was completed and cooled to room temperature, 420 μL of 0.3 M HCl solution was added for neutralization. Dichloromethane was added for extraction. The supernatant was collected. The process was repeated 3 times to obtain the derivatized sample solution and standard solution.

[0067] (3) The derivatized sample solution and standard solution were filtered through a 0.22 μm organic phase membrane. 20 μL of each solution was loaded onto an Agilent 1100 HPLC ZORBAX Eclipse×XDB-C18 column. Mobile phase A was 0.05 M phosphate buffer (pH 6.9), and mobile phase B was acetonitrile (A:B = 87:13). Isocratic elution was performed at a flow rate of 1 mL / min, a detection wavelength of 245 nm, and a column temperature of 25 °C. The HPLC chromatogram of the sample solution is shown below. Figure 1 As shown in the analysis results, the monosaccharide in the characteristic polysaccharide of the laver extract is galactose.

[0068] Example 7: Determination of the molecular weight of characteristic polysaccharides in laver lysate extract

[0069] The polysaccharide extracted from *Porphyra yezoensis* in Example 4 was prepared into a 1 mg / mL sample solution, passed through a 0.22 μm aqueous membrane, and 20 μL was loaded onto the membrane. An Agilent 1260TSK gel GMPWXL GPC column was used, with the mobile phase being 0.01 M phosphate buffer (pH 7.0) containing 0.2 M sodium nitrate, the flow rate being 0.5 mL / min, and the column temperature being 45 °C. The high-performance liquid chromatogram of the sample solution is shown below. Figure 2 As shown, the molecular weight of the laver polysaccharide was measured to be 117.62 kDa.

[0070] Example 8: DPPH scavenging activity of laver lysate extract

[0071] 1. Experimental samples and concentrations

[0072] Experimental sample: Powder of laver cell lysate extract prepared in Example 3.

[0073] Sample concentrations: The lysozyme extract powder of seaweed was dissolved in water to prepare sample solutions with concentrations of 15 mg / mL, 12 mg / mL, 9 mg / mL, 6 mg / mL, and 3 mg / mL.

[0074] 2. Experimental Methods

[0075] Set up sample tubes (T), sample background tubes (T0), DPPH tubes (C), and solvent background tubes (C0) using 10mL test tubes. For each test concentration of the sample, sample tubes (T) should be set up in 3 parallel tubes, and DPPH tubes (C) should also be set up in 3 parallel tubes.

[0076] Add 2 mL of sample solution of the same concentration to both the sample tube (T) and the sample background tube (T0). Replace the DPPH tube (C) and the solvent background tube (C0) with 2 mL of water. Then add 2 × 10⁻⁶ mL of sample solution to both the sample tube (T) and the DPPH tube (C). -4 2 mL of mol / L LPPH methanol solution was used, and the sample background tube (T0) and solvent background tube (C0) were replaced with 2 mL of methanol. The mixture was vortexed and reacted at room temperature in the dark for 30 min. The absorbance at 517 nm was then measured using the solvent background tube (C0) as the zero point.

[0077] 3. Result Calculation

[0078]

[0079] T: Absorbance of the sample tube, i.e., the absorbance of the solution after the sample reacts with DPPH;

[0080] T0: Absorbance of the sample substrate tube;

[0081] C: Absorbance of DPPH tube, i.e., absorbance of DPPH solution without sample added;

[0082] C0: Solvent background absorbance.

[0083] 4. Experimental Results and Analysis

[0084] DPPH is a nitrogen radical compound commonly used in the detection of antioxidant activity. Its alcoholic solution is generally purple, with a maximum absorption peak at 517 nm. DPPH can undergo single-electron pairing with substances possessing antioxidant activity, causing the solution to decolorize and reduce absorbance, thus providing a convenient way to determine the antioxidant activity of a sample. Different concentrations of *Porphyra yezoensis* cell lysate extract reacted with DPPH solution, all resulting in a lighter color in the reaction system. Experimental results are as follows... Figure 3As shown in the figure, the DPPH scavenging ability of the porphyria extract exhibits a significant dose-dependent effect. That is, as the concentration of the porphyria extract increases, the DPPH free radical scavenging rate increases significantly. The sample concentration that reaches the half-maximal scavenging rate is approximately 5.33 mg / mL, indicating that it has a good in vitro DPPH scavenging effect.

[0085] Example 9: Evaluation of the protective effect of Porphyra yezoensis lysate extract against UVB radiation in prokaryotes

[0086] 1. Experimental samples and concentrations

[0087] Experimental sample: Powder of laver cell lysate extract prepared in Example 3.

[0088] Sample concentration: Dissolve the seaweed cell lysate extract powder in water to prepare a seaweed cell lysate extract stock solution with a concentration of 10 mg / mL, sterilize by membrane filtration, and set aside for later use.

[0089] 2. Experimental Methods

[0090] Preparation of bacterial suspension: Escherichia coli were picked from the slant and inoculated into LB liquid medium. After 4-8 hours of shaking culture in a constant temperature shaking incubator at 37°C and 180 rpm, the bacterial count was determined by plate counting. The bacterial suspension required for the experiment was prepared by appropriate dilution.

[0091] The mother liquor of *Porphyra yezoensis* lysate extract was mixed with *E. coli* suspension at a certain volume ratio, resulting in final concentrations of *Porphyra yezoensis* lysate extract of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, and 4.0 mg / mL, respectively, and a final concentration of *E. coli* of 10 mg / mL. 9 CFU / mL. Take 5 mL of the above mixture and place it in a sterile 9 cm diameter petri dish. Irradiate with UVB at the center of the UV chamber. A blank control group containing only E. coli suspension was also set up, with a final E. coli concentration of 10. 9 Take 5 mL of CFU / mL and place it in a sterile petri dish with a diameter of 9 cm. Irradiate the dish with UVB at the center of the UV chamber.

[0092] UVB irradiation method: The irradiation was conducted in a dark-box ultraviolet analyzer at a wavelength of 302 nm and an irradiation intensity of 20 mJ / cm². 2 .

[0093] Colony count detection method: plate count method.

[0094] 3. Result Calculation

[0095]

[0096] A i : The number of surviving colonies after adding laver lysate extract;

[0097] A0: Number of surviving colonies without the addition of seaweed lysate extract.

[0098] 4. Experimental Results and Analysis

[0099] Experimental results are as follows Figure 4 As shown, different concentrations of *Porphyra yezoensis* lysate extract all helped enhance the resistance of *E. coli* to UVB radiation, exhibiting a certain dose-dependent effect. Using the number of surviving colonies in the control group without *Porphyra yezoensis* lysate extract as a reference, the radiation resistance rates of different concentrations of *Porphyra yezoensis* lysate extract were 2.80%, 3.33%, 8.65%, and 8.00%, respectively, with the radiation resistance rate gradually stabilizing at higher concentrations. The 2.0 mg / mL *Porphyra yezoensis* lysate extract showed the best radiation resistance effect, with a radiation resistance rate close to 9%, indicating that *Porphyra yezoensis* lysate extract has a good effect on protecting *E. coli* against UVB radiation.

[0100] Example 10: Evaluation of the protective effect of Porphyra yezoensis lysate extract on eukaryotic cells against UVB radiation

[0101] 1. Experimental samples and concentrations

[0102] Experimental sample: Powder of laver cell lysate extract prepared in Example 3.

[0103] Sample concentration: Dissolve the seaweed cell lysate extract powder in water to prepare a seaweed cell lysate extract stock solution with a concentration of 10 mg / mL, sterilize by membrane filtration, and set aside for later use.

[0104] 2. Experimental Methods

[0105] Preparation of bacterial suspension: Brewer's yeast was picked from the slant and inoculated into YPD liquid medium. After being placed in a constant temperature shaking incubator at 28°C and 180 rpm for 12-24 hours, the number of bacteria was determined by plate counting. The bacterial suspension required for the experiment was prepared by appropriate dilution.

[0106] The mother liquor of *Porphyra yezoensis* lysate extract was mixed with the *Saccharomyces cerevisiae* suspension at a certain volume ratio, resulting in final concentrations of *Porphyra yezoensis* lysate extract of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, and 4.0 mg / mL, respectively, and a final concentration of *Saccharomyces cerevisiae* of 10 mg / mL. 6 CFU / mL. Take 5 mL of the above mixture and place it in a sterile 9 cm diameter petri dish. Irradiate with UVB at the center of the UV chamber. A blank control group containing only brewer's yeast suspension was also set up, with a final brewer's yeast concentration of 10 CFU / mL. 6 Take 5 mL of CFU / mL and place it in a sterile petri dish with a diameter of 9 cm. Irradiate the dish with UVB at the center of the UV chamber.

[0107] UVB irradiation method: The irradiation was conducted in a dark-box ultraviolet analyzer at a wavelength of 302 nm and an irradiation intensity of 30 mJ / cm². 2 .

[0108] Colony count detection method: plate count method.

[0109] 3. Result Calculation

[0110]

[0111] A i : The number of surviving colonies after adding laver lysate extract;

[0112] A0: Number of surviving colonies without the addition of seaweed lysate extract.

[0113] 4. Experimental Results and Analysis

[0114] Experimental results are as follows Figure 5 As shown, different concentrations of *Porphyra yezoensis* lysate extract all helped enhance the resistance of brewer's yeast to UVB radiation, exhibiting a certain dose-dependent effect. Using the number of surviving colonies in the control group without *Porphyra yezoensis* lysate extract as a reference, the radiation resistance rates of different concentrations of *Porphyra yezoensis* lysate extract were 5.11%, 8.35%, 9.71%, and 9.91%, respectively. The increase in radiation resistance rate gradually slowed down and tended to stabilize. The 4.0 mg / mL *Porphyra yezoensis* lysate extract showed the best radiation resistance effect, with a radiation resistance rate close to 10%, indicating that *Porphyra yezoensis* lysate extract has a good effect on protecting yeast against UVB radiation.

[0115] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Bacillus tequilensis T7 was deposited at the China Center for Type Culture Collection (CCTCC) on November 14, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20242561.

2. The application of Bacillus tekirulatus T7 as described in claim 1 in dissolving the cell walls of laver.

3. The application as described in claim 2, characterized in that, The laver includes laver and laver.

4. A method for preparing a laver cell-lysate extract, characterized in that, The supernatant of Bacillus tekirae T7 fermentation was mixed evenly with the laver liquid, and after incubation to lyse the cells, the solid and liquid were separated to obtain the supernatant, which is the laver cell lysate extract.

5. The preparation method according to claim 4, characterized in that, The preparation method of the fermentation supernatant of Bacillus tekirae T7 is as follows: Bacillus tekirae T7 is activated and cultured on a solid culture medium, then inoculated into a liquid culture medium, seed culture is carried out to obtain seed liquid, the seed liquid is inoculated into a fermentation culture medium, fermentation culture is carried out to obtain fermentation broth, and after separation and sterilization, the supernatant is retained to obtain the fermentation supernatant of Bacillus tekirae T7.

6. The preparation method according to claim 5, characterized in that, The method for preparing the fermentation supernatant of Bacillus tekirius T7 meets one or more of the following conditions: i. The solid culture medium is LB solid culture medium, and the liquid culture medium is LB liquid culture medium; ii. The fermentation medium consists of: 5 g / L tryptone, 2 g / L casein, 5 g / L yeast extract, 0.5 g / L soluble starch, 10 g / L sodium chloride, and pH adjusted to 7.

2. iii. The volume ratio of the inoculated seed culture to the fermentation medium is 0.5 to 1:100; iv. The OD of the obtained fermentation broth after 10-fold dilution 600 =0.7~0.8; v. The temperatures for the activation culture, seed culture, and fermentation culture are all 35–40°C; vi. The separation and sterilization are carried out by centrifugation at a speed of 4000-5000 rpm for 20-30 minutes.

7. The preparation method according to claim 4, characterized in that, It also meets one or more of the following conditions: 1) The laver mentioned includes laver and laver; 2) The method for preparing the laver liquid is as follows: after pulverizing dried laver, place it in water with a mass ratio of laver to water of 1:20-30, mix well, sterilize, and then obtain the laver liquid. 3) The mass ratio of the Bacillus tekirius T7 fermentation supernatant to the laver liquid is 2-7:100; 4) The temperature for heat preservation and lysis is 40–50°C, and the time is 3–5 hours; 5) When the solid content in the reaction system is >3%, cell lysis ends; 6) The solid-liquid separation is performed by centrifugation at 4000-5000 rpm for 20-30 minutes.

8. A laver cell lysate extract prepared according to the preparation method of claim 4.

9. The use of the laver lysate extract according to claim 8 in the preparation of products with antioxidant or UV radiation resistance.

10. The application as described in claim 9, characterized in that, The products include: cosmetics, food, beverages, and health products.

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