Breeding and application of ecological high-efficiency low-toxicity high-quality monascus strain

Abstract

The invention discloses breeding and application of an ecological high-efficiency low-toxicity high-quality monascus strain. The invention provides a combined bacterium. The combined bacterium is composed of monascus and salt-tolerant Pentongronchnos sp. The monascus sp. Is monascus sp. CN1001, and the registration number of the monascus sp. CN1001 in the China General Microbiological Culture Collection Center (CGMCC) is CGMCC No.42029; the salt-tolerant Pentannosperm sp. Is a salt-tolerant Pentannosperm sp., and the registration number of the salt-tolerant Pentannosperm sp. In the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms is CGMCC No.30991. The salt-tolerant Pentannosperm sp. Is a salt-tolerant Pentannosperm sp. The strain CN1001 is an eco-friendly high-efficiency low-toxicity high-quality monascus strain, and can be used for fermentation production of lovastatin, and when the strain CN1001 and the salt-tolerant Pentongronoctus XF202 are co-cultured, the yield of lovastatin is further improved.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to the selection and application of an eco-efficient, low-toxicity, high-quality Monascus purpureus strain. Background Technology

[0002] Monascus purpureus is a type of multifunctional filamentous fungus belonging to the phylum Fungi (Monascus). Eumycohyta Ascomycetes ( Ascomycetes ), Subclass Ascomycota ( Euascomycetes Aspergillus ( ) Eurotiales Aspergillus family ( Eurotiaceae Monascus genus () Monascus Based on the morphological characteristics of Monascus during culture, such as the structure of the cleistothecia and spore characteristics, Monascus species were classified, including Monascus fusca (…). M. pilosus ), purple red mold ( M. purpureus ), Red Monascus ( M. ruber ), Smoked red mold ( M. filiginosus Sato ), white red mold ( M. albidus ), rust-colored red mold ( M. rubiginosus ), red Monascus ( M. rubropunctatus Sato ), Orange Monascus ( M. aurantiacus (e.g., Monascus purpureus colonies.) On malt extract agar, Monascus purpureus colonies are initially white, gradually turning yellow, purplish-red, or grayish-black as they grow, with color changes varying depending on the species. Colony structure also varies by species, typically appearing velvety or membranous, with some colonies exhibiting radial striations and fewer folds. The length of hyphae at the colony margin also differs among species. Most Monascus purpureus produce monaxioxins, including monaxioxin red and monaxioxin yellow, which can even be secreted into the culture medium, giving it a red color. The hyphae are septate, multinucleate, and have numerous irregular branches. Young cells contain granules, while mature cells contain vacuoles and oil droplets. Conidia are mainly borne at the hyphal tips, occurring singly or in chains of 2-6, with some reaching up to 10. Cleomestomata are spherical, stalked, and the length of the stalk varies.

[0003] Monascus purpureus is a saprophytic fungus characterized by its acidophilicity (especially its affinity for lactic acid), high temperature tolerance, and tolerance to 10% ethanol. It can grow well in a temperature range of 26-42℃, with an optimal growth temperature of 32-35℃; its optimal pH is 3.5-5, and it can tolerate pH values ​​as low as 2.5. Furthermore, Monascus purpureus can utilize organic acids, sugars, and alcohols as carbon sources, and can utilize nitrate nitrogen, amino nitrogen, and organic nitrogen as nitrogen sources. Secondary metabolites of Monascus purpureus are widely used in the food and pharmaceutical industries (PATAKOVAP. Monascus secondary metabolites: production and biological activity. Journal of Industrial Microbiology and Biotechnology, 2013, 40(2): 169-181; XIA XX, CHEN D, JANG L, et al. Characteristics of pigment production fromfermentation of different varieties of rice by Monascus Purpureus. Mod FoodSci Technol, 2022, 38(5): 33-42).

[0004] In China, Monascus purpureus has a long history of application. With the continuous development of biochemical research methods, a variety of secondary metabolites of Monascus purpureus with high medicinal activity have been discovered and applied, becoming a research hotspot for scholars at home and abroad. Among them, lovastatin produced by Monascus purpureus has multiple functions such as inhibiting cholesterol synthesis, protecting the kidneys, resisting atherosclerosis, and preventing the occurrence and development of cancer (Endo A. Monacolin K, a new hypocholesterolemic agent produced by a Monascus species. The Journal of Antibiotics, 1979, 32(8): 852-854; Pan RH, Fang Y, Tang XY, et al. Research progress on secondary metabolites of Monascus spp. and their pharmacological activities. West China Journal, 2023, 38(3): 345; Hu J, Wang J, Gan X, et al. Impact of red yeastrice on metabolic diseases: A review of possible mechanisms of action. J AgriFood Chem, 2020, 68(39): 10441; CHEN CL, CHANG KY, PAN TM. Monascus purpureusNTU 568 fermented product improves memory and learning ability). in rats with aluminum-induced Alzheimer's disease. Journal of Functional Foods, 2016, 21:167-177).However, in the analysis of secondary metabolites of *Monascus purpureus* and *Monascus redis*, researchers found that, in addition to lovastatin, which has promising medicinal value, these strains also produce toxic substances such as citrinin (Wichmann G, Herbarth O, Lehmann I. The mycotoxins citrinin, gliotoxin, andpatulin affect interferon-gamma rather than interleukin-4 production in human blood cells. Environ Toxicol, 2002, 17: 211-8). Furthermore, the production of lovastatin using *Monascus redis* is hampered by low strain production efficiency and insufficient eco-friendliness, limiting its further development and application. Therefore, the selection and breeding of eco-efficient, low-toxicity, high-quality *Monascus redis* strains is of significant practical importance.

[0005] Peng Zhenrong genus ( Pengzhenrongella ) belongs to the domain Bacteria ( Bacteria ) - Actinobacteria ( Actinomycetota - Actinomycetes ( Actinomycetes )-Micrococcales ( Micrococcales In 2021, Kim et al. isolated a yellow strain LRZ-2T from Arctic tundra soil (Kim MC, Ju YH, Hwang UA, Liu P, Pak SH, Peng F. Pengzhenrongella sicca gen. nov., sp. nov., a new member of suborder Micrococcineae isolated from High Arctic tundra soil. Int J Syst Evol Microbiol 2021; 71:4988.). Based on phylogenetic analysis, phenotypic and chemotaxonomic data using 16S rRNA gene sequence comparison, strain LRZ-2T was identified as a new member of the Micrococcineae suborder, and it was suggested that it be named... Pengzhenrongella sicca gen. nov., sp. nov. To date, there are 3 validly described species in this genus (https: / / lpsn.dsmz.de / genus / pengzhenrongella): Pengzhenrongella sicca(Pengzhenrongella sicca gen. nov., sp. nov., a new member of suborder Micrococcineae isolated from High Arctic tundra soil. Int J Syst Evol Microbiol 2021; 71:4988), isolated from Arctic high tundra soil. Pengzhenrongella phosphoraccumulans (Xie J, Ren L, Wei Z, Peng X, Qin K, Peng F.Pengzhenrongella phosphoraccumulans sp. nov., isolated from high Arcticglacial till, and emended description of the genus Pengzhenrongella (Int JSyst Evol Microbiol 2024; 74:6368.) and samples isolated from the Hailuogou Glacier in Sichuan Province. Pengzhenrongella frigida (Liu Q, Yang LL, Xin YH. Pengzhenrongella frigida sp.nov., isolated from a glacier. Int J Syst Evol Microbiol 2024; 74:6433.).

[0006] Pengzhenrongella This genus of bacteria possesses characteristic morphological and chemical classification features: cells are Gram-positive, corynebacterial, and non-spore-producing. These strains are facultative anaerobic, growing better under aerobic conditions; they are catalase-positive and oxidase-negative. The main methylnaphthoquinone component is MK-9. The most significant polar lipid components include phosphatidylglycerol (PG), phosphatidylinositol mannoside (PIM), and phosphatidylinositol (PI), with anteiso-C being the primary fatty acid. 15:0 C 16:0、 anteiso-C 17:0 iso-C 15:0 The G+C content of genomic DNA is 70.8-72.4%.

[0007] There is currently no use for it. Pengzhenrongella Reports on the production of lovastatin by combining strains of Monascus purpureus and Monascus purpureus. Summary of the Invention

[0008] The purpose of this invention is to provide an eco-friendly, highly efficient, and low-toxicity high-quality Monascus purpureus strain and a mixed preparation of it and salt-tolerant Panax notoginseng, as well as its application in the fermentation production of lovastatin.

[0009] Firstly, the present invention claims protection for a composite bacterium.

[0010] The combined bacteria claimed in this invention consist of Monascus purpureus and salt-tolerant Bacillus thuringiensis; The red mold is Monascus purpureus (Monascus purpureus) Monascus sp. CN1001, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.42029; The salt-tolerant Peng Zhenrong bacteria is a salt-tolerant Peng Zhenrong bacteria ( Pengzhenrongellahalotolerans Its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 30991.

[0011] In some embodiments of the present invention, the Monascus purpureus and the salt-tolerant Bacillus thuringiensis are packaged separately.

[0012] Secondly, this invention claims protection for a salt-tolerant *Peng Zhenrong* bacterium.

[0013] The salt-tolerant Peng Zhenrong bacterium claimed in this invention is a salt-tolerant Peng Zhenrong bacterium ( Pengzhenrongellahalotolerans Its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 30991.

[0014] Thirdly, this invention claims protection for a type of Monascus purpureus.

[0015] The Monascus purpureus claimed in this invention is Monascus purpureus (Monascus purpureus) Monascus sp. CN1001, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.42029.

[0016] Fourthly, the present invention claims protection for a product used in the fermentation production of lovastatin.

[0017] The product for the fermentation production of lovastatin claimed in this invention contains the combined strains described in the first aspect above or the Monascus red mold described in the third aspect above.

[0018] Furthermore, the product may also contain a fermentation medium, such as a solid fermentation medium for producing lovastatin using the combined bacteria or the Monascus purpureus. In some embodiments of the present invention, the solid fermentation medium is a rice-soybean flour medium. Specifically, the rice-soybean flour medium is a mixture of the following components in the following proportions: 180 g of indica rice: 20 g of soybean flour: 200 mL of deionized water; the pH of the rice-soybean flour medium is 7.

[0019] Fifthly, the present invention claims protection for any of the following applications: (A1) The use of the combined bacteria described in the first aspect above or the Monascus purpureus described in the third aspect above in the preparation of products for the fermentation production of lovastatin; (A2) The use of the combined bacteria described in the first aspect above, or the Monascus purpureus described in the third aspect above, or the product described in the fourth aspect above, in the fermentation production of lovastatin.

[0020] Sixthly, the present invention claims a method for fermenting lovastatin, comprising the following steps: (The method utilizes the combined bacteria described in the first aspect above to produce lovastatin.) (A1) The red mold was inoculated into rice-soybean flour culture medium and subjected to the first stage of solid fermentation culture to obtain the first stage fermentation culture; (A2) The salt-tolerant Peng Zhenrong bacteria are inoculated into the fermentation culture obtained in step (A1) and a second-stage solid-state fermentation culture is carried out to obtain a second-stage fermentation culture; (A3) Lovastatin was obtained from the second-stage fermentation culture obtained in step (A2).

[0021] In some embodiments of the present invention, the solid fermentation culture medium is a rice-soybean flour culture medium. Specifically, the rice-soybean flour culture medium is a mixture of the following substances in the following proportions: 180 g of indica rice: 20 g of soybean flour: 200 mL of deionized water; the pH of the rice-soybean flour culture medium is 7.

[0022] In some embodiments of the present invention, in step (A1), the spore concentration is 10 8 The seed culture of Monascus purpureus at a concentration of spores / mL was inoculated into the rice-soybean flour medium, with the inoculation amount calculated as 2 mL of seed culture added per 100 g of medium (calculated as the sum of the masses of rice and soybean flour therein).

[0023] In some embodiments of the present invention, in step (A1), when the first stage of solid fermentation culture is carried out, the culture temperature is 28°C, the culture time is 14-18 days (e.g., 14 days), the humidity is 40%, and the culture is static; during this period, the rice-soybean powder culture medium is shaken once every three days.

[0024] In some embodiments of the present invention, in step (A2), the concentration is 10 8 The seed culture of the salt-tolerant Peng Zhenrong bacterium with a concentration of CFU / mL was inoculated into the first stage fermentation culture, with the inoculation amount calculated as 2 mL of seed culture added per 100 g of culture medium (calculated based on the sum of the masses of rice and soybean flour therein).

[0025] In some embodiments of the present invention, in step (A2), when carrying out the second stage of solid fermentation culture, the culture temperature is 28°C, the culture time is 5-16 days (e.g., 6 days), the humidity is 40%, and the culture is static; during this period, the rice-soybean powder culture medium is shaken once every three days.

[0026] Seventhly, the present invention claims a method for producing lovastatin by fermentation, which utilizes the Monascus purpureus described in the third aspect above to produce lovastatin by fermentation, comprising the following steps: inoculating the Monascus purpureus into a rice-soybean flour culture medium, performing solid-state fermentation culture, and obtaining lovastatin from the resulting fermentation culture.

[0027] In some embodiments of the present invention, the solid fermentation culture medium is a rice-soybean flour culture medium. Specifically, the rice-soybean flour culture medium is a mixture of the following substances in the following proportions: 180 g of indica rice: 20 g of soybean flour: 200 mL of deionized water; the pH of the rice-soybean flour culture medium is 7.

[0028] In some embodiments of the present invention, the concentration is 10 8 The seed culture of Monascus purpureus at CFU / mL was inoculated into the rice-soybean flour medium, with the inoculation amount calculated as 2 mL of seed culture added per 100 g of medium (calculated as the sum of the masses of rice and soybean flour therein).

[0029] In some embodiments of the present invention, when carrying out the solid fermentation culture, the culture temperature is 28°C, the culture time is 18-34 days (e.g., 30 days), the humidity is 40%, and the culture is carried out in a static state, during which the rice-soybean powder culture medium is shaken once every three days.

[0030] The *Monascus purpureus* strain CN1001 provided in this invention is a high-lovastatin-producing strain isolated from soil samples of a rural tofu workshop in Kunming, Yunnan Province, and induced by ultraviolet (UV) irradiation. This strain is a mesophilic bacterium that grows well under neutral or slightly acidic pH conditions. It can utilize inexpensive and readily available biomass as a culture medium. After UV mutagenesis treatment, the lovastatin production capacity of strain CN1001 was significantly improved (the lovastatin production capacity of strain CN1001 was three times that before mutagenesis selection), and it did not produce citric acid. Passage analysis results showed that the genetic traits of the 30th generation strain of strain CN1001 were stable with the original 0th generation strain. Therefore, strain CN1001 is an eco-friendly, highly efficient, low-toxicity, and high-quality *Monascus purpureus* strain. Furthermore, when Monascus purpureus strain CN1001 was co-cultured with salt-tolerant P. pengzhenrong strain XF202 (a new species), the yield of lovastatin was further improved. The highest lovastatin yield was achieved at 20 days of fermentation, reaching (44.9±0.15) mg / g. Compared with fermentation of Monascus purpureus strain CN1001 alone, the lovastatin yield was increased by about 40%.

[0031] Depository Instructions Classification and nomenclature: Monascus purpureus ( Monascus sp.); Biological material of ginseng: CN1001; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: June 10, 2025; Registration number at the Collection Center: CGMCC No.42029.

[0032] Classification and nomenclature: Salt-tolerant Peng Zhenrong bacteria ( Pengzhenrongellahalotolerans ); Biomaterials derived from ginseng: XF202; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: June 18, 2024; Registration number at the Preservation Center: CGMCC No. 30991. Attached Figure Description

[0033] Figure 1These are colonies formed by strain CN1001 after culturing on PDA medium at 28°C for 7 days. A is the front view; B is the back view.

[0034] Figure 2 A phylogenetic tree was constructed based on the ITS sequence of strain CN1001 and its closely related bacteria.

[0035] Figure 3 The colony formed by strain XF202 after culturing at 28°C for 48 hours on ISP2 medium.

[0036] Figure 4 This is a thin-layer chromatography image of the cell polar lipids of strain XF202.

[0037] Figure 5 A phylogenetic tree constructed based on the 16S rRNA gene of strain XF202.

[0038] Figure 6 A phylogenetic tree constructed based on the whole genome sequence analysis of strain XF202. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] Example 1: Isolation, screening, mutagenesis, and strain identification of Monascus purpureus according to the present invention 1. Strain isolation The *Monascus purpureus* strain of this invention was isolated from a soil sample taken from a farmhouse tofu workshop in Kunming, Yunnan Province. The specific procedures for strain isolation are as follows: 2g of soil sample was weighed, and a soil suspension was prepared using sterile physiological saline and serially diluted to obtain a 10-1... -4 Soil suspension diluted to a certain degree. Take 0.2 mL of 10... -4 The diluted soil suspension was evenly spread on PDA agar plates (formulation: potato extract 3 g / L). -1 20 g / L glucose -1 15g / L agar powder -1 Mix with tap water until homogeneous, bring the volume to 1000 mL (pH 6.5), and incubate at 28°C for 7 days to isolate filamentous fungi.

[0042] Single colonies of filamentous fungi were picked from PDA medium and transferred to freshly prepared PDA plates for observation of colony morphology. Strains with different morphologies were selected and numbered accordingly. The pure strain was preserved in liquid nitrogen and frozen at -80°C using 20% ​​(volume percentage) glycerol as a preservative.

[0043] 2. Observation of bacterial cell morphology and detection of physiological and biochemical characteristics Referring to the Flora of Fungi (Volume 35) - Genus *Moldula* and Bergey's Manual of Identification and the Handbook of Fungal Identification, the colony and cell morphology of the filamentous fungi isolated above were observed and their physiological and biochemical characteristics were detected using PDA agar solid medium and liquid medium without agar. The growth temperature range was 4, 10, 15, 20, 28, 30, 32, 35, 37, 40, and 45℃; the growth salt concentration (NaCl) range was 11 concentration gradients (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 g / 100 ml) between 0 and 10% (0-10 g / 100 ml); and the growth pH range was 11 gradients between 4 and 9 (pH 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9). Morphological observation revealed 10 strains of *Monascus purpureus* with the following morphological characteristics: spherical cleistothecia containing ascospores, borne at the tips of hyphae, and partially enclosed by hyphae, consistent with the characteristics of *Monascus purpureus*. Combined with physiological and biochemical characteristic tests, the following four strains were identified: a growth temperature range of 20-35℃, with an optimal growth temperature of 28℃; a tolerance range of 0-8% NaCl, with an optimal NaCl concentration of 0-4%; and the ability to grow in a pH range of 5-7.5, with optimal growth at pH 7.0. These strains underwent ITS identification.

[0044] 3. ITS identification of bacterial strains Total DNA was extracted from *Monascus purpureus* using the CTAB method. PCR amplification was performed using universal fungal primers ITS1 and ITS4. The PCR reaction mixture consisted of: 0.6 μL ITS1 primer, 0.6 μL ITS4 primer, 1 μL template, 4 μL dNTP mix, 10 μL 2×KOD buffer, 0.4 μL KOD FX, and 4.4 μL ddH2O. Amplification conditions were: pre-denaturation: 94℃, 5 min; 31 cycles: 98℃, 10 s; 56℃, 45 s; 68℃, 2 min; extension: 68℃, 5 min. The sequence of primer ITS1 was 5'-TCCGTAGGTGAACCTGCGG-3', and the sequence of primer ITS4 was 5'-TCCTCCGCTTATATGC-3'. The amplified products were validated by agarose gel electrophoresis and then sequenced. Sequencing results were compared using the BLAST algorithm on NCBI to identify closely related strains.

[0045] The ITS sequence alignment results of the above four strains showed that they all belong to the Monascus genus.

[0046] 4. Determination of the strain's ability to produce lovastatin and penicillin The detection of lovastatin and citric acid was performed in accordance with the methods in "QB / T 2847-2023 Functional Red Yeast Rice (Powder)" and the literature (Zhan Ziyue; Zhao Ziwei; Wang Xiuli; Xie Ji'an; Liu Bolin; Identification and Quantitative Analysis of Two Lovastatins in Red Yeast Rice Health Food by High Performance Liquid Chromatography [J]; Analytical Laboratory; 2025(3):378-382; Peng Bining, Yu Chuangbo, Zeng Chuan, et al. Optimization Study on Detection Method of Citric Acid in Red Yeast Rice Pigment. China Food Additives, 2022, 33(8):208-215.) and optimized. The specific operation is as follows: (1) Fermentation culture of Monascus purpureus and pretreatment of fermentation products Seed culture medium formula: 50g glucose, 10g peptone, 10g yeast extract, 1g KH2PO4, 0.01g FeSO4·7H2O, 0.5g MgSO4·7H2O, diluted to 1L with deionized water, pH 7.0.

[0047] Preparation of seed culture medium: Spores of *Monascus purpureus* were scraped from PDA slant culture medium cultured at 28℃ for 7 days and transferred to the above seed culture medium. The medium was then cultured on a shaker at 180 rpm for 7 days to obtain the seed culture medium. The seed culture medium was then adjusted to a spore concentration of 102 using sterile physiological saline. 8 A spore suspension of CFU / mL is referred to as seed solution.

[0048] Rice-soybean flour fermentation medium: 180g indica rice, 20g soybean flour, add 200mL deionized water, stir with a glass rod until fully mixed, and adjust the pH to pH 7. After sterilization, let the rice-soybean flour medium cool (below 40℃), then inoculate with a medium containing 10... 8 4 mL of seed culture with a spore concentration of CFU / mL (i.e., 2 mL of seed culture per 100 g of rice-soybean flour medium, where the medium mass is calculated based on the sum of the masses of rice and soybean flour) was added. The culture was kept statically at 28℃ and 40% humidity, with the medium loosened every 3 days until it reached a loose state, and this process was continued for 40 days. During solid-state fermentation, samples were taken every 5 days in a clean bench (days 5, 10, 15, 20, 25, 30, 35, and 40) and stored for testing. Each time point sampling was performed in triplicate: samples were taken from different culture bottles for testing.

[0049] Sample pretreatment: The solid-state fermentation product was extracted with a 1:1 (w / v) 75% ethanol solution. Specifically, 100 mL of 75% (v / v) ethanol solution was added to 100 g of solid-state fermentation product, vortexed to mix, and soaked overnight. The mixture was then centrifuged at 4000 rpm for 30 minutes. The supernatant was evaporated to dryness under low temperature, dissolved in 2 mL of 75% (v / v) ethanol solution, and transferred to a centrifuge tube. The mixture was vortexed for 5 minutes to ensure thorough mixing; then ultrasonicated at 40 W for 30 minutes; finally, centrifuged at 10000 rpm for 5 minutes, and the supernatant was collected for analysis.

[0050] (2) Preparation of standard solutions Lactone lovastatin standard: CAS No.: 75330-75-5, catalog number B20806-1g, purity HPLC ≥ 98%.

[0051] Citrulline standard: CAS No.: 518-75-2, Catalog No. B25912-1mg, Purity HPLC ≥ 98%.

[0052] Lactone lovastatin stock solution: Accurately weigh 20 mg of lactone lovastatin standard, dissolve it in a small amount of methanol, transfer it to a 100 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain a lactone lovastatin stock solution with a concentration of 0.2 mg / mL, labeled as LL.

[0053] Citrus penicillin stock solution: Prepare a citrus penicillin stock solution with a concentration of 0.2 mg / mL using the same method, and label it as JC.

[0054] Lovastatin acid stock solution: Weigh 20 mg of lovastatin lactone standard, dissolve in 10 mL of methanol, sonicate in 0.2 mol / L NaOH solution for 40 min, cool to room temperature, adjust the pH to 7.7 with 3 mol / L phosphoric acid solution, and add methanol to a final volume of 100 mL to obtain a 0.2 mg / mL lovastatin acid standard solution, labeled HL.

[0055] (3) Detection and calculation results: The sample obtained in step (1) was detected using an Agilent 1200 high performance liquid chromatograph (HPLC) with a Shiseido C18 column. The detection wavelength was set to 238 nm, the column temperature was 30 °C, and the mobile phase was as shown in Table 1.

[0056] Table 1. Mobile phase (% represents volume percentage)

[0057] Note: Water-0.1% phosphoric acid is an aqueous solution containing 0.1% (volume percentage) phosphoric acid.

[0058] Record the peak areas and calculate the contents of lactone lovastatin (LL) and acid lovastatin (HL) in the test samples. The sum of these values ​​is the total lovastatin yield. Simultaneously, detect the presence of penicillin in the test samples. Measure the samples from three replicates and calculate the average of the three measurements as the result.

[0059] One strain of Monascus purpureus, CN100, was found to have a total lovastatin yield of (10.7 ± 0.2) mg / g, meaning approximately 10.7 mg of lovastatin was obtained per gram of dry weight of culture medium. The lovastatin yield produced by strain CN100 from rice-soybean flour fermentation was significantly higher than that of other strains, and this strain did not produce citric acid. The highest yield of strain CN100 in rice-soybean flour fermentation was observed on day 30, with the results on days 35 and 40 remaining largely consistent with those on day 30.

[0060] 5. Ultraviolet mutagenesis irradiation of Monascus purpureus strains Using Monascus purpureus CN100 as the starting strain, a high-lovastatin-producing mutant strain was screened through ultraviolet mutagenesis. The specific procedures are as follows: Spores of Monascus purpureus strain CN100, grown at 28°C for 7 days on PDA solid medium, were collected and suspended in sterile physiological saline to prepare a solution with a concentration of 1×10⁻⁶. 8A spore suspension of 1 spore / mL was prepared. 5 mL of this spore suspension was placed in a 9 cm diameter petri dish (containing two sterile small stir bar), stirred evenly on a magnetic stirrer, and irradiated with a 15W UV lamp (preheated for 30 min) at a distance of 30 cm for 1 h. The irradiated bacterial solution was then placed in a dark chamber for 2 h.

[0061] Then, 0.2 mL of bacterial suspension was spread onto PDA medium and incubated at 28°C for 7 days. Well-grown single colonies were selected and numbered CN1001, CN1002, ..., CN1019. The production of lovastatin and citric acid by strains CN1001, CN1002, ..., CN1019 obtained through UV mutagenesis was determined according to the method described in step 4 above. The yields of lovastatin and citric acid obtained by strains CN100, CN1001, CN1002, ..., CN1019 on day 30 of fermentation in rice-soybean flour medium are shown in Table 2.

[0062] Table 2. Statistics on the production of lovastatin and penicillin by the test strains on rice-soybean flour medium up to day 30.

[0063] Note: - indicates not detected, LL indicates lactone lovastatin, and HL indicates acid lovastatin.

[0064] The results showed that strain CN1001, obtained through UV mutagenesis, had the highest lovastatin yield among the 19 mutagenized strains, at (32.1±0.1) mg / g, approximately three times that of the original strain CN100; none of the 19 mutagenized strains produced citric acid. The total lovastatin yield of strain CN1001 reached its peak on day 30 of fermentation of rice-soybean flour.

[0065] 6. Genetic stability test of Monascus purpureus CN1001 Strain CN1001 was passaged using PDA slant medium, and the passages were designated CN1001, CN1001-1, CN1001-2, ..., CN1001-30. Morphological observation and culture condition testing were performed on CN1001 and its 1st to 30th generations using PDA medium. The production of lovastatin and citric acid was determined according to the methods described in "4. Determination of Lovastatin and Citric Acid Production by the Strains" above. The ITS (Intracytoplasmic Strain) of CN1001 and its passages was measured, and the results were compared and analyzed.

[0066] The test results showed that the genetic traits of strain CN1001 were stable between the 1st to 30th generations of the strain CN1001 and strain CN1001.

[0067] 7. Identification results of strain CN1001 After incubating strain CN1001 on PDA medium at 28°C for 7 days, the resulting colonies are as follows: Figure 1 The colonies are orange-red and do not produce soluble pigments. They form spherical cleistothecia containing ascospores, which are borne at the tips of the hyphae and partially enclosed by them. The growth temperature range is 20-35℃, with an optimum growth temperature of 28℃; it tolerates NaCl concentrations of 0-8%, with an optimum concentration of 0-4%; it can grow in a pH range of 5-7.5, with optimal growth at pH 7.0. The ITS sequence of strain CN1001 (containing the nucleotide sequence shown in SEQ ID NO:1) and *Monascus purpureus* are compared. Monascus purpureus IFO 4513 and Monascus purpureus Monascus pilosus ATCC16363 showed the highest similarity, at 99.6% and 99.2%, respectively. In the phylogenetic tree constructed based on the ITS sequences of strain CN1001 and Monascus spp. and its closely related species, strain CN1001 is similar to Monascus spp. Monascus pilosus ATCC 16363, Monascus purpureus Monascus purpureus IFO 4513 clustered on the same evolutionary subbranch, and strain CN1001 was associated with Monascus purpureus. Monascus pilosus ATCC 16363 is the closest in evolutionary distance ( ).

[0068] Based on the morphological characteristics and phylogenetic analysis of strain CN1001, it was confirmed that strain CN1001 is a member of the genus Monascus. Monascus (… Figure 2 sp.) CN1001 was deposited at the China General Microbiological Culture Collection Center on June 10, 2025, with the accession number CGMCC No. 42029. Hereinafter referred to as strain CN1001 or Monascus purpureus strain CN1001.

[0069] Example 2: Isolation and identification of salt-tolerant *P. pengzhenrong* XF202 I. Strain Isolation Soil samples were collected from a rural tofu workshop in Kunming, Yunnan Province. The isolation medium for strain XF2022 was ISP2 medium. The solvent for ISP2 medium was water, and the solutes and concentrations were as follows: yeast extract 4.0 g / L, malt extract 10.0 g / L, glucose 4.0 g / L, agar 15.0 g / L, pH 7.5. Inhibitors were added to the medium at the following concentrations: potassium dichromate 30 μg / L, nystatin 50 μg / L.

[0070] Methods and procedures for strain isolation, purification, and preservation: Collected soil samples were placed in a sterile, ventilated area and air-dried at room temperature for 2 weeks, followed by dry heat treatment at 120 °C for 15 min. 2 g of the dry heat-treated soil sample was then serially diluted with sterile physiological saline. A dilution gradient of 10 was obtained from the prepared samples. -4 Take 0.3 mL of the soil suspension and spread it onto an isolation plate, then incubate at 28°C for 4 weeks. Pick single colonies of different morphological bacteria and actinomycetes from the same plate and transfer them to a pre-prepared ISP2 slant medium; isolate single colonies using the quartering method. Store the purified bacterial strains on slants at 4°C for short-term preservation; or store them in tubes containing 20% ​​(v / v) glycerol at -80°C for long-term preservation.

[0071] In this experiment, a strain numbered XF202 was obtained.

[0072] II. Identification of strain XF202 1. Observation of cell morphology and detection of physiological and biochemical characteristics of strain XF202 The growth temperature range for strain XF202 was 4, 10, 15, 28, 30, 32, 37, 40, 42, and 45 °C; the growth salt concentration (NaCl) range was 12 concentration gradients (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15) between 0 and 15% (0-15 g / 100 ml); and the growth pH range was 8 gradients (4, 5, 6, 7, 8, 9, 10, 11) between pH 4 and 11. The physiological and biochemical characteristics of the strain were determined using API 50CH, API ZYM, and BIOLOG GEN III carbon source assay kits. Other physiological and biochemical characteristics of the strain, including Gram staining properties, oxygen requirement, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity, and cellulose hydrolysis activity, were mainly determined according to the *Handbook of Actinomycete Systematics* (Xu L H. *Actinomycetesystematics: principles, methods and practices* [M]. Beijing: Science Press, 2007, 93-108.). The identification results showed that strain XF202 is a chemoheterotrophic aerobic bacterium, and it showed a positive Gram staining reaction. The cells are short rod-shaped, non-motile, and do not produce endospores. It forms pale yellow, round colonies on ISP2 medium, with a dry colony surface, neat edges, and a maximum colony diameter of 0.9-1.0 mm. MonascusIt can grow in a pH range of 6.0-10.0, a NaCl concentration range of 0-8.0%, and a temperature range of 15-30℃; the optimal growth conditions are pH 7.0, a NaCl concentration of 6-7%, and a temperature range of 25-28℃. It can grow on ISP (International Streptomyces Research Programme) media. It can ferment the following carbon sources to produce acids: glucose, fructose, mannose, sucrose, inulin, raffinose, gluconate, and 2-keto-gluconate. It exhibits alkaline phosphatase, lipoesterase (C4), leucine amidase, α-glucosidase, and N-acetyl-β-glucosidase activities, and is positive for starch hydrolysis. It is negative for aescin hydrolysis, hydrogen sulfide production, gelatin liquefaction, oxidase, VP test, and methyl red test. The distinguishing characteristics of strain XF202 and other valid descriptive species of the genus *P. pengzhenrong* are shown in Table 3.

[0073] Table 3. Distinguishing features of strain XF202 and other valid descriptive species of the genus *Peng Zhenrong*

[0074] 2. Detection of cytochemical components of strain XF202 The cytochemical components of strain XF202, including fatty acids, quinone types, and polar lipids, were detected by gas chromatography (GC), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC) (Sasser M. Identification of bacteria by gasghromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE:MIDI inc;1990. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye Met al. An integrated procedure for the extraction of bacterial isoprenoidquinones and polar lipids. J Microbiol Methods 1984;2:233–241.). The fatty acid composition of strain XF202 is shown in Table 4. The results showed that the main fatty acid of strain XF202 was antesio-C. 15: 0 C 16:0 and anteiso-C 15 : 1A; In strain XF202, the dominant methylnaphthoquinone is MK-9(H4); polar lipids are mainly composed of phosphatidylglycerol (PG) and phosphatidylinositol (PI), such as Figure 3 As shown.

[0075] Table 4. Fatty acid composition of strain XF202 and its closely related bacteria

[0076] Note: TR indicates <1.0%; - indicates not detected.

[0077] In summary, the analysis results of the dominant fatty acid composition, major respiratory quinones, and polar lipid components of strain XF202 support the classification of strain XF202 into the genus *Peng Zhenrong*. Furthermore, the specific fatty acid composition of strain XF202 can be distinguished from other species in the genus *Peng Zhenrong*. For example, the type strains of closely related species in the genus *Peng Zhenrong* share the same dominant fatty acid composition, all containing antesio-C. 15:0 and C 16:0 As the main component. However, strain XF202 also contains a large amount of antesio-C. 15:1 A; while iso-C 16:0 The content was high in other strains but low in strain XF202 (Table 4). These results support the view that strain XF202 represents a new species of the genus *Peng Zhenrong*.

[0078] 3. Determination of the phylogenetic position of strain XF202 Genomic DNA was extracted from strain XF202 and sequenced. The 16S rRNA gene sequence was then compared online in an internationally authoritative bacterial taxonomy database (http: / / www.ezbiocloud.net / ) (Kim OS, ChoYJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA genesequence database with phylotypes that represent uncultured species. Int JSyst Evol Microbiol, 62: 716-721.). The similarity values ​​of the 16S rRNA gene sequence (1423 bp, as shown in SEQ ID NO:2) of strain XF202 with known bacterial species are listed in Table 5. The results showed that strain XF202 of this invention had the highest similarity to species of the genus *Peng Zhenrong*, with a similarity range of 97.6-98.8%. Figure 4The 16S rRNA gene similarity of CGMCC 1.11116 was higher than the threshold for distinguishing prokaryotes (98.65%) (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequences similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014;64:346–351.). Therefore, the average nucleotide identity (ANI) of the whole genome sequences of strain XF202 and its closely related strains was further compared and analyzed (https: / / www.ezbiocloud.net / tools / orthoani). The ANI values ​​of strain XF202 and its closely related bacteria were 78.2-78.6% (Table 5), all lower than the gold standard value of 95-96% for defining prokaryotic species (Chun, J., Oren, A., Ventosa, A., Christensen, H., Arahal, DR, Da Costa, MS, Rooney, AP, Yi, H., Xu, XW, De Meyer, S., and Trujillo, ME (2018). Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst EvolMicrobiol 68, 461-466.). Based on this, strain XF202 was confirmed to represent a new species within the genus *Peng Zhenrong*, distinct from the other three species.

[0079] Table 5. Information on Close Relatives of Strain XF202

[0080] Phylogenetic trees were constructed using the 16S rRNA gene sequences of all valid species of the genus *P. pengzhenrong* and some species of species from neighboring genera in the order Micrococcidales. Pengzhenrongella frigida The results show that strain XF202 is included within the genus *P. pengensis*, and that within the genus *P. pengensis*, effectively descriptive species cluster on a stable evolutionary branch, with strain XF202 occupying a species position. This is shown in the phylogenetic tree constructed from whole-genome sequencing.Figure 5 Strain XF202 is closely related to three other species in the genus *Peng Zhenrong*, occupying a separate subbranch within the order Micrococciales, stably holding a species position. Whether in a phylogenetic tree constructed based on 16S rRNA genes or a phylogenetic tree constructed based on whole-genome sequence analysis, strain XF202 occupies an independent evolutionary subbranch of a species. Therefore, strain XF202 is confirmed to represent a new species within the genus *Peng Zhenrong*.

[0081] Genome sequence analysis of strain XF202 revealed that the G+C content of the strain XF202 genome was 72.8%.

[0082] Based on the analysis of polymorphic taxonomic research data, the strain XF202 of this invention exhibits some significant differences from existing scientifically studied effective descriptive species of the genus *Peng Zhenrong*, including phenotypic, physiological and biochemical, and cytochemical components. Furthermore, phylogenetic analysis at the gene level further demonstrates that strain XF202 can be distinguished from existing effective species of the genus *Peng Zhenrong*, fully proving that strain XF202 of this invention represents a new species of the genus *Peng Zhenrong*, named *Salt-Tolerant *Peng Zhenrong*. Figure 6 ).

[0083] Salt-tolerant Peng Zhenrong bacteria ( Pengzhenrongella halotolerans Pengzhenrongella halotolerans XF202 was deposited at the China General Microbiological Culture Collection Center on June 18, 2024, with the accession number CGMCC No. 30991. Hereinafter referred to as strain XF202 or salt-tolerant Peng Zhenrong strain XF202.

[0084] Example 3: Test for lovastatin production by co-fermentation of Monascus purpureus CN1001 and salt-tolerant Bacillus thuringiensis XF202. 1. The production of lovastatin by strain XF202 was detected by culturing it alone on rice-soybean flour medium. Seed culture medium formula: 50g glucose, 10g peptone, 10g yeast extract, 1g KH2PO4, 0.01g FeSO4·7H2O, 0.5g MgSO4·7H2O, diluted to 1L with deionized water, pH 7.0.

[0085] Preparation of seed culture medium: Mycelial growth of strain XF202 was picked from ISP2 slant culture medium cultured at 28℃ for 4 days and transferred to the above seed culture medium. The culture was then incubated on a shaker at 180 rpm for 4 days to obtain the seed culture medium. The seed culture medium was then adjusted to a cell concentration of 102 using sterile physiological saline. 8 A bacterial suspension of CFU / mL is referred to as seed culture.

[0086] Rice-soybean flour fermentation medium: 180g indica rice, 20g soybean flour, add 200mL deionized water, stir with a glass rod until fully mixed, and adjust the pH to pH 7. After sterilization, cool the rice-soybean flour medium (below 40℃) and inoculate with a medium containing 10... 8 4 mL of seed culture with a spore concentration of CFU / mL (i.e., 2 mL of seed culture per 100 g of rice-soybean flour medium, where the medium mass is calculated based on the sum of the masses of rice and soybean flour) was added. The culture was kept statically at 28℃ and 40% humidity, and the medium was loosened by tapping every 3 days until it was in a loose state. This culture was continued for 40 days. During solid-state fermentation, samples were taken every 5 days in a clean bench (5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, and 40 days) and stored for testing.

[0087] The sample pretreatment and specific detection methods are the same as in step 4 of Example 1.

[0088] The results showed that when strain XF202 was inoculated alone on rice-soybean flour medium and fermented for 40 days, neither lovastatin nor penicillin was detected.

[0089] 2. Detection of lovastatin produced by co-fermentation of strain CN1001+XF202 (1) Following the method in step 4 of Example 1, the concentration of spores containing Monascus purpureus CN1001 was 10. 8 CFU / mL seed culture was inoculated into rice-soybean flour medium (2 mL of seed culture was added to every 100 g of rice-soybean flour medium, where the medium mass was calculated based on the sum of the masses of indica rice and soybean flour), and incubated statically at 28°C and 40% humidity for 14 days. The medium was shaken every three days.

[0090] (2) On day 14, the concentration of cells containing strain XF202 was 10 8 Inoculate the above fermentation culture with CFU / mL seed culture (2 mL of seed culture per 100 g rice-soybean flour medium, where the medium is calculated based on the sum of the masses of indica rice and soybean flour). Continue static culture at 28°C and 40% humidity for 30 days. Shake the medium every three days.

[0091] (3) During the above fermentation process, samples were taken on days 3, 6, 9, 12, 14, 17, 18, 19, 20, 21, 24, 27 and 30 to detect the content of lovastatin and penicillin in the fermentation products. (4) Detect according to the method described in step 4 of Example 1.

[0092] The results showed that the highest total lovastatin yield was achieved by co-fermenting strain CN1001+XF202 on day 20, with a yield of (44.9±0.15) mg of lovastatin per gram of dry weight of culture medium, which is equivalent to a lovastatin yield of (44.9±0.15) mg / g. Specific data are shown in Table 6.

[0093] Table 6. Statistical table of lovastatin and penicillin yield during co-fermentation of strain CN1001+XF202

[0094] Note: - indicates not detected.

[0095] This invention demonstrates that a *Monascus* species producing lovastatin but not citric acid, and a novel actinomycete strain XF202, were isolated from soil samples from a rural tofu workshop in Kunming, Yunnan Province. Through ultraviolet irradiation mutagenesis, a high-lovastatin-producing strain CN1001 was screened. Genotypic and phenotypic data of strain XF202 confirmed it to be a new species within the *Peng Zhenrong* genus, named *Peng Zhenrong* tolerant. *Monascus* strain CN1001 alone produced 32.1 mg / g of lovastatin from rice-soybean flour fermentation without producing citric acid. Co-fermentation of rice-soybean flour with strains CN1001 and XF202 produced 44.9 mg / g of lovastatin without producing citric acid, and shortened the fermentation time (from 30 days to 20 days). The rice-soybean flour culture medium does not contain expensive raw materials, and is inexpensive and readily available. Therefore, the combined microbial agent CN1001+XF202 is an eco-friendly, highly efficient, and low-toxicity high-quality microbial agent with good application potential in the pharmaceutical and food industries.

[0096] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A combination of bacteria, characterized in that: The combined bacteria consist of Monascus purpureus and salt-tolerant Peng Zhenrong bacteria; The red mold is Monascus purpureus (Monascus purpureus) Monascus sp. CN1001, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.42029; The salt-tolerant Peng Zhenrong bacteria is a salt-tolerant Peng Zhenrong bacteria ( Pengzhenrongellahalotolerans Its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 30991.

2. Salt-tolerant *Peng Zhenrong* bacteria, characterized by: The salt-tolerant Pengzhenrong bacterium is Pengzhenrongella halotolerans, and its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 30991.

3. Monascus purpureus, characterized by: The red mold mentioned is Monascus sp. CN1001, which has the registration number CGMCC No.42029 at the China General Microbiological Culture Collection Center.

4. A product for the fermentation production of lovastatin, comprising the combined strains of claim 1 or the Monascus purpureus of claim 3.

5. The product according to claim 4, characterized in that: The product also contains a rice-soybean flour culture medium.

6. Any of the following applications: (A1) The use of the combined bacteria of claim 1 or the Monascus purpureus of claim 3 in the preparation of a product for the fermentation production of lovastatin; (A2) The use of the combined bacteria of claim 1, the Monascus purpureus of claim 3, or the product of claim 4 or 5 in the fermentation production of lovastatin.

7. A method for fermenting lovastatin, comprising the following steps: (1) Using the combined bacteria of claim 1 to ferment lovastatin. (A1) The red mold was inoculated into rice-soybean flour culture medium and subjected to the first stage of solid fermentation culture to obtain the first stage fermentation culture; (A2) The salt-tolerant Peng Zhenrong bacteria are inoculated into the fermentation culture obtained in step (A1) and a second-stage solid-state fermentation culture is carried out to obtain a second-stage fermentation culture; (A3) Lovastatin was obtained from the second-stage fermentation culture obtained in step (A2).

8. The method according to claim 7, characterized in that: In step (A1), during the first stage of solid-state fermentation culture, the culture temperature is 28℃ and the culture time is 14-18 days; and / or In step (A2), during the second stage of solid-state fermentation culture, the culture temperature is 28°C and the culture time is 5-16 days.

9. A method for producing lovastatin by fermentation, comprising the following steps: inoculating the red mold into a rice-soybean flour culture medium, performing solid-state fermentation culture, and obtaining lovastatin from the resulting fermentation culture.

10. The method according to claim 9, characterized in that: When carrying out the solid-state fermentation culture, the culture temperature is 28℃ and the culture time is 18-34 days.

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