Application of rhizopus arrhizus in fermentation and deastringency of roxburgh rose

Abstract

The invention belongs to the technical field of microbial fermentation, discloses application of a strain of rhizopus arrhizus in fermentation and deastringency of rosa roxburghii tratt, and particularly discloses a strain of rhizopus arrhizus CLF001. The invention discloses the rhizopus arrhizus CLF001 capable of adapting to the fermentation of the roxburgh rose normal juice and reducing the tannin content in the roxburgh rose normal juice for the first time, and the rhizopus arrhizus CLF001 can rapidly grow in a low pH value environment and enrich the types of dominant strains for roxburgh rose fermentation. When the strain is used for fermenting roxburgh rose normal juice for 3 days, the content of tannin in the roxburgh rose normal juice can be remarkably reduced (the highest degradation rate can reach 85% or above), and the content of polysaccharide can be remarkably increased (increased by 80% or above), so that the fermented roxburgh rose normal juice does not feel obvious astringent taste, the bitter taste of the roxburgh rose normal juice is greatly improved, the fermentation time is shortened, and the production cost is reduced. The method has important significance in roxburgh rose normal juice fermentation processing.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to application of a strain of Rhizopus arrhizus in fermentation and astringency removal of roxburghii. Background Art

[0002] Rosa roxburghii is a unique and innovative fruit that combines medicinal, health-promoting, edible, and ornamental uses. It boasts a unique aroma and is rich in nutrients, particularly vitamin C, rutin (VP), and superoxide dismutase (SOD), earning it the nickname "Three King Fruits." However, rosa roxburghii juice contains a large amount of polyphenols, such as tannins, resulting in a strong bitter taste that significantly impacts the quality and taste of the juice. Currently, common methods for removing bitterness from juice include encapsulation, addition of bitterness inhibitors, and enzyme-based methods.

[0003] Microbial fermentation can effectively remove the astringency of sea buckthorn juice and also has a synergistic effect. Microbial fermentation is an effective way to improve the astringency of sea buckthorn juice. However, since sea buckthorn juice is rich in VC and tannin polyphenols, which have a significant inhibitory effect on the growth of most microorganisms, and has a low pH value, the fermentation of the juice is limited. Existing sea buckthorn fermented products are almost all diluted or blended before fermentation. Therefore, screening and obtaining dominant strains that are suitable for sea buckthorn juice fermentation and applying the obtained dominant strains to sea buckthorn juice fermentation are of great significance for enriching the variety of sea buckthorn products, increasing the economic benefits of sea buckthorn processing enterprises, and promoting the sustainable development of the sea buckthorn industry. Summary of the Invention

[0004] The first aspect of the present invention aims to provide a strain of Rhizopus arrhizus CLF001.

[0005] The second aspect of the present invention aims to provide a method for preparing a culture of Rhizopus arrhizus CLF001.

[0006] The third aspect of the present invention aims to provide a culture of Rhizopus arrhizus CLF001.

[0007] The fourth aspect of the present invention aims to provide a microbial agent.

[0008] The purpose of the fifth aspect of the present invention is to provide the Rhizopus arrhizus CLF001 of the first aspect of the present invention, the culture of Rhizopus arrhizus CLF001 of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention.

[0009] The sixth aspect of the present invention aims to provide a method for degrading tannins.

[0010] The seventh aspect of the present invention aims to provide a method for preparing a roxburghii drink.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] In a first aspect of the present invention, a strain of Rhizopus arrhizus CLF001 is provided. The Rhizopus arrhizus CLF001 is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC No: 66063 and a deposit date of March 27, 2025.

[0013] Without diluting or changing the pH of the sea buckthorn juice, the Rhizopus arrhizus CLF001 can quickly ferment and efficiently degrade tannins in the sea buckthorn, playing a role in removing astringency and enhancing synergy.

[0014] A second aspect of the present invention provides a method for preparing a culture of Rhizopus arrhizus CLF001, comprising:

[0015] Provide liquid culture system;

[0016] The Rhizopus arrhizus CLF001 according to the first aspect of the present invention is inoculated into the liquid culture system for culture.

[0017] In some embodiments of the present invention, the liquid culture system contains nutrients, including protein (peptone), sugar, phosphorus, magnesium, etc. For example, the nutrients can be provided in the form of a liquid culture medium selected from any one of PDB, YPD, WORT, DRBC, and DTM.

[0018] In some embodiments of the present invention, the pH value of the liquid culture system is 2-9, preferably 3-6.

[0019] In some embodiments of the present invention, the culture condition is culturing at 20-40° C. for 20-50 h, preferably culturing at 30° C. for 48 h.

[0020] The third aspect of the present invention provides a culture of Rhizopus arrhizus CLF001, which is prepared by the preparation method of the second aspect of the present invention.

[0021] A fourth aspect of the present invention provides a microbial agent, comprising the culture of Rhizopus arrhizus CLF001 according to the first aspect of the present invention or the culture of Rhizopus arrhizus CLF001 according to the third aspect of the present invention.

[0022] The active ingredient of the above-mentioned microbial agent may be the above-mentioned Rhizopus arrhizus CLF001, the culture of the above-mentioned Rhizopus arrhizus CLF001 (such as a fermentation metabolite), and the active ingredient of the above-mentioned microbial agent may also contain other biological components or non-biological components. The other active ingredients of the above-mentioned microbial agent can be determined by those skilled in the art according to the effect of the microbial agent.

[0023] The term "culture" refers to any liquid or solid product (including all materials within a culture vessel, fermentation products) containing a microbial population following artificial inoculation and cultivation. This refers to a product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of the microorganism or contain a certain amount of culture medium, metabolites, or other components produced during the culture process.

[0024] In the above-mentioned microbial agent, in addition to the active ingredient, the microbial agent further contains a carrier. The carrier can be a commonly used biologically inert carrier in the field (such as microbiology, agriculture). The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material, or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of bagasse powder, corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent or water; the organic solvent can be decane and / or dodecane.

[0025] The microbial agents mentioned above may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, capsule, tablet, wettable powder or water dispersible granule.

[0026] The fifth aspect of the present invention provides the use of the Rhizopus arrhizus CLF001 of the first aspect of the present invention, the culture of Rhizopus arrhizus CLF001 of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention in at least one of (1) to (5):

[0027] (1) Degradation of tannins;

[0028] (2) preparing a product of degraded tannin;

[0029] (3) removal of astringency;

[0030] (4) preparing astringent products;

[0031] (5) Preparation of sea buckthorn products.

[0032] In some embodiments of the present invention, the degrading tannins comprises degrading tannins in roxburghii juice having a pH value of 2-9.

[0033] In some embodiments of the present invention, the astringency removal includes achieving the purpose of astringency removal by degrading tannins and increasing polysaccharides.

[0034] A sixth aspect of the present invention provides a method for degrading tannins, comprising the step of treating a substance containing tannins using the Rhizopus arrhizus CLF001 of the first aspect of the present invention, the culture of Rhizopus arrhizus CLF001 of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention.

[0035] In some embodiments of the present invention, the substance includes tannins, and plant tissues containing tannins (eg, fruits, juices).

[0036] The seventh aspect of the present invention provides a method for preparing a sea buckthorn beverage, comprising the following steps: mixing sea buckthorn juice with sugar, inoculating the Rhizopus arrhizus CLF001 of the first aspect of the present invention, the Rhizopus arrhizus CLF001 culture of the third aspect of the present invention, or the microbial agent of the fourth aspect of the present invention, and fermenting to obtain a sea buckthorn fermented beverage.

[0037] In some embodiments of the present invention, the added amount of the sugar is 0.1 w / v% to 5 w / v% of the roxburghii juice.

[0038] In some embodiments of the present invention, the added amount of the sugar is 0.5 w / v% to 3 w / v% of the roxburghii juice.

[0039] In some embodiments of the present invention, the inoculation amount of Rhizopus arrhizus CLF001 is 1 v / v% to 5 v / v%.

[0040] In some embodiments of the present invention, the inoculation amount of Rhizopus arrhizus CLF001 is 1 v / v% to 3 v / v%.

[0041] In some embodiments of the present invention, the fermentation conditions are 20-50° C., 100-200 r / min, and culturing for 2-7 days.

[0042] In some embodiments of the present invention, the fermentation conditions are 25-40° C., 100-200 r / min, and culturing for 2-6 days.

[0043] The beneficial effects of the present invention are:

[0044] The present invention discloses for the first time a strain of Rhizopus arrhizus CLF001 that can adapt to the fermentation of roxburghii raw juice and reduce the tannin content in the roxburghii raw juice. The strain can grow rapidly in a low pH environment, enriching the types of dominant strains for roxburghii fermentation. The strain is used to ferment roxburghii raw juice for 3 days, which can significantly reduce the tannin content in the roxburghii raw juice (the degradation rate can reach more than 85%) and significantly increase the polysaccharide content (by more than 80%). This makes the fermented roxburghii raw juice have no obvious astringency, greatly improves the bitter taste of the roxburghii raw juice, and shortens the fermentation time. The present invention is of great significance in the fermentation and processing of roxburghii raw juice. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 These are the results of strain screening based on tannin degradation.

[0046] Figure 2 This is the result of strain morphological identification of strain CLF001.

[0047] Figure 3 This is the phylogenetic tree of strain CLF001.

[0048] Figure 4 The results are from a single-factor experiment. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below through specific examples.

[0050] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0051] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0052] Preparation process of sea buckthorn juice: select fresh, moderately mature (yellow-green or golden yellow peel), pest-free and rot-free sea buckthorn fruits, wash with high-pressure water to remove surface impurities, squeeze, filter and clarify, and pasteurize at 80-85℃ for 5-10 minutes to obtain sea buckthorn juice.

[0053] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0054] Example 1

[0055] Screening of dominant strains: Spread the original juice of roxburghii from the bottle mouth, ferment under natural conditions for 5 days, and dilute the natural fermentation liquid to 10 -3 , 10 -4 , 10 -5 , dilute and spread on PDA, MRS, and LB culture medium plates, culture at 30℃ for 72h, and pick out single strains for purification and culture.

[0056] Strain purification: For bacteria, pick a single colony and inoculate it into liquid culture medium. Incubate at 30°C for 48 hours. Streak a loopful of culture medium several times, repeating until the colonies are uniform in morphology. Mix the culture medium from the single colony with an equal volume of glycerol and store at -80°C. For fungi, pick the mycelial tip onto a PDA plate and incubate at 28°C for 3-5 days. Repeatedly remove the tip for purification and store on a slant.

[0057] A total of 13 bacterial strains (number: CLB001-13), 5 yeast strains (number: CLS001-005) and 2 fungal strains (number: CLF001-2) were isolated from naturally fermented sea buckthorn juice. The screened strains were tested for their ability to ferment sea buckthorn juice, and the dominant strains were selected based on the tannin degradation rate in the sea buckthorn juice.

[0058] The results showed that the degradation rate of fungus CLF001 was the strongest, followed by fungus CLF002, yeast CLS002, CLS004 and bacteria CLB005, CLB008 ( Figure 1 Among them, the fungus CLF001 was the most effective at degrading tannins, achieving a tannin degradation rate of over 60% during the initial screening process and also yielding the best sensory evaluation of the fermented juice. Therefore, strain CLF001 was selected for subsequent experiments.

[0059] The strain CLF001 was identified by morphology. The morphological characteristics of the strain CLF001 are as follows Figure 2 As shown. After 2 days of incubation at 30°C on PDA, the colony filled a 9-cm petri dish. The colony was loose, initially white, and later dark brown. Branching was finger-like or root-like, and the creeping hyphae were poorly differentiated. Sporangia were erect or curved, solitary, and some grew in bundles of 2-3. Sporangia were spherical or subspherical, turning black with aging. Strain CLF001 exhibited typical morphological characteristics of Rhizopus.

[0060] Further molecular characterization of strain CLF001 involved the following steps: extracting the isolated strain, inoculating the dominant strain CLF001 onto PDA plates, incubating at 25°C for 5 days, scraping the hyphae, extracting fungal DNA, and amplifying the target ITS sequence. Primers for the ITS sequence were selected: ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO: 1); ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 2). Sequencing yielded the ITS sequence shown in SEQ ID NO: 3. This sequence was uploaded to NCBI for sequence alignment, and 10 sequences with high homology were downloaded. An ML phylogenetic tree was constructed using MEGA.

[0061] (SEQ ID NO: 3).

[0062] According to the ITS of strain CLF001, the ML phylogenetic tree was constructed using MEGA. Figure 3 As shown in Figure 3, strain CLF001 was 100% similar to Rhizopus arrhizus and clustered into one branch. Therefore, based on morphology and ITS analysis, strain CLF001 was identified as Rhizopus arrhizus.

[0063] The strain (Rhizopus arrhizus CLF001) is deposited in the Guangdong Provincial Microbial Culture Collection Center, the storage address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is GDMCC No: 66063, and the storage time is March 27, 2025.

[0064] Example 2

[0065] A fermentation process for roxburghii juice comprises the following steps:

[0066] 1 w / v% glucose was added to the roxburghii juice (pH 3.4), mixed, and 2 mL of Rhizopus arrhizus CLF001 seed solution was inoculated into every 100 mL, and cultured at 30° C. and 150 r / min for 3 days.

[0067] Preparation of the above-mentioned Rhizopus arrhizus CLF001 seed liquid: Remove the slant culture from the 4°C refrigerator, cut a piece (3mm*3mm) in the center of the PDA plate, and culture at 28-30°C for 24-48 hours until the mycelium is plump and spores are formed. Directly pick the bacterial block (5mm in diameter) and inoculate it into PDB liquid culture medium at 28-30°C and 150-200rpm for 24 hours until the liquid becomes turbid and a uniform mycelial suspension is formed, of which about 1-3g of mycelial dry weight per 100mL is formed.

[0068] The above fermentation process was further optimized, including single factor experiments and orthogonal experiments.

[0069] 1. Single-factor experiment

[0070] Fermentation pH: 2 w / v% glucose was added to the roxburghii juice (pH 3.4), and the pH of the roxburghii juice was adjusted to 4.0, 5.0, 6.0, and 7.0 with NaOH, respectively. 5 mL of Rhizopus arrhizus CLF001 seed solution was inoculated into every 100 mL. The mixture was cultured at 30 ° C and 150 r / min for 3 days. The fermentation was stopped and the tannin content in the fermentation broth was determined (refer to "NY / T 1600-2008 Determination of tannin content in fruits, vegetables and their products", the same below) to confirm the optimal pH.

[0071] Glucose addition amount: 1w / v%, 2w / v%, 3w / v%, 4w / v%, and 5w / v% glucose were added to the original juice of sea buckthorn, respectively. 5 mL of Rhizopus arrhizus CLF001 seed liquid was inoculated into every 100 mL. The culture was carried out at 30°C and 150 r / min for 3 days. The fermentation was stopped and the tannin content in the fermentation broth was tested to confirm the optimal glucose addition amount.

[0072] Inoculation amount: 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of Rhizopus arrhizus CLF001 seed solution were added to every 100 mL of juice, respectively. The mixture was cultured at 30 ° C and 150 r / min for 3 days. The fermentation was stopped and the tannin content in the fermentation liquid was tested to confirm the optimal inoculation amount.

[0073] Fermentation temperature: Rosa roxburghii juice, glucose addition 1%, every 100mL juice inoculated with 2mL Rhizopus arrhizus CLF001 seed liquid, respectively, at 20℃, 25℃, 30℃, 35℃ and 40℃, 150r / min culture for 3 days, stop fermentation, and detect the tannin content in the fermentation liquid to confirm the optimal fermentation temperature.

[0074] Fermentation time: Rosa roxburghii juice, glucose addition rate 1%, every 100mL juice inoculated with 2mL Rhizopus arrhizus CLF001 seed liquid, cultured at 30℃, 150r / min for 2, 3, 4, 5, 6 days respectively, stopped fermentation, the tannin content in the fermentation liquid was tested to confirm the optimal fermentation time.

[0075] The results are as follows Figure 4 As shown in the figure, the results of the single-factor experiment showed that the optimal process for fermenting sea buckthorn juice with Rhizopus arrhizus CLF001 was as follows: 1w / v% glucose was added to the sea buckthorn juice, and 2v / v% Rhizopus arrhizus CLF001 seed solution was inoculated (i.e., 2 mL of Rhizopus arrhizus CLF001 seed solution was inoculated for every 100 mL of sea buckthorn juice), and the culture was shaken at 30°C and 150 r / min for 3 days.

[0076] 2. Orthogonal test

[0077] On the basis of single factor experiment, the inoculation amount, fermentation temperature and fermentation time were used as factors to analyze the effect of L9(3 3 )Orthogonal.

[0078] The experimental design used tannin degradation rate (Y1), polysaccharide content (Y2), and sensory evaluation (Y3) as comprehensive evaluation indicators. The comprehensive scoring method was: tannin degradation rate accounted for 40%, polysaccharide content and sensory evaluation accounted for 30%, and the total score was weighted. The factor level design is shown in Table 1. Tannin content was determined according to "NY / T 1600-2008 - Determination of Tannin Content in Fruits, Vegetables and Their Products", and polysaccharide content was determined according to "TGZCX019-2022 - Determination of Polysaccharide Content in Sea Rosa Rosa and Its Products". The sensory evaluation criteria are shown in Table 2.

[0079] Table 1 Orthogonal factor level table

[0080]

[0081]

[0082] Table 2 Sensory evaluation

[0083]

[0084] The orthogonal test optimization results are shown in Table 3. The results show that the combination with the highest tannin degradation effect is A1B3C2, that is, the inoculation amount is 1.5v / v%, the fermentation temperature is 32°C, and the fermentation time is 3 days. Factor A and factor B have large ranges and are the main influencing factors. The optimal process parameters of the orthogonal test are to add 1w / v% glucose to the sea buckthorn juice and 1.5v / v% Rhizopus CLF001 seed liquid as the inoculation amount, and to shake and culture at 32°C and 150r / min for 3 days. Using this process, the tannin degradation rate reached 88.64±2.36%, the polysaccharide content increased by 83.54±3.24%, and the sea buckthorn juice had almost no astringency after fermentation, and the taste was significantly improved.

[0085] Table 3 Orthogonal test optimization results

[0086]

[0087] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A strain of Rhizopus arrhizus CLF001, characterized in that: The Rhizopus arrhizus CLF001 is deposited in the Guangdong Provincial Microbial Culture Collection Center with a deposit number of GDMCC No: 66063 and a deposit date of March 27, 2025.

2. A method for preparing a culture of Rhizopus arrhizus CLF001, comprising: Provide liquid culture system; The Rhizopus arrhizus CLF001 according to claim 1 is inoculated into the liquid culture system for cultivation.

3. The preparation method according to claim 2, characterized in that The pH value of the liquid culture system is 2-9.

4. A culture of Rhizopus arrhizus CLF001, prepared by the preparation method according to claim 2 or 3.

5. A microbial agent, characterized in that: The microbial agent includes the Rhizopus arrhizus CLF001 according to claim 1 or the Rhizopus arrhizus CLF001 culture according to claim 4.

6. Use of the Rhizopus arrhizus CLF001 according to claim 1, the culture of Rhizopus arrhizus CLF001 according to claim 4, or the microbial agent according to claim 5 in at least one of (1) to (5): (1) Degradation of tannins; (2) preparing a product of degraded tannin; (3) removal of astringency; (4) preparing astringent products; (5) Preparation of sea buckthorn products.

7. A method for degrading tannins, comprising the step of treating a substance containing tannins using the Rhizopus arrhizus CLF001 according to claim 1, the Rhizopus arrhizus CLF001 culture according to claim 4, or the microbial agent according to claim 5.

8. A method for preparing a roxburghii drink, comprising the following steps: The roxburgh juice is mixed with sugar, and the mixture is inoculated with the Rhizopus arrhizus CLF001 according to claim 1, the Rhizopus arrhizus CLF001 culture according to claim 4, or the microbial agent according to claim 5, and fermented to obtain a roxburgh fermented drink.

9. The preparation method according to claim 8, characterized in that The added amount of the sugar is 0.1 w / v% to 5 w / v% of the roxburghii juice; and / or the inoculation amount of the Rhizopus arrhizus CLF001 is 1 v / v% to 5 v / v%.

10. The preparation method according to claim 8, characterized in that The fermentation conditions are 20-50° C. and 100-200 r / min for 2-7 days.

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