Paracoccus Wheatstone and application thereof

Abstract

The invention belongs to the technical field of biology, and provides a paracoccus Wheatstone strain and application thereof, the paracoccus Wheatstone strain can produce pyrroloquinoline quinine at high yield, the strain is named as YJY25-02, and the preservation number is CGMCC NO.34258. The strain is subjected to shake-flask aerobic culture for 83h, and the titer of the pyrroloquinoline quinone in fermentation liquor is 176.7 mg / L; the titer of pyrroloquinoline quinine in fermentation liquor during aerobic culture in a fermentation tank is 1796-1863 mg / L, the methanol conversion rate is 16.19-18.12 mg / g, the method has the advantages of short fermentation time, high titer and high methanol conversion rate, and the problem of large-scale production can be solved; pure pyrroloquinoline quinine with the purity larger than or equal to 98% is prepared through separation and purification of fermentation liquor, the yield ranges from 80% to 85%, the high yield and high purity of the pure pyrroloquinoline quinine are achieved, the separation and purification process is simple, the cost is low, organic solvents difficult to remove are not introduced, and the safety of the product is guaranteed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and provides a Parvularubellus sp. and application thereof, and particularly relates to application of the Parvularubellus sp. in production of pyrroloquinoline quinone. BACKGROUND

[0002] Pyrroloquinoline quinone (hereinafter referred to as PQQ) has the chemical name of 4,5-dihydro-4,5-dioxo-1-hydrogen pyrrole (2,3f) quinone-2,7,9-tricarboxylic acid, and an alias of Methoxatin. In 1979, Salisbury et al. first isolated it from methylotrophic bacteria, and determined that it is an oxidation-reduction cofactor of membrane-bound dehydrogenase in bacterial cells, and it is the third auxiliary group found after flavin nucleotides and nicotinamide nucleotides, and is called the fourteenth vitamin in the world medical field. PQQ, as a new type of water-soluble vitamin, is an oxidation-reduction enzyme auxiliary group, and its source is very rare and can only be synthesized in some gram-negative bacteria, and mammals cannot synthesize it by themselves, and mainly obtain it through the dietary route. PQQ exists in daily vegetables and fruits, such as natto, celery and kiwi fruit, etc.

[0003] Studies have shown that PQQ has physiological functions such as participating in the growth and differentiation of mammalian cells, enhancing the tolerance of cells to extreme environments, and increasing the availability of insoluble phosphate. Some recent studies have found that PQQ, as a necessary factor for human development, stimulates human cell growth, such as human B cells and T cells, so that antibodies are produced, thereby improving human immune function; and PQQ has an exceptionally high free radical scavenging ability, and can prevent and treat the damage to the body caused by excessive free radicals and trigger various diseases such as various inflammations and cancers. In 2003, Japanese scientist Kasahara published a paper in Nature suggesting that PQQ should be classified as a vitamin, and in 2018, American cancer and anti-aging expert Bruce N. Ames published a paper in PNAS defining PQQ as a “longevity vitamin”. At present, PQQ is classified as a vitamin in the United States, Japan and the European Union, and is sold as a dietary supplement. In recent years, the distribution, mechanism, structure, function and biological properties of PQQ have been very active in international research, and the research on pyrroloquinoline quinone as a new drug also has important practical significance.

[0004] Currently, the production methods of PQQ are mainly chemical synthesis and fermentation. The chemical synthesis method has complex process, serious pollution, and production scale is strictly controlled, which cannot meet the rapidly growing market demand. The microbial fermentation method has gradually become a new research direction, which has the advantages of green environmental protection, low cost, few steps and easy separation, but there is a problem of lack of excellent industrial production strains. Patent CN 103898011 B discloses a methylo pilas p producing pyrroloquinoline quinone and its application, and the PQQ shake flask production capacity is 50-113 mg / L, the titer is relatively low, and it is difficult to realize industrial scale production; patent CN 106282044 B discloses a kind of raw silk microorganism (Hyphomicrobium sp) and a preparation method of pyrroloquinoline quinone. The strain can reach 1783 mg / L on the fermentation tank, but the consumption of methanol is too much, and the conversion rate is low. In patent CN 113981021 B, when the paracoccus amnioniphilus is used for fermentation synthesis of PQQ, the final PQQ yield is more than 1 g / L after 6-8 days of fermentation tank culture, the titer is relatively low, and the culture time is long, which is not conducive to industrial production. 3

[0005] Therefore, breeding a high-yield PQQ production strain with high methanol conversion rate has important significance for accelerating the industrialization process of microbial fermentation method for producing PQQ. SUMMARY

[0006] The present application provides a Paracoccus haeundaensis, which can produce pyrroloquinoline quinone in high yield. The inventors named the strain YJY25-02, and the preservation number is CGMCC NO.34258. Verification found that the pyrroloquinoline quinone titer of the fermentation broth was 176.7 mg / L after 83 h of shake flask aerobic culture; the pyrroloquinoline quinone titer in the fermentation broth was 1796-1863 mg / L, and the methanol conversion rate was 16.19-18.12 mg / g under the conditions of maintaining pH and flow adding methanol to supplement carbon source during the fermentation process after 120-144 h of aerobic culture in the fermentation tank, which has the advantages of short fermentation time, high titer and high methanol conversion rate, and can solve the problem of large-scale production; pyrroloquinoline quinone pure product with a purity of ≥98% can be prepared by separating and purifying the above-mentioned fermentation broth, and the yield is 80%-85%, which realizes high yield and high purity of pyrroloquinoline quinone pure product, and the separation and purification process is simple, the cost is low, no organic solvent is introduced in the production process, and the safety of the product is ensured. The immune-enhancing and anticancer product prepared by compounding the above-mentioned pyrroloquinoline quinone pure product with grifola frondosa extract has significant immune-enhancing and anticancer effects, and has a broad market prospect.​

[0007] The specific technical solutions provided by the present application are as follows: The inventors first provide a Paracoccus zeaxanthinifaciens which can produce pyrroloquinoline quinone at a high yield, and name the strain YJY25-02, and the preservation number is CGMCC NO.34258.

[0008] The morphological characteristics of the strain are as shown in the following table: Figure 1 And 2 The flat plate culture colony is round and convex, the surface is smooth and moist, the edge is neat, and the color is milky yellow.

[0009] The source of the above-mentioned strain YJY25-02 is that the inventors isolate and purify single colonies from Malus micromalus Fuji ferment, and repeatedly mutate by using conventional mutagenesis methods such as UV, DMS, MMS, NTG, etc., combined with ARTP mutagenesis system. After conventional methanol enrichment medium, methanol solid plate primary screening, methanol liquid shake flask rescreening, combined with HPLC analysis, and in-depth research on its physiological and biochemical characteristics and genetic stability, the Paracoccus zeaxanthinifaciens is finally screened out. The inventors have biologically preserved the strain in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.34258. It is in a viable state after detection. It is easy to culture and has genetic stability, and can be used for the preparation of PQQ pure product and compound product.

[0010] The inventors have sequenced the 16S rDNA of the strain YJY25-02, and the sequence is as shown in SEQ ID No:1. The sequence is the full sequence of the 16S rDNA of the strain. The measured 16S rDNA sequence is subjected to BLAST comparison, and the comparison result shows that the nucleotide sequence of the 16S rDNA of the strain YJY25-02 has more than 99% homology with the nucleotide sequences of different strains of Paracoccus (genus) Paracoccus sp. ), and has 100% homology with the strain marked as Paracoccus zeaxanthinifaciens (genus) Paracoccus huijuniae ). It is identified as Paracoccus zeaxanthinifaciens (genus) Paracoccus huijuniae ).

[0011] The inventors have biologically preserved the above-mentioned strain, and the preservation information is as follows: Preservation time: April 17, 2025 Preservation unit name: China General Microbiological Culture Collection Center Preservation number: CGMCC NO.34258 Preservation unit address: No.1, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences Classification name: Paracoccus zeaxanthinifaciens (genus) Paracoccus huijuniae ).

[0012] After obtaining the above strain, the inventors further provided its application in the preparation of pyrroloquinoline quinone and immunity-enhancing and anti-cancer products. The specific preparation steps are as follows: (1) Preparation of fermentation broth of PQQ by Paracoccus hyedii YJY25-02, the specific steps are as follows: The activated Paracoccus huiyi YJY25-02 test tube slant culture was scraped with an inoculating needle to obtain a loopful of the culture medium and inoculated into a seed culture medium for aerobic culture at 30° C. for 18 to 26 hours, and the culture liquid OD600 was cultured to 0.6 to 1.0 to obtain a seed solution; the prepared seed solution was inoculated into a sterilized fermentation medium at an inoculum size of 5 to 10% (v / w), and the fermentation temperature was 30° C. and the culture was aerobic for 120 to 144 hours. During the fermentation production process, 25% ammonia water was added to maintain the pH of the system at 6.5 to 7.0, and methanol was added to maintain the methanol concentration in the fermentation system at not less than 0.5 to 1 g / L, thereby obtaining a fermentation liquid of PQQ fermented by Paracoccus huiyi YJY25-02; The PQQ titer in the fermentation broth was as follows: Figure 3 As shown, it is 1796-1863 mg / L, the volume of added methanol accounts for 11-13% of the total volume of the fermentation broth, and the methanol conversion rate is 16.19-18.12 mg / g, which is much higher than the methanol conversion rate of the existing PQQ production strain.

[0013] (2) Preparation of pure PQQ, specifically: The fermentation broth obtained in the previous step was adjusted to pH 2.5-3.5 with hydrochloric acid, and a volume ratio of 0.5‰-2‰ of a swollen, clear, and transparent flocculant chitosan was added, and the mixture was allowed to stand at 10°C-35°C for 30-60 minutes, and then a filter aid, diatomaceous earth, was added, and the filtrate was obtained after plate and frame filtration; the filtrate was passed through a 5KD-10KD ultrafiltration membrane, and the ultrafiltration clear liquid was collected; the ultrafiltration clear liquid was passed through a resin column (OH-type weakly basic anion exchange resin, specifically Shandong Dongda Resin Co., Ltd.) at a flow rate of 1BV / h. The company's weak base acrylic resin D301) was eluted from the resin column with a desorbent of the same volume as the ultrafiltration clear liquid at a flow rate of 2BV / h. The desorbent used was a 0.5wt% sodium hydroxide solution; the red and dark red effluents were collected; the pH of the effluent was then adjusted to 6.5-7.0, and the effluent was passed through a 100D-300D nanofiltration membrane to collect the nanofiltration concentrate; the nanofiltration concentrate was concentrated under reduced pressure until crystals precipitated, and the temperature was lowered for crystallization and filtration to obtain crude PQQ. Purified water was added to the crude PQQ product and dissolved at 40°C to 60°C. Activated carbon was then added, stirred for 30 minutes, filtered, and the filtrate was cooled for recrystallization, and wet PQQ was obtained by suction filtration. The wet PQQ product was washed with cold water, filtered again, and dried to obtain pure PQQ. The purity was tested to be ≥98%, and the PQQ yield was 80% to 85%.

[0014] After obtaining the PQQ pure product, the inventors further disclose its application in the preparation of an immunity-enhancing and anticancer product, which can be one of a medicine, a health product or a functional food; and its dosage form can be a capsule, a tablet or a solid or liquid beverage.

[0015] As an optional mode, the product comprises a pharmaceutically, health product or functional food acceptable carrier selected from one or more of a filler, a binder, a disintegrant, a lubricant, a suspending agent, a wetting agent, a pigment, a flavoring agent, a solvent and a surfactant.

[0016] Preferably, the inventors provide a tablet as the dosage form of the product, and the excipients used in the tablet are stachyose, malt dextrin, microcrystalline cellulose, magnesium stearate and silicon dioxide.

[0017] In order to achieve better immunity-enhancing and anticancer effects, the inventors find that the immunity-enhancing and anticancer product can further comprise a Grifola frondosa extract, which can be obtained by biological purification from Grifola frondosa mycelium or purchased from a commercial product.

[0018] The main component of the Grifola frondosa extract is Grifola frondosa polysaccharide, which is a long-chain glucan with β-1, 6-glucan as the main chain and β-1, 3-branched chains. Research shows that this unique structure makes Grifola frondosa polysaccharide have special tumor inhibition activity and immunity-enhancing effects. Its mechanism of action is to activate the immune system function of the body, activate immune effector cells, produce a large amount of immune factors, and through the host mediation of the body, the number and activity of immune cells in tumor patients can be maximized, and macrophages, natural killer cells (NK), dendritic cells, etc. are activated, and the secretion of cytokines (such as IL-12, IFN-γ) is promoted, the innate and adaptive immune responses are enhanced, and tumor cells are targeted killed; on the other hand, Grifola frondosa polysaccharide has a strong apoptosis induction effect, which can cause programmed necrosis of tumor cells. At the same time of completely removing the immune substances around the cancer cells, the activity of Myc gene is strongly inhibited to reduce the production of Myc protein, so that the cancer cells die and the cells with cancerous tendency become harmless normal cells; the Grifola frondosa extract can enhance the immunity-enhancing and anticancer effects of PQQ.

[0019] PQQ promotes the generation of new mitochondria by activating key pathways related to mitochondrial biogenesis (such as AMPK / PGC-1α); at the same time, it provides energy and antioxidant protection for the efficient operation of immune cells, while Grifola frondosa polysaccharide directly activates and regulates immune cells, and the combination of the two can more comprehensively support the overall health and response capacity of the immune system; at the same time, PQQ directly destroys the mitochondria, the energy factory of cancer cells, and Grifola frondosa polysaccharide activates immune cells to recognize and remove PQQ-damaged cancer cells, and the combination of the two can form a "physical destruction + immune cleaning" double blow, so that the Grifola frondosa polysaccharide in the technical solution provided by the present application can enhance the immune-enhancing and anticancer effects of PQQ.

[0020] As an optional mode, the weight ratio of the PQQ pure product with a purity of ≥98% to the Grifola frondosa extract is 1:2 to 1:5; preferably 1:3. Preferably, the Grifola frondosa polysaccharide content in the Grifola frondosa extract is 10wt% to 30wt%, and as long as the Grifola frondosa polysaccharide content is within the above range, the product can be applied to the above combination of the present application.

[0021] Compared with the prior art, the technical solution of the present application has the following advantages: (1) The Paracoccus yeei YJY25-02 strain has the ability to produce PQQ at a high yield, and the PQQ titer is 176.7mg / L after 83h of aerobic culture in a fermentation flask, which has a short culture time and a high titer.

[0022] (2) The Paracoccus yeei YJY25-02 strain is aerobically cultured in a fermentation tank for 120 to 144h, the pH is maintained during the fermentation process, and methanol is added to supplement the carbon source, and the PQQ titer is 1796 to 1863mg / L, and the methanol conversion rate is 16.19 to 18.12mg / g; the fermentation time in the fermentation tank is short, the titer is high, and the methanol conversion rate is high, which can solve the problem of large-scale production.

[0023] (3) The PQQ pure product with a purity of ≥98% is prepared by separating and purifying the fermentation broth, and the yield is 80% to 85%, the process is simple, the cost is low, no organic solvent difficult to remove is introduced in the production process, the safety of the product is ensured, and high yield and high purity of the PQQ pure product are achieved.

[0024] (4) The product prepared by compounding the PQQ pure product and the Grifola frondosa extract is proved to have a significant immune-enhancing and anticancer effect by the immune regulation effect on mice, the influence on the growth cycle of tumor cells in vitro, and the inhibition effect on tumor cells in vivo of nude mice, and has a broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The figure is a photograph of the colony morphological characteristics of the Paracoccus yeei YJY25-02 strain in the present application. Figure 2 Figure 1 is a photograph of the morphological characteristics of Parvibaculum lavendulae YJY25-02 under 400x magnification; Figure 3 Figure 4 is a graph showing the relationship between PQQ titer, time and biomass in the preparation of PQQ fermentation broth provided by the present application. DETAILED DESCRIPTION

[0026] The above content of the present application is further explained in detail by way of specific embodiments in the form of examples, but this should not be understood as limiting the scope of the above subject matter of the present application to the following examples. Any technology realized based on the above content of the present application falls within the scope of the present application, and the following examples are completed by using conventional prior art unless otherwise specified. The specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents, instruments or cells used are not specified by the manufacturer, and are conventional products that can be purchased on the market.

[0027] The Grifola frondosa extract used in the following examples is purchased from Zhejiang Fangge Pharmaceutical Co., Ltd.; the Grifola frondosa polysaccharide content is 10wt%-30wt%; the OH type weak alkaline anion exchange resin is specifically a weak alkaline acrylic acid resin D301 from Shandong Dongda Resin Co., Ltd.

[0028] The medium formula used is as follows: Seed medium (g / L): methanol 10, (NH4)2SO4 1, KH2PO4 1, Na2HPO4 4, MgSO4·7H2O 1, trace element solution 1 mL / L, vitamin solution 1 mL / L, pH 6.8; Fermentation medium (g / L): methanol 20, (NH4)2SO4 2, KH2PO4 1, Na2HPO4 6, MgSO4·7H2O 1, trace element solution 1 mL / L, vitamin solution 2 mL / L, pH 6.8; Trace element solution (mg / L) formula: FeSO4·7H2O 80, ZnSO4·7H2O 22.5, MnSO4·4H2O 40, CuSO4·5H2O 5, NaCl 15, Na2MoO4·2H2O 0.3, KCl 0.3, CoCl2·6H2O 0.03, H3BO3 3, CaCl2·2H2O 300; Vitamin solution (mg / L) formula: riboflavin 200, pyridoxine hydrochloride 400, thiamine hydrochloride 400, folic acid 2, nicotinic acid 400, p-aminobenzoic acid 200, calcium pantothenate 400, biotin 2, myo-inositol 2000.

[0029] HPLC detection chromatographic conditions: detection wavelength 248 / 330 nm, BEH C18 chromatographic column (2.1 mm x 50 mm, 1.7 m), column temperature 35℃, mobile phase is water: acetonitrile (95:5), volume flow rate 0.1 mL / min.

[0030] Example 1 Shake flask verification of PQQ-producing strain Paracoccus haeundaensis YJY25-02 The activated Paracoccus haeundaensis YJY25-02 test tube slant strain was scraped with a inoculation needle to 1 ring and inoculated into seed culture medium for aerobic culture at 30℃ for 24h, and the culture was cultured to OD600=0.8 to obtain a seed liquid; the prepared seed liquid was inoculated into sterilized fermentation medium at an inoculation amount of 7% (v / w), the fermentation temperature was 30℃, the rotation speed was 200 rpm, and aerobic culture was carried out for 83h; HPLC detection showed that the PQQ titer in the fermentation broth of the fermentation shake flask was 176.7mg / L. 600 =0.8, to obtain a seed liquid; the prepared seed liquid was inoculated into sterilized fermentation medium at an inoculation amount of 7% (v / w), the fermentation temperature was 30℃, the rotation speed was 200 rpm, and aerobic culture was carried out for 83h; HPLC detection showed that the PQQ titer in the fermentation broth of the fermentation shake flask was 176.7mg / L.

[0031] Example 2 Fermentation of Paracoccus haeundaensis YJY25-02 to prepare PQQ pure product (1) Preparation of PQQ fermentation broth of Paracoccus haeundaensis YJY25-02, specifically: The activated Paracoccus haeundaensis YJY25-02 test tube slant strain was scraped with a inoculation needle to 1 ring and inoculated into seed culture medium for aerobic culture at 30℃ for 24h, and the culture was cultured to OD600=0.8 to obtain a seed liquid; the prepared seed liquid was inoculated into sterilized fermentation medium at an inoculation amount of 7% (v / w), the fermentation temperature was 30℃, the rotation speed was 200 rpm, and aerobic culture was carried out for 83h; HPLC detection showed that the PQQ titer in the fermentation broth of the fermentation shake flask was 176.7mg / L. (2) Preparation of PQQ pure product, specifically: The fermentation broth of Paracoccus zeaxanthinifaciens YJY25-02 fermenting PQQ is adjusted to pH 2.5 with hydrochloric acid, 2‰ of the swelled flocculating agent chitosan is added, and then the mixture is placed at 10 ℃ for 45 min, then the filter aid diatomite is added, and the plate and frame filtration is performed to obtain the filtrate; the filtrate is subjected to ultrafiltration through a 10 KD ultrafiltration membrane, and the ultrafiltration clear liquid is collected; the ultrafiltration clear liquid is subjected to column chromatography at a flow rate of 1 BV / h, and the same volume of the eluent as the ultrafiltration clear liquid is used to elute the column at a flow rate of 2 BV / h, the eluent used is 0.5 wt% sodium hydroxide solution, and the red and dark red effluent is collected; the pH of the effluent is adjusted to 7.0, and the effluent is subjected to nanofiltration through a 300D nanofiltration membrane, and the nanofiltration concentrate is collected; the nanofiltration concentrate is subjected to vacuum concentration until crystals are precipitated, and then the temperature is reduced for crystallization and suction filtration to obtain the PQQ crude product; the PQQ crude product is dissolved in purified water at 40 ℃, then activated carbon is added, and the mixture is stirred for 30 min, filtered, and the filtrate is cooled and recrystallized, and then suction filtered to obtain the PQQ wet product; the PQQ wet product is washed with cold water, and then suction filtered again, and dried to obtain the PQQ pure product with a purity of 98.5%, and the PQQ yield is 80%.

[0032] Example 3: Preparation of PQQ pure product by fermentation of Paracoccus zeaxanthinifaciens YJY25-02 (1) Preparation of fermentation broth of Paracoccus zeaxanthinifaciens YJY25-02 fermenting PQQ, specifically as follows: The activated Paracoccus zeaxanthinifaciens YJY25-02 test tube slant strain is scraped with a inoculation needle to obtain 1 ring, which is inoculated into seed culture medium and subjected to aerobic culture at 30 ℃ for 18 h, and the culture is continued until the OD600 of the bacterial liquid is 1.0 to obtain the seed liquid; the prepared seed liquid is inoculated into sterilized fermentation medium at a inoculation amount of 5% (v / w), and the fermentation temperature is 30 ℃, and the aerobic culture is performed for 130 h, and 25% ammonia water is added during the fermentation to maintain the pH at 6.5-7.0, and methanol is added to maintain the methanol concentration in the fermentation system at not less than 0.5-1 g / L, and thus the fermentation broth of Paracoccus zeaxanthinifaciens YJY25-02 fermenting PQQ is obtained; HPLC detection shows that the PQQ titer in the fermentation broth is 1796 mg / L, the volume ratio of the added methanol to the total volume of the fermentation broth is 11%, and the methanol conversion rate is 18.12 mg / g; (2) Preparation of PQQ pure product, specifically as follows: The fermentation liquor of Paracoccus zeaxanthinifaciens YJY25-02 fermenting PQQ is adjusted to pH 3.5 with hydrochloric acid, 1.5‰ swelled flocculating agent chitosan is added, and then the mixture is placed at 20°C for 60 min, then filter aid diatomite is added, and the filtrate is obtained by plate and frame filtration; the filtrate is passed through an 8KD ultrafiltration membrane, and the ultrafiltration clear liquor is collected; the ultrafiltration clear liquor is passed through a resin column (OH type weak alkaline anion exchange resin) at a flow rate of 1 BV / h, the same volume of desorbent as the ultrafiltration clear liquor is used to elute the resin column at a flow rate of 2 BV / h, the desorbent used is 0.5wt% sodium hydroxide solution, and the red and dark red effluent is collected; the pH of the effluent is adjusted to 6.5, the effluent is passed through a 100D nanofiltration membrane, and the nanofiltration concentrate is collected; the nanofiltration concentrate is concentrated under reduced pressure until crystals are precipitated, the temperature is lowered for crystallization and suction filtration, and the PQQ crude product is obtained; the PQQ crude product is added with purified water, and dissolved at 50°C, then activated carbon is added, stirred for 30 min, filtered, the filtrate is cooled and recrystallized, and suction filtered to obtain PQQ wet product; the PQQ wet product is washed with cold water, suction filtered again, and dried to obtain PQQ pure product with a purity of 99.3%, and the PQQ yield is 82%.

[0033] Example 4 Preparation of PQQ pure product by fermentation of Paracoccus zeaxanthinifaciens YJY25-02 (1) Preparation of fermentation liquor of Paracoccus zeaxanthinifaciens YJY25-02 fermenting PQQ, specifically: Activated Paracoccus zeaxanthinifaciens YJY25-02 test tube slant strain is scraped with a inoculation needle to 1 ring, which is inoculated into seed culture medium and aerobically cultured at 30°C for 26 h, and the seed liquid is obtained when the OD600 of the culture is 0.7; the prepared seed liquid is inoculated into sterilized fermentation medium at an inoculation amount of 10% (v / w), and the fermentation temperature is 30°C, and the aerobic culture is carried out for 144 h; during the fermentation production process, 25% ammonia water is added to maintain pH 6.5-7.0, and methanol is added to maintain the methanol concentration in the fermentation system not less than 0.5-1 g / L, and the fermentation liquor of Paracoccus zeaxanthinifaciens YJY25-02 fermenting PQQ is obtained; HPLC detection shows that the PQQ titer in the fermentation liquor is 1863 mg / L, the volume ratio of the added methanol to the total volume of the fermentation liquor is 13%, and the methanol conversion rate is 16.19 mg / g; (2) Preparation of PQQ pure product, specifically: The fermentation liquid of Paracoccus zeaxanthinifaciens YJY25-02 fermenting PQQ is adjusted to pH 2.8 with hydrochloric acid, 0.5 ‰ swelled flocculating agent chitosan is added, and then the mixture is placed at 35 ℃ for 30 min, then filter aid diatomite is added, and then the mixture is filtered by a plate and frame filter to obtain a filtrate; the filtrate is filtered through a 5 KD ultrafiltration membrane to collect an ultrafiltration clear liquid; the ultrafiltration clear liquid is filtered through a resin column (OH type weak alkaline anion exchange resin) at a flow rate of 1 BV / h, the same volume of desorbent as the ultrafiltration clear liquid is used to elute the resin column at a flow rate of 2 BV / h, the desorbent used is 0.5 wt% sodium hydroxide solution, and red and dark red effluent is collected; the pH of the effluent is adjusted to 6.7, the effluent is filtered through a 200 D nanofiltration membrane to collect a nanofiltration concentrate; the nanofiltration concentrate is concentrated under reduced pressure until crystals are precipitated, the temperature is reduced for crystallization and suction filtration, and PQQ crude product is obtained; the PQQ crude product is dissolved in purified water at 60 ℃, then activated carbon is added, the mixture is stirred for 30 min, filtered, the filtrate is cooled and recrystallized, and suction filtration is performed to obtain PQQ wet product; the PQQ wet product is washed with cold water, suction filtration is performed again, and drying is performed to obtain PQQ pure product with a purity of 98.8%, and the yield of PQQ is 85%.

[0034] Comparative Example 1: The strains of the patent CN 103898011 B and CN 106282044 B are used for fermentation according to the seed culture medium and the fermentation medium described in the specification, and HPLC is used for detecting the PQQ titer, and the corresponding PQQ production capacity is compared with the technical solutions described in Examples 1-4 of the present application as shown in Table 1. Table 1: Comparison of PQQ production capacity of the strains obtained by the present application and the strains of the patent CN 103898011 B and CN 106282044 B According to the comparison of the PQQ production capacity of the strains obtained by the present application and the strains of the patent CN 103898011 B and CN 106282044 B, it can be seen that the strains obtained by the present application have short fermentation time, high titer and high methanol conversion rate, and have obvious progress compared with the existing strains.

[0035] Example 5: Preparation of an immune-enhancing and anticancer product The immune-enhancing and anticancer product group and preparation method are as follows: according to weight parts, 20 parts of PQQ pure product prepared in Example 2, 60 parts of Grifola frondosa extract, and 150 parts of stachyose are taken; the above components are uniformly mixed, then 152 parts of malt dextrin, 117.65 parts of microcrystalline cellulose, 0.25 parts of magnesium stearate, and 0.1 parts of silicon dioxide are added and uniformly mixed to obtain an immune-enhancing and anticancer tablet product.

[0036] Test Example 1: Experiment of immune-enhancing and anticancer product on immune regulation Take 48 mice, and randomly divide into 4 groups, respectively, the first group of normal control group, the second group of tumor-bearing control group and the third group of PQQ pure product prepared in Example 2 group, the fourth group of product prepared in Example 5 group, 12 mice in each group; the first group is the conventional feeding mice, the second, third and fourth groups are all the mice inoculated with A549 lung cancer cells, 48 hours after inoculation, the second group is not given drug; the third group is gavaged with PQQ pure product prepared in Example 2 33.3ug (equivalent to 5 times of the recommended daily amount of human body); the fourth group is gavaged with product prepared in Example 5 833.3 ug (equivalent to 5 times of the recommended daily amount of human body), and the mice are taken blood from the eyeball after 2 weeks. The contents of IgA, IgG and IgM are determined, and the specific experimental results are shown in Table 2.

[0037] Table 2 Effect of each experimental group on IgA, IgG and IgM of mice Group IgA IgG IgM Group 1 89±2.9 866±15.8 78±4.2 Group 2 60±4.2 509±16.9 47±3.8 Group 3 73±3.5 672±15.4 63±4.3 Group 4 83±3.2 815±16.1 72±3.6 From Table 2, it can be seen that the contents of immunoglobulin IgA, IgG and IgM of the mice inoculated with A549 lung cancer cells are obviously lower than those of the normal control mice; the PQQ pure product prepared in Example 2 and the product prepared in Example 5 can improve the low contents of IgA, IgG and IgM of the mice, and the product prepared in Example 5 has stronger immune promoting effect. It is proved that the PQQ pure product prepared in the application and the product prepared by compounding PQQ pure product and Grifola frondosa extract can improve the immune function of tumor patients.

[0038] Test Example 2 Effect of in vitro immune-enhancing and anticancer product on tumor cell growth cycle Resuscitate gastric cancer cells MGC-803, liver cancer cells HepG2 and lung cancer cells A549, and subculture in vitro with serum-free RPMI-1640 culture medium, and incubate in a 37℃, 5% CO2 incubator for 48-72h, and replace the culture medium every 24h. After the culture bottle is covered with tumor cells, discard the culture medium, and digest the tumor cells with 0.25% trypsin.

[0039] Adjust the cell concentration to 1x10 6After 24 hours, 0.04 ug / mL PQQ prepared in Example 2 and 1 ug / mL product prepared in Example 5 were inoculated into the culture medium of each experimental group, respectively. The experiment was divided into three groups: blank control group, PQQ prepared in Example 2 group, and product prepared in Example 5 group. The first group was blank control group, and the RPMI-1640 culture medium without serum was used; the second group was PQQ prepared in Example 2 group, and the RPMI-1640 culture medium without serum containing 0.04 ug / mL PQQ was used; the third group was product prepared in Example 5 group, and the RPMI-1640 culture medium without serum containing 1 ug / mL product was used. After two generations of co-culture, the cells were digested with 0.25% trypsin to prepare a single cell suspension, washed with PBS, fixed with pre-cooled ethanol at -20°C, and then stained with iodine pyridine (PI) before being detected by flow cytometry to determine the changes in cell cycle.

[0040] The statistical detection results showed that the proportions of different cycle cells in each group of gastric cancer cells MGC-803 were shown in Table 3, the proportions of different cycle cells in each group of liver cancer cells HepG2 were shown in Table 4, and the proportions of different cycle cells in each group of lung cancer cells A549 were shown in Table 5.

[0041] Table 3 Proportions of different cycle cells in each group of gastric cancer cells MGC-803 Cell cycle G0 / G1 phase S phase Group 1 38.36±1.61 37.59±2.34 Group 2 49.27±1.85* 30.58±2.05 Group 3 62.53±2.17*# 24.86±1.95 Note: compared with the first group, *P<0.05; compared with the second group, #P<0.05.

[0042] As shown in Table 3, compared with the control group, the PQQ prepared in Example 2 and the product prepared in Example 5 had a blocking effect on the cell cycle of MG-803 gastric cancer cells, which mainly showed that the cells were blocked in the G0 / G1 phase and did not enter or delayed entering the S phase. In addition, the product prepared in Example 5 had a more obvious blocking effect on the gastric cancer cells MGC-803 in the G0 / G1 phase, indicating that the prepared PQQ and the product prepared by compounding the PQQ with the extract of Grifola frondosa could help to improve the ability to inhibit the proliferation and growth of gastric cancer cells MGC-803.

[0043] Table 4 Proportions of different cycle cells in each group of liver cancer cells HepG2 Cell cycle S phase G2 / M phase Group 1 24.72±1.21 9.38±2.02 Group 2 35.05±1.93* 12.86±1.47 Group 3 42.19±2.45*# 15.87±1.63 Note: compared with the first group, *P<0.05; compared with the second group, #P<0.05.

[0044] As shown in Table 4, compared with the control group, the PQQ prepared in Example 2 and the product prepared in Example 5 had a blocking effect on the cell cycle of HepG2 liver cancer cells, which mainly showed that the cells were blocked in the S and G2 phases. In addition, the product prepared in Example 5 had a greater blocking effect on the liver cancer cells HepG2 in the S and G2 phases, indicating that the prepared PQQ and the product prepared by compounding the PQQ with the extract of Grifola frondosa could help to improve the ability to inhibit the proliferation and growth of liver cancer cells HepG2.

[0045] Table 5 Proportion of different cycle cells of lung cancer cell A549 in each group Cell cycle G0 / G1 phase S phase Group 1 65.85±2.56 15.04±1.93 Group 2 73.97±2.49* 12.01±1.76 Group 3 83.56±1.84*# 8.12±2.52 Note: compared with the first group, *P<0.05; compared with the second group, #P<0.05.

[0046] As shown in Table 5, compared with the control group, the PQQ pure product prepared in Example 2 and the product prepared in Example 5 have a blocking effect on the A549 lung cancer cell cycle, mainly showing that the cells are blocked in the GO / G1 phase, and the product prepared in Example 5 has a more effective ability to block the lung cancer cell A549 in the G0 / G1 phase, indicating that the prepared PQQ pure product and the product prepared by compounding the PQQ pure product with the Grifola frondosa extract can help to improve the ability to inhibit the proliferation and growth of the lung cancer cell A549.

[0047] Test Example 3 Inhibition of tumor cells in vivo of nude mice by the product for enhancing immunity and resisting cancer in vitro The A549 cells in the logarithmic growth phase were taken and adjusted to a cell concentration of 5×10 7 The A549 cells were inoculated subcutaneously on the back of the mice at 0.1 mL, and when the volume of the subcutaneously transplanted tumor reached 70mm 3 The mice were randomly divided into a blank control group, a PQQ pure product prepared in Example 2 group and a product prepared in Example 5 group, totally 3 groups.

[0048] The volume of the tumor basically reached 70mm 3 After 48h of inoculation, the first group was given deionized water (10mL / Kg•Bw), the second group was given the deionized water (10mL / Kg•Bw) added with the PQQ pure product prepared in Example 2 at 33.3ug (equivalent to 5 times of the daily recommended amount for human body), the filtered clear solution after sufficient shaking and dissolution; the third group was given the deionized water (10mL / Kg•Bw) added with the product prepared in Example 5 at 833.3ug (equivalent to 5 times of the daily recommended amount for human body), the filtered clear solution after sufficient shaking and dissolution; the above-mentioned dosages can ensure the equivalent dosage of PQQ; the drug was given once a day, continuously for 3 weeks, and the mice were sacrificed by dislocation 48h after the last administration, the transplanted tumor was removed, the tumor weight was weighed and the tumor inhibition rate was calculated, as shown in Table 6. Tumor inhibition rate (%) = (1-experimental group tumor weight mean / control group tumor weight mean) x 100%.

[0049] Table 6 Effect of each group on the growth of A549 cell tumor in nude mice Group Tumor weight (mg) Average tumor inhibition rate (%) Group 1 438.72±46.38 / Group 2 318.56±35.76* 28.47 Group 3 136.24±40.13*# 68.91 Note: compared with the first group, *P<0.05; compared with the second group, #P<0.05.

[0050] From Table 6, compared with the control group, the PQQ pure product prepared in Example 2 and the product prepared in Example 5 both have significant differences in inhibiting the growth of A549 lung cancer cell transplanted tumors in nude mice, and the product prepared in Example 5 has more obvious inhibiting effect on lung cancer cells A549, which indicates that the PQQ pure product and the product prepared by compounding the PQQ pure product and the grifola frondosa extract can help to enhance the tumor inhibiting effect.

[0051] In summary, the mouse experiments on the immune regulation, the influence on the growth cycle of tumor cells in vitro and the inhibiting effect on tumor cells in vivo show that the PQQ pure product and the product prepared by compounding the PQQ pure product and the grifola frondosa extract have the effects of enhancing immunity and resisting cancer, and the product prepared by compounding the PQQ pure product and the grifola frondosa extract has more obvious effect.

[0052] The above examples are convenient for those skilled in the art to better understand the present application, and the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement modes and shall be included in the protection scope of the present application.

Claims

1. A strain of Paracoccus huiyi ( Paracoccus huijuniae ), characterized in that, The strain was named YJY25-02, with a collection number of CGMCC NO.34258. It was deposited in the General Microbiology Center of the China Culture Collection Administration on April 17, 2025. The strain is used to produce pyrroloquinoline quinone.

2. The Paracoccus huiyi according to claim 1, characterized in that The 16S rDNA sequence is shown in SEQ ID No:

1.

3. Use of the Paracoccus huiyi according to claim 1 in the preparation of pyrroloquinoline quinone.

4. The use of Paracoccus huiyi in the preparation of pyrroloquinoline quinone according to claim 3, characterized in that: The specific steps of the preparation method are as follows: (1) Preparation of fermentation broth of pyrroloquinoline quinone by Paracoccus huitschmidt YJY25-02, the specific steps are as follows: The activated Paracoccus huiebengyi YJY25-02 test tube slant culture was scraped with an inoculating needle to obtain a loopful of culture medium, which was aerobically cultured at 30°C for 18-26 hours until the bacterial solution OD600 was 0.6-1.0 to obtain a seed solution; the prepared seed solution was inoculated into a sterilized fermentation medium at a v / w inoculum rate of 5-10%, and the fermentation temperature was 30°C for aerobic culture for 120-144 hours. During the fermentation production process, 25% ammonia water was fed to maintain the pH at 6.5-7.0, and methanol was fed to maintain the methanol concentration in the fermentation system at not less than 0.5-1 g / L, thereby obtaining a fermentation solution of pyrroloquinoline quinone by Paracoccus huiebengyi YJY25-02; (2) Preparation of pure pyrroloquinoline quinone, specifically: The fermentation broth obtained in the previous step is adjusted to pH 2.5-3.5 with hydrochloric acid, and a volume ratio of 0.5‰-2‰ of swollen flocculant chitosan is added, and the mixture is allowed to stand at 10°C-35°C for 30-60 minutes, and then a filter aid diatomaceous earth is added, and the filtrate is obtained after plate and frame filtration; the filtrate is passed through a 5KD-10KD ultrafiltration membrane, and the ultrafiltration clear liquid is collected; the ultrafiltration clear liquid is passed through a resin column of an OH type weakly basic anion exchange resin at a flow rate of 1BV / h, and the resin column is eluted with a desorbent having the same volume as the ultrafiltration clear liquid at a flow rate of 2BV / h. The desorbent is a 0.5 wt% sodium hydroxide solution, and red and dark red effluents are collected; the pH of the effluent is adjusted to 6.5-7.0, the effluent is passed through a 100D-300D nanofiltration membrane, and the nanofiltration concentrate is collected; the nanofiltration concentrate is concentrated under reduced pressure until crystals precipitate, the temperature is lowered, crystallized, and filtered to obtain a crude pyrroloquinoline quinone product; the crude pyrroloquinoline quinone is added to purified water and dissolved at 40° C.-60° C., and then activated carbon is added, stirred for 30 minutes, filtered, and the filtrate is cooled and recrystallized, and filtered to obtain a wet pyrroloquinoline quinone product; The wet pyrroloquinoline quinone was washed with cold water, filtered again, and dried to obtain the pure pyrroloquinoline quinone.

5. Use of the pure pyrroloquinoline quinone prepared according to claim 3 or 4 in the preparation of immunity-enhancing and anti-cancer products.

6. The use according to claim 5, wherein the immunity-enhancing and anti-cancer product is a mixture of pure pyrroloquinoline quinone and maitake mushroom extract, with a weight ratio of 1:2 to 1:5, and the purity of the pure pyrroloquinoline quinone is ≥98%.

7. The use according to claim 6, wherein the weight ratio of the pure pyrroloquinoline quinone to the Grifola frondosa extract is 1:3, and the Grifola frondosa polysaccharide content in the Grifola frondosa extract is 10 wt% to 30 wt%.

8. The use according to claim 5, wherein the immunity-enhancing and anti-cancer product is a capsule, tablet or solid beverage; wherein the carrier comprises one or more of a filler, a binder, a disintegrant, a lubricant, a suspending agent, a wetting agent, a pigment, a flavoring agent, a solvent and a surfactant that is acceptable in pharmacy, health care products or functional foods.

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