Fermentation strain with high yield of tocopherol and application of fermentation strain in coarse cereal fermentation

Abstract

The invention belongs to the field of microorganisms, and relates to a high-yield tocopherol fermentation strain and application thereof in coarse cereal fermentation. The invention provides a strain M.rbg-2M, and the strain M.rbg-2M is obtained by carrying out ultraviolet mutation breeding on an original strain M.rbg. The strain forms a form beneficial to enrichment of tocopherol after mutagenesis, and fermentation verifies that the enrichment amounts of total tocopherol and alpha-tocopherol produced by the strain are 1.21 times and 1.93 times higher than those of an original strain respectively. Meanwhile, a high-quality coarse cereal substrate capable of promoting the high yield of tocopherol is selected and inoculated with M.rbg-2M for fermentation, and the total tocopherol content is increased by 3.2 times or above compared with that of a CK group. The growth and metabolism of fermentation strains are regulated and controlled by utilizing the metabolism characteristics of microorganisms, the metabolic flux is effectively increased, the enrichment amount of metabolites is increased, the optimal fermentation period and the optimal active matter harvesting time are determined, and at the moment, alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol are obviously increased compared with those before optimization. The total tocopherol enrichment amount is more than 15 times of the total tocopherol enrichment amount in the early stage of research, and finally high-efficiency and high-amount enrichment of total tocopherol and abundant tocopherol types are realized. And an excellent strain, a feasible scheme and technical parameters are provided for industrial production of tocopherol through monascus fermentation and enrichment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and relates to a fermentation strain with high production of tocopherol and application of the fermentation strain in fermentation of coarse cereals. BACKGROUND

[0002] Red koji, also known as red koji and Dan Qu, refers to a medicinal and edible fermentation product prepared by solid-state fermentation of rice by Monascus. Monascus is a filamentous fungus of the genus Monascus, which is the production strain of red koji. In 2001, Monascus was included in the "List of Fungal Strains for Health Foods", and is commonly used as a fermentation fungus to prepare red koji powder, red koji wine, red koji vinegar and other foods. Monascus can produce a variety of secondary metabolites, mainly including red pigment, Monacolin K, ergosterol, gamma-aminobutyric acid and unsaturated fatty acids, which endow the Monascus fermentation product with medicinal and health values. For example, the red pigment, as a natural dyeing agent, is widely used as a coloring agent in food, textile industry, pharmaceuticals, medicine and cosmetics, and is also used as a folk medicine, which has the effects of improving food digestion, blood circulation, antioxidant and reducing blood cholesterol level. Monacolin K extracted from red koji, as a new type of cholesterol-lowering drug, can inhibit cholesterol synthesis and has important effects in reducing blood lipids, anti-cancer, neuroprotection and anti-inflammatory and antibacterial effects. With the continuous in-depth theoretical research and deep analysis of fermentation products, the types and abundance of Monascus secondary metabolites are also expanding and enriching, such as tocopherol, gamma-oryzanol, and uric acid.

[0003] Tocopherol is the hydrolysis product of vitamin E, which has 8 different configurations, including alpha, beta, gamma, delta tocopherol and corresponding tocotrienol, and is the most important antioxidant. It can protect polyunsaturated / saturated fatty acids and cholesterol in cell membranes from damage caused by free radicals, has antioxidant, fat-burning, cholesterol-lowering, immune cell function restoration, anticancer and anticancer effects in the human body, and has become one of the widely consumed nutritional supplements and antioxidants by humans. Tocopherol mainly exists in oil plants, among which alpha-tocopherol and gamma-tocopherol are the main types, alpha-tocopherol is the most abundant and active form of vitamin E, and is the most utilized form of tocopherol structure in the human body. It is also the most synthesized tocopherol structure by humans, and is mainly present in vegetable oil, with high antioxidant, anti-aging, and fertility-promoting physiological functions. Alpha-tocopherol has very good heat resistance, and as a nutritional supplement and antioxidant, it has outstanding effects and wide application prospects in food thermal processing. At present, alpha-tocopherol is mainly obtained by extraction distillation or chemical synthesis from vegetable oil, which has problems such as difficult extraction, low yield, poor purity, high cost, and environmental unfriendliness; while the enzyme catalysis method has technical complexity and difficulty in popularization. Therefore, in order to obtain a safe, efficient, and low-cost microbial fermentation method for enriching tocopherol, strain selection has become a focus. The present application takes the enrichment of tocopherol as the core target, and optimizes the screening of high-tocopherol-producing Monascus rubiginosus as the purpose, uses microbial fermentation of coarse grain substrate, and uses multi-level and multi-dimensional fermentation regulation as a means to explore the optimal strategy and parameters for high-tocopherol-producing Monascus rubiginosus fermentation of coarse grain substrate, realize high-yield of tocopherol, increase of active ingredients, and richness of nutritional structure, and greatly improve the functional characteristics and utilization degree of coarse grains.

[0004] Therefore, finding a safe strain of yeast that can produce high amounts of tocopherol is one of the current research directions. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application provides a fermentation strain (M.rbg-2M) that produces high amounts of tocopherol, as follows:

[0006] A fermentation strain that produces high amounts of tocopherol, Monascus rubiginosus M.rbg-2M, is deposited at the China Center for Type Culture Collection, with the accession number CCTCC NO:M 20251375, and the deposit date is June 12, 2025.

[0007] Further, the fermentation strain that produces high amounts of tocopherol is Monascus rubiginosus, with the strain number M.rbg-2M, which is specifically obtained by UV mutagenesis selection from the starting strain M.rbg.

[0008] Further, the high-tocopherol-producing fermentation strain grows on potato glucose agar medium at 30 DEG C, and its colony is slightly orange yellow with yellow mycelium edge; the mycelium is white, flocculent and short, and the conidium is more, and the mycelium is less branched.

[0009] Application of the high-tocopherol-producing fermentation strain M.rbg-2M in the fermentation of mixed grains.

[0010] Further, the mixed grains are any one or more of rice, millet, Job's tears and oat in any proportion.

[0011] Application of the high-tocopherol-producing fermentation strain in the fermentation of mixed grains to enrich tocopherol.

[0012] The method for fermenting the high-tocopherol-producing fermentation strain M.rbg-2M to produce tocopherol, specifically, the strain is fermented on Job's tears substrate with 10% water at 28 DEG C for 10-12 days.

[0013] The beneficial effects of the present application are as follows:

[0014] (1) In the previous research, by comparing the metabolic products of purple red koji, black koji, mould and penicillium in the fermentation of mixed grains, it is found that the purple red koji fermented mixed grains can promote the significant increase of lipophilic compounds, and has outstanding advantages in the enrichment of tocopherol, and compared with black koji, mould and penicillium and other common fungi, there is significant strain dependence. Six red koji strains from health food and koji are used as test materials, according to the morphological comparison and the evaluation of the enrichment ability of functional active substances (tocopherol, Monacolin K, gamma-oryzanol, red koji pigment) of fermented Job's tears, the high-quality rusty red koji strain M.rbg is screened out for fermentation and enrichment of tocopherol. The results show that the tocopherol enrichment ability of the rusty red koji strain M.rbg is significantly stronger than that of other strains.

[0015] (2) The strain M.rbg-2M described in the application is obtained by ultraviolet mutagenesis of the starting strain M.rbg. The strain forms a morphology conducive to tocopherol enrichment after mutagenesis, and the enrichment amount of total tocopherol and a-tocopherol produced by the strain is 1.21 times and 1.93 times higher than that of the starting strain, respectively. Meanwhile, high-quality coarse grain substrates that promote high production of tocopherol are selected, inoculated with M.rbg-2M for fermentation, and the total tocopherol content is increased by more than 3.2 times compared with the CK group. By utilizing the metabolic characteristics of microorganisms, regulating the growth and metabolism of the fermentation strain, effectively increasing the metabolic flux, and improving the enrichment amount of metabolic products, the optimal fermentation period and active substance harvesting time are determined, at which time the a-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol are all significantly increased compared with before optimization. The total tocopherol enrichment amount is more than 15 times that at the beginning of the study, and finally the efficient and high-yield enrichment of total tocopherol and the richness of tocopherol types are realized. This provides an excellent strain and a feasible scheme and technical parameters for industrialized Monascus fermentation for tocopherol production. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the tocopherol enrichment characteristics of the starting strain M.rbg in fermentation of different coarse grain substrates.

[0017] Figure 2 is the tocopherol enrichment characteristics of the starting strain M.rbg under optimized fermentation conditions.

[0018] Figure 3 is the tocopherol enrichment characteristics of the mutagenized strain M.rbg-2M in fermentation of Job's tears.

[0019] Figure 4 is the SEM morphology of the mutagenized strain M.rbg-2M.

[0020] Figure 5 is the tocopherol enrichment characteristics of the mutagenized strain M.rbg-2M under optimized fermentation conditions.

[0021] Figure 6 is the colony morphology of the mutagenized strain M.rbg-2M.

[0022] Figure 7 is the spore morphology of the mutagenized strain M.rbg-2M.

[0023] Figure 8 is the mycelial morphology of the mutagenized strain M.rbg-2M.

[0024] Figure 9 is the fermentation of Job's tears of the mutagenized strain M.rbg-2M.

[0025] Figure 10is an agarose gel electrophoresis map of a conserved sequence fragment of Basidiomycota fungi (a phylogenetic marker molecule commonly used in analysis of fungal community structure) for identifying fungal populations. DETAILED DESCRIPTION

[0026] The application will be further described in detail below with specific examples, which are an explanation of the application rather than a limitation.

[0027] Example 1 Fermentation substrate optimization

[0028] According to the fermentation optimization culture conditions of Monascus, four kinds of common coarse grain substrates were used for fermentation (the specific results are shown in Table 1). Figure 1 The total phytosterol enrichment of M. rbg fermented substrate was most significant when the substrate was Job's tears, reaching 62.30 mg / kg. Compared with other fermented substrates, the several types of phytosterols of fermented Job's tears were relatively rich, and the total was 3.51 times, 1.59 times and 3.65 times that of fermented rice, millet and oat, respectively. The characteristics of Job's tears substrate provided a good environment and material basis for the enrichment of phytosterols by Monascus fermentation.

[0029] Example 2 Optimization of fermentation conditions

[0030] The total phytosterol enrichment of M. rbg fermented substrate was most significant when the substrate was Job's tears, reaching 62.30 mg / kg. Compared with other fermented substrates, the several types of phytosterols of fermented Job's tears were relatively rich, and the total was 3.51 times, 1.59 times and 3.65 times that of fermented rice, millet and oat, respectively. The characteristics of Job's tears substrate provided a good environment and material basis for the enrichment of phytosterols by Monascus fermentation. Figure 2 The results (see Table 2) show that the total phytosterol content of M. rbg fermented Job's tears was the highest when the fermentation conditions were optimized, i.e., the substrate was Job's tears, the water addition amount was 10%, the fermentation temperature was 28℃, and the fermentation time was 10-12 days. Under these conditions, the total phytosterol content of M. rbg fermented Job's tears was up to 67.39 mg / kg, the α-phytosterol enrichment was up to 23.56 mg / kg, and the γ-phytosterol was up to 43.83 mg / kg. The contents of β-phytosterol and δ-phytosterol were low and not detected.

[0031] Example 3 Mutagenic breeding of high phytosterol-producing Monascus strains

[0032] (1) Preparation of spore suspension: inoculate the Monascus strain on a modified PDA medium slant, and incubate in a 30℃ constant temperature incubator for 6-8 days. Add a certain amount of sterile water to the PDA slant, scrape the mycelium with an inoculation loop, and transfer to a triangular flask containing sterile water. Shake at 200 rpm / min for 30 min to disperse the spores, and then filter them into a new 50 mL triangular flask using 4 layers of gauze to prepare a spore suspension.

[0033] (2) UV mutagenesis: transfer the prepared Monascus spore suspension and sterile rotor to a sterile glass culture dish. Place the culture dish on a magnetic stirrer at a distance of about 30 cm from the UV lamp (30 W), and stir with a sterile magnetic stirrer while irradiating with UV for a certain period of time.

[0034] (3) Inoculation and calculation of mortality rate: the exposed spore solution is diluted with sterile water and inoculated in a dark environment for 3d, and the number of colonies is counted and the mortality rate is calculated. The qualified mutant strains are screened according to the mortality rate, and the strains meeting the mortality rate are inoculated on PDA medium for 5 generations to ensure stable properties.

[0035] (4) Plate separation and screening: the treated bacterial suspension is diluted and then coated on PDA plates, and the pH is natural. Then the bacterial plates are placed in a 30℃ constant temperature incubator and cultured for 6-8d. The strains with changed colony morphology (colony, color, size, etc.) are selected, single colony streaking separation is performed, and then multiple separation and purification are performed to obtain purified strains. The mutant strains are rechecked and microstructure observation is performed, and compared with the original strain for evaluation.

[0036] (5) Fermentation culture: the spore suspension prepared in step (2) is inoculated into the yam substrate at a certain inoculation amount, mixed uniformly, and fermented at 28℃ for 10-12d. The tocopherol enrichment of the fermented yam is compared with that of the original strain, and the mutant Monascus strain is selected to realize efficient and high-yield enrichment of total tocopherol and rich types of tocopherol.

[0037] Example 1: Fermentation of yam by starting strain M.rbg to enrich tocopherol

[0038] The total tocopherol enrichment of yam fermented by the starting strain M.rbg was 23.37mg / kg, the α-tocopherol content was 8.89mg / kg, the γ-tocopherol enrichment was 14.48mg / kg, the β-tocopherol and δ-tocopherol contents were low, and no detection was made.

[0039] Example 2: Fermentation of yam by mutant strain M.rbg-30s to enrich tocopherol

[0040] The mutant strain M.rbg-30s screened according to the mortality rate. Under electron microscope observation, the mycelium of M.rbg-30s strain showed deep indentation, and the conidium was less. The mycelium was thin and branched. The results showed that compared with the CK group, the tocopherol enrichment of the mutant strain M.rbg-30s was significantly reduced.

[0041] Example 3: Fermentation of yam by mutant strain M.rbg-60s to enrich tocopherol

[0042] The mutagenic strain M.rbg-60s screened according to the lethality. Under the electron microscope observation, the mycelium of M.rbg-60s strain presents deeper indentation, less conidium, and less thick branches. The results show that the total tocopherol enrichment amount of the mutant M.rbg-60s is reduced by 0.68 times compared with the CK group.

[0043] The tocopherol enrichment amount of the mutant M.rbg-90s fermented with the Coix fermented by the mutant M.rbg-90s of the comparative example 4

[0044] The mutagenic strain M.rbg-90s screened according to the lethality. Under the electron microscope observation, the mycelium of M.rbg-90s strain presents deeper indentation, less conidium, and more thick branches. The results show that the total tocopherol enrichment amount of the mutant M.rbg-90s is not significantly changed compared with the CK group.

[0045] The tocopherol enrichment amount of the mutant M.rbg-2M fermented with the Coix fermented by the mutant M.rbg-2M of the comparative example 5

[0046] The mutagenic strain M.rbg-2M screened according to the lethality. Under the electron microscope observation, the mycelium of M.rbg-2M strain presents more folded wrinkles on the surface, more obvious spore folding degree, and deeper indentation. The results show that (see the specific data in Table 1) Figure 3 ), the tocopherol enrichment amount of the mutant M.rbg-2M fermented with the Coix is most significantly increased, in which the enrichment amount of α-tocopherol is 1.9 times of the CK group, and the total tocopherol is increased by 1.2 times. In summary, the moderate mutagenesis of M.rbg-2M is more successful in the enrichment of total tocopherol and α-tocopherol.

[0047] The tocopherol enrichment amount of the mutant M.rbg-2M fermented with the Coix fermented by the mutant M.rbg-2M of the comparative example 6 under the optimized conditions

[0048] The UV mutagenesis was used to select the strain M.rbg-2M as the test strain, the fermentation temperature, fermentation period, key regulatory factor L-tyrosine (L-Tyr), L-phenylalanine (L-Phe) and surfactant (SDS) as factors, the optimization experiment was designed, and the total tocopherol enrichment of M.rbg-2M fermented coix seed was comprehensively analyzed. The results showed that under the optimized fermentation conditions of M.rbg-2M strain, on the coix seed substrate with 10% water addition, the α-tocopherol enrichment could reach 81.66 mg / kg, the β-tocopherol could reach 162.1 mg / kg, the γ-tocopherol could reach 43.83 mg / kg, and the δ-tocopherol could reach 41.05 mg / kg. Compared with the optimization, the total tocopherol enrichment was significantly increased. After 12 days of fermentation, the total tocopherol enrichment could reach 354.16 mg / kg, which was more than 15 times of the initial study (see Figure 5 ).

[0049] The obtained different strains will be compared as follows

[0050] (1) Mutagenesis lethality of strains

[0051] Table 1 Mutagenesis lethality of strains

[0052]

[0053]

[0054] From Table 1, the mutagenesis lethality of mutagenized strains M.rbg-30s, M.rbg-60s, M.rbg-90s and M.rbg-2M all reached more than 80%. The mutagenesis lethality reached more than 80%, the better the mutagenesis effect, the stronger the genetic damage tolerance, which could maximize the activation of cell mutation and cause high-level expression of some genes. Therefore, the mutagenized strains M.rbg-30s, M.rbg-60s, M.rbg-90s and M.rbg-2M can be used as coix seed fermentation safety preliminary screening strains for standby.

[0055] (2) Description of colony morphology and microscopic mycelium morphology characteristics of mutagenized strains

[0056] Table 2 Colony morphology and microscopic mycelium morphology characteristics of mutagenized strains

[0057]

[0058] Table 2 shows that the mycelial morphology of the strains treated with different durations of mutagenesis exhibited regular changes. The M. rbg-60s mutagen showed a certain degree of thickening of the mycelium, while the M. rbg-90s and M. rbg-2M mutagens began to thin again with prolonged mutagenesis. Spore size also changed, showing a similar pattern to the mycelial changes, initially increasing in size and then decreasing. Notably, the M. rbg-2M mutagen generally exhibited thinner mycelium and thinner colonies.

[0059] (3) SEM morphological changes of the mutant strain M.rbg-2M

[0060] from Figure 4 It was observed that the hyphae of the mutant strains all exhibited varying degrees of folding and swelling. Hyphae folding and swelling are highly likely to affect the pathways and efficiency of intracellular material transport, interfering to some extent with the intensity of protein synthesis and energy metabolism-related activities, and enhancing the efficiency of fermentation metabolites. In contrast, the hyphal surface of the M.rbg-2M mutant strain showed more folds and wrinkles, and its spores not only exhibited more pronounced folding but also deeper indentations.

[0061] (4) Tocopherol enrichment characteristics of mutant strain M.rbg-2M under optimized fermentation conditions

[0062] Depend on Figure 5 It was determined that the mutant strain M.rbg-2M, selected based on lethality, was the test strain and inoculated into a Job's tears substrate for fermentation under optimized conditions. The results showed that by utilizing the metabolic characteristics of the microorganisms and regulating the growth and metabolism of the fermentation strain, the metabolic flux was effectively increased, the accumulation of metabolites was enhanced, and the optimal fermentation cycle and harvest time of active substances were determined. At this point, the levels of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol were significantly increased compared to before optimization. The total tocopherol accumulation was more than 15 times that at the beginning of the study, ultimately achieving efficient and high-quantity accumulation of total tocopherol and a rich variety of tocopherol types. In conclusion, the moderate mutagenesis of strain M.rbg-2M was quite successful in targeting the accumulation of total tocopherol and tocopherol types.

[0063] (5) Comparison of literature on tocopherol enrichment levels

[0064] The literature "Bacillus tekirae QH1 Producing α-Tocopherol and Its Application" (Liu Qinhua) reported that the α-tocopherol level in rice straw and piglet feed fermented with Bacillus tekirae QH1 was 50.72 mg / L. However, the optimized fermentation of Job's tears by our patented strain M.rbg-2M yielded significantly higher levels of α-tocopherol and total tocopherol. The maximum enrichment of α-tocopherol reached 81.66 mg / kg, β-tocopherol reached 162.1 mg / kg, and γ-tocopherol reached 43.83 mg / kg. δ-tocopherol reached a maximum of 41.05 mg / kg. All levels showed significant increases compared to the initial results. At 12 days of fermentation, the total tocopherol enrichment reached its maximum of 354.16 mg / kg, more than 15 times that of the initial research. The higher total tocopherol levels in this technology compared to the literature data are due to three main reasons: the screening method, the difference in strains, and the technical objectives. Bacillus QH1 was obtained through enrichment culture and dilution plate culture screening. In this technical solution, the mutagenic strain M.rbg-2M is a beneficial and safe strain obtained through methods such as screening of superior strains, safety performance testing, comparison of mutagenic mortality rate, substrate optimization, fermentation condition optimization, and tocopherol level detection. It can achieve efficient and high-quantity enrichment of total tocopherols and a rich variety of tocopherol types.

[0065] In this technical solution, the high-yielding tocopherol-producing strain M.rbg-2M is a safe and beneficial fermentation strain obtained through methods such as mutagenesis of *Monascus purpureus* strains enriched by fermentation, selection, calculation of mutagenesis mortality rate, retesting of the mutagenized strains, SEM morphological change observation, substrate screening, optimization of fermentation conditions, and determination of tocopherol enrichment. Tocopherol is one of the most widely consumed nutritional supplements and antioxidants, and is widely used in food, pharmaceuticals, and cosmetics. Currently, natural tocopherol is mainly extracted from plants, but the production cycle of plant tocopherol is long and the yield is low, which cannot adequately meet current demand. Fermentation production of tocopherol using microorganisms as a "factory" has advantages such as economy, high efficiency, and environmental friendliness, with excellent market prospects and huge development potential. The purpose of this technical solution is to achieve efficient and high-volume enrichment of total tocopherol and to diversify the types of tocopherol. It provides excellent strains, feasible solutions, and technical parameters for the industrial-scale fermentation of *Monascus purpureus* to enrich and produce tocopherol from Job's tears.

[0066] In summary, the use of the beneficial yeast safe mutagenesis strain M.rbg-2M, developed under this invention, in the fermentation production of miscellaneous grains not only improves product quality and achieves efficient and high-quantity enrichment of total tocopherols and a diverse range of tocopherol types, resulting in high-quality fermented miscellaneous grain products, but also demonstrates significant advantages and strong application prospects as a beneficial yeast safe production strain.

[0067] Finally, it should be noted that the above embodiments are merely representative examples of this application. Obviously, the technical solutions of this application are not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this application should be considered within the scope of protection of this application.

Claims

1. A fermentation strain with high tocopherol production, characterized in that, It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 20251375; the deposit date is June 12, 2025.

2. The high-tocopherol-producing fermentation strain according to claim 1, characterized in that, The fermentation strain that produces high levels of tocopherol is a mutant strain of Monascus rubiginosus, with the strain number M.rbg-2M.

3. The high-tocopherol-producing fermentation strain according to claim 1, characterized in that, The fermentation strain that produces high levels of tocopherol has colonies that are slightly orange-yellow with yellow ribbons at the edges; the hyphae are white, fluffy, and short, with numerous conidia and few fine branches.

4. The application of the high tocopherol-producing fermentation strain described in claim 1 in the fermentation of miscellaneous grains.

5. The application according to claim 4, characterized in that, The coarse grains mentioned are any one or more of rice, millet, Job's tears, and oat groats, combined in any proportion.

6. The application according to claim 4, characterized in that, The grain mentioned is Job's tears.

7. The application of the high tocopherol-producing fermentation strain of claim 1 in the fermentation production of tocopherol.

8. The specific fermentation enrichment method for tocopherol by the high-tocopherol-producing fermentation strain according to claim 1, characterized in that, The strain was fermented on a Job's tears substrate with 10% water at 28°C for 10–12 days.

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