Use of marine actinomycetes of the genus saccharithrix in improving heat tolerance and / or promoting growth of algae

Abstract

The application discloses application of marine actinomycetes of Saccharothrix sp. in improving heat resistance of algae and / or promoting growth of algae, and relates to the technical field of microbial mutation breeding. The marine actinomycetes of Saccharothrix sp. is screened by using a method of ultraviolet mutation. Compared with a wild type, the bacteria can obviously improve the high-temperature resistance of algae, and has the characteristics of promoting the growth of algae. It is indicated that the bacteria strain has good application prospect of improving the heat resistance of algae and / or promoting the growth of algae.

Description

Technical Field

[0001] This invention relates to the field of microbial mutagenesis breeding technology, and more specifically, to the application of marine actinomycetes of the genus *Saccharomyces* in improving the heat resistance of algae and / or promoting algal growth. Background Technology

[0002] Porphyra yezoensis is one of the most economically valuable cultivated seaweeds in my country, accounting for over 70% of the country's total seaweed production annually. It is characterized by its delicious taste and rich nutritional value, making it a popular edible seaweed. Furthermore, Porphyra yezoensis also possesses significant medicinal and ecological value.

[0003] The growing season for laver (Porphyra yezoensis) is approximately from October to March of the following year, with a suitable growth temperature of 16-24℃ and an optimum temperature of around 21℃. In recent years, due to global climate change factors such as the greenhouse effect, frequent high temperatures and warm spells have occurred during the early stages of laver cultivation, causing water temperatures to reach as high as 30℃. These high temperatures damage laver, leading to frequent diseases and ultimately resulting in widespread seedling rot. This high-temperature seedling rot has had a significant impact on the coastal laver cultivation industry, seriously affecting its sustainable development.

[0004] In 1972, Bell and Mitchell proposed the concept of "algal microhabitat," providing a new perspective for understanding the healthy growth of algae. Throughout the long evolution of nature, different bacteria have always lived in the growth environment of algae, and the two groups have complex and unique interactions.

[0005] Marine algae provide a habitat for microorganisms while also secreting large amounts of carbohydrates, lipids, and polypeptides. The organic debris produced after algal death can be used as nutrients by microorganisms in the surrounding environment. The algal microbiome simultaneously provides algae with carbon and nitrogen sources, as well as various important growth factors. These diverse and complex algal microorganisms are not simply pathogens or beneficial bacteria; some can disrupt the normal structure and function of algae, negatively impacting their growth. Probiotics, on the other hand, can promote algal growth by secreting substances related to algal growth, such as vitamins and auxin analogs. Some secondary metabolites can also protect algae under stress.

[0006] However, simply isolating and purifying microorganisms in nature, and selecting the best strains, is unlikely to yield superior strains, and generally cannot meet the needs of production. Microbial breeding, on the other hand, refers to inducing variations in the genetic characteristics of microorganisms under artificial conditions using physical and chemical factors, and then selecting superior strains with specific traits that meet the requirements for cultivation, so that the microorganisms can be effectively applied to industrial production.

[0007] Currently, most scholars both domestically and internationally employ natural selection methods. However, natural selection is time-consuming and often fails to yield the desired microorganisms. Ultraviolet (UV) mutagenesis remains one of the most commonly used and effective methods in microbial breeding, offering advantages such as a high mutation frequency and low reversibility. However, there are currently no reports on using UV-induced mutagenesis alone to enhance the performance of actinomycetes.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide the application of marine actinomycetes of the genus *Saccharomyces* in improving the heat resistance of algae and / or promoting algal growth, thereby broadening the application scope of actinomycetes and better meeting the needs of production practice.

[0010] This invention is implemented as follows:

[0011] In a first aspect, the present invention provides the application of a marine actinomycete of the genus *Saccharothrix* sp. in improving the heat resistance of algae and / or promoting algal growth.

[0012] The inventors isolated a marine actinomycete from the algae of a heat-sensitive strain of *Porphyra yezoensis* (material from the Fujian Provincial *Porphyra yezoensis* Germplasm Resource Bank). Sequencing revealed its 16S rDNA sequence, which, when compared with known bacterial information in a database, showed a 98% similarity to the closest bacterium (*Saccharothrix* sp.). Therefore, the isolated bacterium was identified as a marine actinomycete belonging to the genus *Saccharothrix* sp. Incidentally, it was discovered that this bacterium significantly enhances the heat resistance of algae and promotes algal growth.

[0013] Based on the aforementioned strain, the inventors used ultraviolet mutagenesis to screen and obtain a marine actinomycete species of the genus *Saccharothrix*. Compared with the wild type, this bacterium significantly improves the high-temperature resistance of algae and also promotes algal growth. This indicates that the strain has good application prospects in improving the heat resistance of algae and / or promoting algal growth.

[0014] The inventors have for the first time discovered that marine actinomycetes of the genus *Saccharothrix* possess properties that enhance the heat resistance of algae and / or promote algal growth. For microorganisms, the species is the basic unit of classification. Since a species is a fundamental taxonomic unit, it is a collective term for a large group of strains with highly similar phenotypic characteristics, extremely close phylogenetic relationships, and significant differences from other species within the same genus. The same microorganism will share the same or highly similar phenotypic characteristics and functional properties; furthermore, different strains of the same microorganism, when used for classification and identification, have highly similar genomes, identical main traits, and highly similar functions. Therefore, those skilled in the art will readily recognize that other marine actinomycetes belonging to the genus *Saccharothrix* possess highly similar properties that enhance the heat resistance of algae and / or promote algal growth.

[0015] In a preferred embodiment of the present invention, the 16S rDNA sequence of marine actinomycetes has at least 90% identity with the sequence shown in SEQ ID NO.2.

[0016] SEQ ID NO.2:

[0017]

[0018] The 16S rDNA sequence has at least 90%, or at least 91%, at least 92%, or at least 93%, at least 94%, at least 94.5%, or at least 95%, at least 96%, or at least 97%, at least 98%, at least 98.65%, or at least 99% identity with the sequence shown in SEQ ID NO.2.

[0019] In an alternative embodiment, the 16S rDNA sequence of the marine actinomycete has at least 99% identity with the sequence shown in SEQ ID NO.2.

[0020] In an alternative embodiment, the 16S rDNA sequence of the marine actinomycete has at least 99% identity with the sequence shown in SEQ ID NO.2.

[0021] In a preferred embodiment of the present invention, the marine actinomycete is a strain or culture thereof with accession number GDMCC NO: 63741, the biological material name is Saccharothrix sp.SA01, which is deposited at the Guangdong Provincial Microbial Culture Collection Center; the strain classification is: Saccharothrix sp., the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology, the deposit date is August 23, 2023, and the identification result is viable.

[0022] In one alternative embodiment, the culture is selected from at least one of the following: cell fragments, fermentation supernatant, fermentation precipitate, and metabolites of marine actinomycetes belonging to the genus *Saccharomyces*.

[0023] Cell fragments include, but are not limited to, cell fragments obtained by at least one of the following methods: ultrasound, mechanical disruption, enzyme, physical or chemical treatment.

[0024] The fermentation supernatant can be prepared by the following method: inoculating marine actinomycetes, culturing, and removing the bacterial cells.

[0025] The fermentation precipitate can be prepared by the following method: inoculating marine actinomycetes for culture, centrifuging or allowing it to settle naturally, and removing the supernatant to obtain the fermentation precipitate.

[0026] In one alternative implementation, the metabolites are primary metabolites and / or secondary metabolites.

[0027] The term "metabolite" refers to primary and / or secondary metabolites produced during microbial metabolism. Primary metabolites are substances produced by microorganisms from various nutrients absorbed from the external environment, generating substances and energy necessary for their life activities through catabolism and anabolism. These include substances essential for their growth and reproduction, such as amino acids, nucleotides, polysaccharides, lipids, and vitamins. The types of primary metabolites are generally similar across different types of microbial cells. Furthermore, the synthesis of primary metabolites is continuous; any disruption in the synthesis of any product will affect the normal life activities of the microorganism. Secondary metabolites are substances with complex chemical structures produced only at a certain stage of microbial growth. These substances have no obvious physiological function for the microorganism or are not essential for its growth and reproduction, such as antibiotics, toxins, hormones, and pigments. Different types of microorganisms produce different secondary metabolites, which may accumulate within the cell or be excreted into the external environment. Antibiotics are a class of organic compounds with specific antibacterial and bactericidal effects; there are many types, with commonly used ones including streptomycin, penicillin, erythromycin, and tetracycline.

[0028] In a preferred embodiment of the present invention, the algae is selected from laver.

[0029] In one optional embodiment, the laver is selected from at least one of Porphyra yezoensis, Porphyra tenera, Porphyra yezoensis, Porphyra yezoensis, and Porphyra longifolia;

[0030] In one alternative implementation, the laver is a heat-sensitive strain of laver.

[0031] In a preferred embodiment of the present invention, promoting algal growth refers to: improving the photosynthetic efficiency of algae, extending the growth cycle of algae, increasing the daily growth rate of algae, and reducing the number of dead algal cells.

[0032] The improvement in photosynthetic efficiency of algae is manifested in the following: compared with the control group without added bacterial agent, the Fv / Fm (maximum quantum yield) value of algae is significantly increased.

[0033] Photosynthesis is crucial for plant matter transformation and energy metabolism, and it is highly sensitive to heat stress. The main targets of plant heat damage are the oxygen evolution complex and related cofactors in photosystem II. The Fv / Fm ratio reflects the maximum quantum yield of photosystem II, thus providing feedback on the stress status of the algae.

[0034] In a preferred embodiment of the present invention, improving the heat resistance of algae means improving the heat resistance of algae at temperatures 17-21°C above the optimal growth temperature.

[0035] The marine actinomycetes provided by this invention can significantly improve the heat resistance of algae and avoid high-temperature sequences with at least 90% identity.

[0036] The 16S rDNA sequence has at least 90%, or at least 91%, at least 92%, or at least 93%, at least 94%, at least 94.5%, or at least 95%, at least 96%, or at least 97%, at least 98%, at least 98.65%, or at least 99% identity with the sequence shown in SEQ ID NO.2.

[0037] In one alternative embodiment, the 16S rDNA sequence of the marine actinomycetes has at least 99% identity with the sequence shown in SEQ ID NO.2;

[0038] In one alternative embodiment, the marine actinomycete is a strain with accession number GDMCC NO: 63741.

[0039] Thirdly, the present invention also provides a bacterial agent or culture comprising the aforementioned marine actinomycetes.

[0040] In one alternative implementation, the culture is in the form of a liquid, solid, or semi-solid.

[0041] Microbial agents can be formulated in various forms, such as liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0042] In one optional embodiment, the amount of marine actinomycetes in the solid bacterial agent or culture is at least 3 × 10⁻⁶. 5 CFU / g; the amount of marine actinomycetes in the liquid inoculum or culture is at least 3 × 10⁻⁶. 5 CFU / mL.

[0043] Further, the preparation is specifically carried out according to the following steps: Saccharothrix sp. is inoculated into a bacterial culture medium and cultured to obtain a bacterial suspension (3 × 10⁻⁶) with an OD₆₀ value of 0.5-1.0 (e.g., 1.0). 5 -3×10 7 CFU / mL is the bacterial agent.

[0044] Furthermore, the bacterial culture medium was Gao's No. 1 liquid culture medium.

[0045] In the preparation method of the microbial agent, the cultivation conditions can be: 25-35℃ (e.g., 25-30℃, 30-35℃, 25℃, 30℃ or 35℃), 100-180r / min (e.g., 100-130r / min, 130-150r / min, 100r / min, 130r / min or 150r / min) for 30-60h (e.g., 30-48h, 48-60h, 30h, 48h or 60h).

[0046] Fourthly, the present invention also provides a composition comprising the above-described marine actinomycetes.

[0047] Fifthly, the present invention also provides a method for improving the heat resistance of algae and / or promoting algal growth, by applying the above-mentioned marine actinomycetes, the above-mentioned bacterial agents or cultures, or the above-mentioned combinations to the test algae.

[0048] In one optional embodiment, when the thallus of the test algae grows to a length of 3.8-4.2 cm, marine actinomycetes, bacterial agents, cultures, or compositions are co-cultured with the thallus of the test algae.

[0049] At the aforementioned time points, co-culturing marine actinomycetes, bacterial agents, cultures, or combinations with the thallus of the test algae can better enhance the efficacy of marine actinomycetes.

[0050] This algae-bacterial co-culture system can improve the problems of seedling rot and harmful bacterial growth in heat-sensitive varieties of Porphyra yezoensis during high-temperature exposure, resulting in a higher daily growth rate and greater vitality of the algae when Porphyra yezoensis and beneficial bacteria are co-cultured.

[0051] In the above-mentioned algae-bacteria co-culture system, the bacterial culture medium used can be a solid culture medium or a liquid culture medium.

[0052] The bacterial culture medium is specifically Gao's No. 1 medium.

[0053] The term "culture" refers to a liquid or solid product (all substances within the culture container) that has grown a microbial community after artificial inoculation and cultivation. It is a product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0054] In one alternative implementation, the culture is carried out for 1 to 10 days; for example, for 1 day, 2 days, 5 days, 8 days or 10 days.

[0055] In one alternative implementation, non-aerated co-culture is performed.

[0056] In one alternative embodiment, each thallus is treated with 1 mL of OD600 value 3 × 10⁻⁶. 5 -3×10 7 A mixture of marine actinomycetes, bacterial agents, cultures, or compositions at CFU / mL.

[0057] The present invention has the following beneficial effects:

[0058] This invention isolated a marine actinomycete from the algae of a heat-sensitive *Porphyra yezoensis* strain from the Fujian Provincial *Porphyra yezoensis* germplasm resource bank. Sequencing yielded its 16S rDNA sequence, which was compared with known bacterial information in a database. The sequence showed a 98% similarity to the closest bacterium (*Saccharothrix* sp.), thus identifying the isolated bacterium as a marine actinomycete belonging to the genus *Saccharothrix* sp. Incidentally, it was discovered that this bacterium significantly enhances the heat resistance of algae and promotes algal growth.

[0059] Furthermore, based on the strain with the aforementioned accession number, this invention used ultraviolet mutagenesis to screen and obtain a marine actinomycete species of the genus *Saccharothrix*. Compared to the wild type, this bacterium significantly improves the high-temperature resistance of algae and exhibits algal growth-promoting properties. This suggests that this strain has promising application prospects in improving the heat resistance of algae and / or promoting algal growth. Attached Figure Description

[0060] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0061] Figure 1 The colony morphology of the wild-type strain on Gao's No. 1 solid medium;

[0062] Figure 2 This is a statistical graph showing the effect of microbial agents on the Fv / Fm value of Porphyra yezoensis under high temperature stress.

[0063] Figure 3 A statistical graph showing the effects of wild-type fungal inoculum and blank control on the daily growth rate of Porphyra yezoensis under high temperature stress.

[0064] Figure 4 A statistical graph showing the effects of wild-type and mutant fungal agents on the daily growth rate of Porphyra yezoensis under high-temperature stress.

[0065] Figure 5 Figure 1 shows the effect of bacterial agent on the growth of Porphyra yezoensis thallus and cells under high temperature stress.

[0066] Figure 6 A graph showing the statistical results of SOD activity in the heat-sensitive strain of *Porphyra yezoensis* under high temperature conditions;

[0067] Figure 7A graph showing the statistical results of proline content in the thallus of the heat-sensitive strain of *Porphyra yezoensis* under high temperature conditions;

[0068] Figure 8 The phylogenetic tree alignment results for the 16S rRNA of wild-type actinomycete (Saccharothrix sp.);

[0069] Figure 9 This is a diagram showing the phylogenetic tree alignment results of the 16S rRNA of mutant actinomycetes. Detailed Implementation

[0070] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0071] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. This technique is well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Protocols in Molecular Biology* (edited by F.M. Mausubel et al., 1987); and *PCR: The Polymerase Chain Reaction*. The references cited in the references are: "Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (JEColigan et al., ed., 1991), each of which is explicitly incorporated herein by reference.

[0072] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0073] Gao's No. 1 liquid culture medium: Add 20g soluble starch, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.01g ferrous sulfate, and 0.5g sodium chloride to sterilized seawater to adjust the pH to 7.3±0.1 (25℃), bring the volume to 1L, then sterilize at 121℃ for 15min and cool before use.

[0074] Gao's No. 1 solid medium: Add agar to Gao's No. 1 liquid medium to a concentration of 15 g / L; then sterilize at 121°C for 15 min. Pour the Gao's No. 1 liquid medium, cooled to approximately 55°C, into petri dishes and allow to cool naturally.

[0075] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0076] Example 1

[0077] This embodiment provides the process for isolating, identifying, and preserving actinomycetes (Saccharothrix sp.).

[0078] I. Isolation of Actinomycetes (Saccharothrix sp.)

[0079] 1. After briefly rinsing the heat-sensitive *Porphyra yezoensis* thalassemia strain treated at 30℃ with sterilized seawater to remove surface impurities, add a small amount of sterilized seawater and vortex for 10 minutes to remove loose bacteria on the surface. Place the vortexed thalassemia in a sterilized mortar, add a small amount of sterile quartz sand, and grind thoroughly. Filter through a sterile silk sieve and collect the filtrate. Add 5 mL of the treated grinding solution to 45 mL of sterilized seawater, shake well for 5 minutes, and let stand for 10 minutes. Take 1 mL of the supernatant and dilute it 10 times (this dilution is recorded as 10). -1 Continue to dilute in a 10-fold serial dilution series to achieve a dilution of 10. -2 10 -3 10 -4 10 -5 10 -6 10 -7 The bacterial suspension was prepared. 0.1 mL of each of the above dilutions was evenly spread on Gao's No. 1 solid culture medium and incubated at 30°C for 2 days.

[0080] 2. After completing step 1, pick a single colony from Gao's No. 1 solid culture medium and repeat the streak culture purification at least 3 times. Identify the screened bacteria.

[0081] II. Identification of Bacteria

[0082] The method for 16S rDNA sequence homology analysis is as follows:

[0083] Bacterial genome was extracted, 16S rDNA sequence was amplified using specific primers, PCR product was purified, 16S rDNA sequence was obtained by DNA sequencing, and the sample species information was obtained by comparison with known bacterial information in the database. The bacterial 16S rDNA is shown in SEQ ID No. 1.

[0084] SEQ ID NO.1:

[0085] gacgaacgctggcggcgtgcttaacacatgcaagtcgagcggtaaggcccttcggggtac

[0086] acgagcggcgaacgggtgagtaacacgtgggtaacctgccccgtactctgggataagcct

[0087] gggaaactaggtctaataccggatatgaactccttgggcatccagggggttgtaaagttc

[0088] cggcggtacgggatgggcccgcggcctatcagcttgttggtggggtgatggcctaccaag

[0089] gcgacgacgggtagccggcctgagagggtgaccggccacactgggactgagacacggccc

[0090] agactcctacgggaggcagcagtggggaatattgcacaatgggcgaaagcctgatgcagc

[0091] gacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagcagggacgaagcg

[0092] caagtgacggtacctgcagaagaagcaccggctaactacgtgccagcagccgcggtaata

[0093] cgtagggtgcgagcgttgtccggaattattgggcgtaaagagctcgtaggcggtttgttg

[0094] cgtcggccgtgaaaacttcacgcttaacgtggagcctgcggtcgatacgggcagacttga

[0095] gttcggcaggggagactggaattcctggtgtagcggtgaaatgcgcagatatcaggagga

[0096] acaccggtggcgaaggcgggtctctgggccgatactgacgctgaggagcgaaagcgtggg

[0097] gagcgaacaggattagataccctggtagtccacgccgtaaacggtgggtgctaggtgtgg

[0098] gggacttccacgtcctccgtgccgcagctaacgcattaagcaccccgcctggggagtacg

[0099] gccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcggcggagcatgtgg

[0100] attaattcgatgcaacgcgaagaaccttacctgggcttgacatgcactggaaaccagtag

[0101] agatattggcccccttgtggccggtgtacaggtggtgcatggctgtcgtcagctcgtgtc

[0102] gtgagatgttgggttaagtcccgcaacgagcgcaaccctcgttccatgttgccagcgcgt

[0103] aatggcggggactcatgggagactgccggggtcaactcggaggaaggtggggatgacgtc

[0104] aagtcatcatgccccttatgtccagggcttcacacatgctacaatggccggtacagaggg

[0105] ctgctaagccgtgaggtggagcgaatcccaaaaagccggtctcagttcggatcggggtct

[0106] gcaactcgaccccgtgaagtcggagtcgctagtaatcgcagatcagcaacgctgcggtga

[0107] atacgttcccgggccttgtacacaccgcccgtcacgtcacgaaagtcggtaacacccgaa

[0108] gcccgtggcccaacccgcaagggggggagcggtcgaaggtgggactggcgattgggac.

[0109] Reference image for 16S rRNA phylogenetic tree alignment results Figure 8 As shown (n1 represents the isolated target bacterium), the closest species was *Saccharothrix ecbatanensis*. Therefore, this bacterium was identified as a marine actinomycete belonging to the genus *Saccharothrix* sp.

[0110] III. Preservation

[0111] Strain preservation: Single colonies of actinomycetes (Saccharothrix sp.) were inoculated into Gao's No. 1 liquid medium and cultured at 30°C for 12 hours to obtain the culture solution.

[0112] The 20% glycerol solution was sterilized at 121°C for 15 minutes before use.

[0113] Using a sterile dropper, mix 20% glycerol with the bacterial solution at a 1:1 volume ratio, then dispense the mixture into containers and store at -80°C.

[0114] Example 2

[0115] Preparation of actinomycete inoculants

[0116] 1. Take out the Saccharothrix sp. actinomycete strain stored at -80℃ in Example 1, and immediately place it in a water bath at 37-45℃ to thaw rapidly and shake appropriately until the glycerol bacterial solution in the tube is completely dissolved. Under aseptic conditions, open the glycerol tube and use an inoculation loop to pick up the bacterial solution and activate it on Gao's No. 1 solid culture medium.

[0117] 2. After activation, a single colony of Gao's No. 1 solid culture medium was picked and inoculated into 5 mL of Gao's No. 1 liquid culture medium. The culture was then incubated at 28℃ and 180 r / min for 24 h to obtain the OD. 600 A bacterial solution with an nm value of approximately 1. This bacterial solution is an inoculum of Saccharothrix sp.

[0118] Reference for single colony morphology of Gao's No. 1 solid culture medium Figure 1 As shown.

[0119] Example 3

[0120] This embodiment uses ultraviolet light to induce mutations in the strain obtained in Example 1. The specific steps are as follows:

[0121] 1. Stable light waves

[0122] In a dark room, place a 15W ultraviolet lamp above the work surface and turn on the ultraviolet light source 30 minutes in advance to stabilize the light wave.

[0123] 2. Aseptic-induced mutation

[0124] Five mL of the culture medium containing the actinomycete *Saccharothrix* sp. from Example 1, cultured to the logarithmic growth phase, was centrifuged at 5000 × g for 20 min at 4 °C. The supernatant was then centrifuged again at 12000 × g for 20 min at 4 °C. The precipitate was resuspended in 10 mL of Gao's No. 1 liquid culture medium. Gao's No. 1 culture medium consisted of 20 g soluble starch, 1 g potassium nitrate, 0.5 g dipotassium hydrogen phosphate, 0.01 g ferrous sulfate, and 0.5 g sodium chloride. The solution was added to sterile seawater to adjust the pH to 7.3 ± 0.1 (25 °C), and the volume was brought to 1 L. The medium was then sterilized at 121 °C for 15 min and cooled before use.

[0125] Under the condition of filtering visible light, the above 1×10 5-7 Add 5 mL of CFU / mL actinomycete concentrate to a 9 cm sterile culture dish, add a sterile magnetic rotor and turn on magnetic stirring. While stirring slowly, place the culture dish directly under the UV lamp at a distance of 20 cm to begin inducing mutations. The mutagenesis time is 2 min.

[0126] (3) Ice bath preservation

[0127] Turn off the UV lamp, remove the bacterial solution, immediately wrap it in aluminum foil to protect it from light, and place it in an ice bath for at least 1 hour to inhibit repair.

[0128] (4) Cumulative mutagenesis

[0129] Using the actinomycete mutant strain that has been enhanced in step 3, repeat steps 2 and 3 six times to cumulatively induce mutagenesis.

[0130] Cell growth was observed under a microscope, and the effect of mutant strains on the heat resistance of *Porphyra yezoensis* was tested, thereby screening for actinomycete mutant strains.

[0131] The 16S rDNA of the selected strain (SA01) was identified, and the results are as follows: Figure 9 As shown.

[0132] Example 4

[0133] Preparation of actinomycete mutant strain inoculum.

[0134] After activating the actinomycete mutant strain from Example 3, a single colony was picked from Gao's No. 1 solid culture medium and inoculated into 5 mL of Gao's No. 1 liquid culture medium. The culture was then incubated at 28°C and 180 r / min for 24 h to obtain OD. 600 A bacterial solution with an nm value of approximately 1. This bacterial solution is the bacterial agent to be tested.

[0135] Experimental Example 1

[0136] Application of Saccharothrix sp. in improving the high-temperature resistance of Porphyra yezoensis

[0137] Thick thallus selection: Using the high-temperature sensitive strain of Porphyra yezoensis (wo49-1) as experimental material, three healthy thallus plants with a length of 3.8-4.2 cm were selected and inoculated with 1 mL of the bacterial agent from Example 2 into 500 mL of seaweed culture medium (P+T group).

[0138] Artificial seawater formula: NaCl 24.54g / L -1 SrCl2:6H2O 0.017g L -1 KC1 0.7g L -1 1.11 g / L CaCl2 -1 MgCl2 6H2O 11.1g L -1 H3BO3 0.003g L -1 4.09 g L of Na2SO4 -1 KBr 0.1g L -1 0.003 g / L NaF -1 0.185g / L NaHCO3 -1

[0139] Set up a mutant group (UV group): Using the heat-sensitive strain of Porphyra yezoensis (wo49-1) as the experimental material, three healthy thallus plants with a length of 3.8-4.2cm were selected and inoculated with 1mL of mutant bacteria into 500mL of seaweed culture medium.

[0140] The culture conditions were as follows: temperature (high temperature: 30℃; control: 21℃); aeration-free culture was preferred. 12h light / 12h dark; light intensity 40-50 μmol / (m²). 2 ·s).

[0141] The test method for determining whether actinomycete mutants enhance the heat resistance of *Porphyra yezoensis* is as follows:

[0142] The physiological activities of heat-sensitive *Porphyra yezoensis* thallus were measured daily under high temperature conditions, including SOD activity, free proline content, Fv / Fm ratio, relative growth rate, and cell growth status. The thallus cell status was observed after 5 days of culture (the experiment was repeated three times, and the average value was taken).

[0143] The method for determining superoxide dismutase (SOD) activity is as follows: Superoxide anions (O2-) are generated using a xanthine and xanthine oxidase reaction system. O2- reacts with WST-8 to produce the water-soluble dye formazan, which absorbs at 450 nm. SOD can scavenge O2-, thereby inhibiting the formation of formazan. The deeper the yellow color of the reaction solution, the lower the SOD activity, and vice versa. Homogenize the tissue (g) and extract volume (mL) at a ratio of 1:5-10 on ice. Centrifuge at 8000g, 4℃ for 10 min, collect the supernatant, and place it on ice for testing. Measurement procedure: Preheat the microplate reader for at least 30 min and adjust the wavelength to 450 nm. Dilute reagent 3 50 times with distilled water, add reagent 1 to 100 μL of reagent 2, and mix thoroughly. Dissolve one bottle of reagent 4 in 5 mL of distilled water and measure the absorbance (A) at 450 nm. Finally, the SOD activity was calculated based on the fresh weight of the sample: SOD activity (U / g fresh weight) = [inhibition percentage ÷ (1 - inhibition percentage) × Vtotal] ÷ (W × Vsample ÷ Vtotal) = 20 × inhibition percentage ÷ (1 - inhibition percentage) ÷ W

[0144] The method for determining proline (Pro) content is as follows: Pro is extracted using sulfosalicylic acid (SA). After heating, Pro reacts with acidic ninhydrin solution to form a red color. After extraction with toluene, the absorbance is measured at 520 nm. The tissue mass (g) to extraction volume (mL) is homogenized in an ice bath at a ratio of 1:5–10. Extraction is then carried out at 90℃ with shaking for 10 min. The sample is then centrifuged at 10000g at 25℃ for 10 min, and the supernatant is collected, cooled, and ready for analysis. The assay steps are as follows: First, the microplate reader is preheated for at least 30 min, and the wavelength is adjusted to 520 nm. Second, 0.25 mL of sample extract + 0.25 mL of glacial acetic acid + 0.25 mL of reagent II are placed in a capped EP tube and incubated in a boiling water bath for 30 min (the tube is tightly capped to prevent moisture loss), shaking every 10 min. After cooling, add 0.5 mL of toluene, shake for 30 s, and let stand for a moment to allow the pigment to transfer to the toluene. Pipette 0.2 mL of the upper layer solution into a 96-well plate and measure the absorbance (A) at 520 nm. Finally, calculate the Pro content (μg / g fresh weight) based on the sample mass: [(A+0.0021)÷0.02605×V1]÷(W×V1÷V2)=38.4×(A+0.0021)÷W.

[0145] The effect of adding bacterial agent on the Fv / Fm value of Porphyra yezoensis thallus under high temperature conditions was referenced. Figure 2 As shown, UV: UV-mutated strain from Example 4 added; P+T: bacterial agent from Example 2 added; CK: blank control (no bacterial agent added).

[0146] The effect of microbial agents on the relative growth rate of *Porphyra yezoensis* under high temperature conditions is shown in the figure below. Figure 3 As shown, P+T: Added bacterial agent of Example 2; CK: Blank control.

[0147] The effect of mutagenic agents on the relative growth rate of Porphyra yezoensis under high temperature conditions (refer to...) Figure 4 As shown, UV: UV-mutated strain from Example 4; P+T: bacterial agent from Example 2; CK: blank control.

[0148] The effect of microbial agents on the growth of Porphyra yezoensis thallus and cells under high temperature stress is shown in the figure. Figure 5 As shown, UV: UV-mutated strain from Example 4; P+T: bacterial agent from Example 2; CK: blank control.

[0149] The effect of microbial agents on SOD enzyme activity of Porphyra yezoensis thallus under high temperature stress is shown in the figure below. Figure 6 The effect of the inoculant on the proline concentration of *Porphyra yezoensis* thallus under high temperature stress is shown in the figure below. Figure 7 As shown.

[0150] The results showed that the inoculant from Example 2 enabled heat-sensitive *Porphyra yezoensis* varieties to tolerate 5 days of 30°C high-temperature treatment. Specifically, under high-temperature conditions, the addition of the inoculant from Example 2 significantly increased the daily growth rate of thallus and the fractional vegetative growth rate (Fv / Fm) of the heat-sensitive *Porphyra yezoensis* varieties, and reduced the number of dead cells. This indicates that the aforementioned actinomycetes can mitigate the negative impact of high temperatures on the yield of *Porphyra yezoensis*.

[0151] Compared to the wild-type inoculum of Example 2, the addition of the mutagenic inoculum of Example 3 significantly increased the daily growth rate of thallus in the heat-sensitive Porphyra yezoensis strain and reduced the number of dead cells after 72 hours of high-temperature treatment. The UV-mutated strain exhibited higher SOD enzyme activity and proline metabolic activity.

[0152] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. *Saccharomyces* genus ( Saccharothrix Application of marine actinomycetes of species sp. in improving the heat resistance of algae, wherein the marine actinomycetes are strains or cultures thereof with accession number GDMCC NO: 63741, which are deposited at Guangdong Provincial Microbial Culture Collection Center, and the improvement of the heat resistance of algae refers to: improving the heat resistance of algae at temperatures 17-21℃ above the optimum growth temperature.

2. Use according to claim 1, characterized in that, The culture is at least one selected from the group consisting of cell disruption, fermentation supernatant, fermentation precipitate and metabolite of the marine actinomycete of the Saccharothrix sp.

3. Use according to claim 1, characterized in that, The algae is selected from Porphyra.

4. Use according to claim 3, characterized in that, The Porphyra is at least one selected from the group consisting of Porphyra haitanensis, Porphyra yezoensis, Porphyra tenera, Porphyra dentata and Porphyra sp.

5. Use according to claim 4, characterized in that, The Porphyra haitanensis is a heat-sensitive strain.

6. A marine actinobacterium of the species Saccharimonas sp. characterized in that, Saccharothrix sp. ) characterized in that, The marine actinomycete is a strain with the accession number of GDMCC NO: 63741 preserved in Guangdong Microbial Culture Collection Center.

7. An inoculant or culture, characterized in that, It comprises the marine actinomycete of claim 6.

8. The inoculant or culture of claim 7, characterized in that, The form of the bacterial agent or culture is liquid, solid or semi-solid.

9. A method of improving the heat tolerance of algae, characterized by, The marine actinomycete of claim 6 or the bacterial agent or culture of any one of claims 7-8 is applied to the test algae, and the improved heat tolerance of the algae refers to the improved heat tolerance of the algae at 17-21℃ higher than the optimum growth temperature.

10. The method of improving heat tolerance in algae according to claim 9, wherein, The marine actinomycete, the bacterial agent or the culture is co-cultured with the thallus of the test algae when the growth length of the thallus of the test algae is 3.8-4.2 cm.

11. The method for improving heat tolerance of algae according to claim 10, wherein, The co-culture is performed for 1-10 days.

12. The method of improving heat tolerance in algae according to claim 11, wherein, The co-culture is performed without aeration.

13. The method of improving heat tolerance in algae according to claim 10, wherein, 3 x 10 5 -3 x 10 7 CFU / mL of marine actinomycetes, inoculum or culture mixture per plant.

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