Method for multi-bacterium synergistic fermentation of roxburgh rose juice

Abstract

The invention belongs to the technical field of microbial fermentation, discloses a method for multi-bacterium synergistic fermentation of roxburgh rose juice, and particularly discloses a microbial agent which comprises rhizopus arrhizus CLF001 and at least one of pediococcus pentosaceus, lactobacillus plantarum, acetic acid bacteria, saccharomycetes and lactobacillus acidophilus. According to the microbial agent provided by the invention, the content of polysaccharide, polyphenol, brass and the like can be remarkably increased and the content of tannin in the roxburgh rose normal juice can be reduced by fermenting the roxburgh rose normal juice for 3 days by using the microbial agent, so that the fermented roxburgh rose normal juice does not feel obvious astringent taste, and the bitterness of the roxburgh rose normal juice is greatly improved; when the microbial agent is used for fermentation, polyphenol substances such as tannin with heavy astringent taste in the roxburgh rose normal juice are converted into non-astringent polyphenol substances, the taste of the normal juice is improved, the content of active ingredients is increased, for example, key ingredients (such as polyphenol, flavone and polysaccharide) capable of remarkably improving the antioxidant activity in the roxburgh rose normal juice can be used for improving the antioxidant performance of the roxburgh rose normal juice.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for multi-bacteria cooperative fermentation of roxburghii juice. 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, VP (rutin), 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. Microbial fermentation can effectively remove the astringency of Rosa roxburghii juice while also providing a synergistic effect. However, the high concentration of vitamin C and antimicrobial substances, such as tannins and polyphenols, in Rosa roxburghii juice limits its fermentation.

[0003] Rhizopus is an important type of industrial fungus. It has high amylase activity and can produce organic acids such as fumaric acid, lactic acid, succinic acid, etc. It can also produce aromatic esters. It is also an important fungus for converting alcohol compounds. It has strong ability to secrete pectinase and is often used in glucose manufacturing and brewing industries. It is rarely used in fruit and vegetable fermentation, especially in removing astringency and improving taste.

[0004] Multi-bacteria synergistic fermentation provides an efficient and environmentally friendly solution for removing astringency from fruits and vegetables through multi-target and multi-pathway effects, while taking into account flavor enhancement and safety, and has important application value. Multi-bacteria synergistic fermentation can also promote the production of prebiotics (oligosaccharides) and the proliferation of probiotics, enhancing the health effects of products such as regulating intestinal flora and anti-oxidation. Multi-bacteria synergistic fermentation achieves "1+1>2" synergy through complex interactions between microorganisms, providing an important strategy for the development of high-quality, functional fermented fruit and vegetable products. However, its complexity and the dynamic interactions between multiple bacterial species also bring many challenges and difficulties. Summary of the Invention

[0005] The first aspect of the present invention aims to provide a microbial agent.

[0006] The purpose of the second aspect of the present invention is to provide the use of the microbial agent of the first aspect of the present invention in the preparation of a roxburghii fermented product.

[0007] The third aspect of the present invention aims to provide a method for preparing a roxburghii fermented product.

[0008] The fourth aspect of the present invention aims to provide a roxburghii fermented product.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides a microbial agent comprising Rhizopus arrhizus CLF001 and at least one of Pediococcus pentosaceus, Lactobacillus plantarum, acetic acid bacteria, yeast, and Lactobacillus acidophilus;

[0011] 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.

[0012] In some embodiments of the present invention, the microbial agent includes Rhizopus arrhizus CLF001 and Pediococcus pentosaceus; or

[0013] The microbial agent includes Rhizopus arrhizus CLF001 and Lactobacillus plantarum; or

[0014] The microbial agent includes Rhizopus arrhizus CLF001 and acetic acid bacteria; or,

[0015] The microbial agent includes Rhizopus arrhizus CLF001 and Lactobacillus acidophilus; or

[0016] The microbial agent includes Rhizopus arrhizus CLF001 and yeast.

[0017] In some embodiments of the present invention, the microbial agent includes Rhizopus arrhizus CLF001, Pediococcus pentosaceus and Lactobacillus plantarum; or

[0018] The microbial agent includes Rhizopus arrhizus CLF001, Pediococcus pentosaceus and acetic acid bacteria; or,

[0019] The microbial agent includes Rhizopus arrhizus CLF001, Lactobacillus plantarum and acetic acid bacteria.

[0020] In some embodiments of the present invention, the microbial agent includes Rhizopus arrhizus CLF001, Pediococcus pentosaceus, Lactobacillus plantarum and Acetobacter.

[0021] In some embodiments of the present invention, the Pediococcus pentosaceus includes Pediococcus pentosaceus JYPR-9330 or Pediococcus pentosaceus HH-PP57; preferably Pediococcus pentosaceus JYPR-9330.

[0022] In some embodiments of the present invention, the Lactobacillus plantarum includes Lactobacillus plantarum JYLP-002 or Lactobacillus plantarum HH-LP56; preferably Lactobacillus plantarum JYLP-002.

[0023] The active ingredients of the above-mentioned microbial agent can be the above-mentioned microorganisms (i.e., Rhizopus arrhizus CLF001, Pediococcus pentosaceus, Lactobacillus plantarum, acetic acid bacteria, yeast and / or Lactobacillus acidophilus), the culture of the above-mentioned microorganisms (such as fermentation metabolites), and the active ingredients of the above-mentioned microbial agent can also contain other biological components or non-biological components. Those skilled in the art can determine the other active ingredients of the above-mentioned microbial agent according to the effect of the microbial agent.

[0024] 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.

[0025] 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 and biologically inert carrier in a field (such as microbiology or 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.

[0026] 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.

[0027] The second aspect of the present invention provides use of the microbial agent of the first aspect of the present invention in the preparation of a roxburghii fermented product.

[0028] The third aspect of the present invention provides a method for preparing a roxburghii fermented product, comprising the step of treating roxburghii juice with the microbial agent of the first aspect of the present invention.

[0029] In some embodiments of the present invention, the pH value of the roxburghii juice is 3-5.

[0030] In some embodiments of the present invention, the pH value of the roxburghii juice is 3-4.

[0031] In some embodiments of the present invention, the preparation method comprises the following steps: mixing roxburghii raw juice with sugar, inoculating the microbial agent of the first aspect of the present invention, and fermenting to obtain a roxburghii fermented product.

[0032] In some embodiments of the present invention, the fermentation conditions are 20-50° C., 100-200 r / min, and culturing for 2-5 days.

[0033] In some embodiments of the present invention, the fermentation conditions are 20-40° C. and 100-200 r / min for 2-3 days.

[0034] In some embodiments of the present invention, each microorganism in the microbial agent is activated separately before inoculation.

[0035] In some embodiments of the present invention, the activation treatment includes inoculating each bacterial species to an activation culture medium (MRS broth culture medium is used for Pediococcus pentosaceus, Lactobacillus plantarum and Lactobacillus acidophilus, potato dextrose water culture medium is used for yeast and Rhizopus arrhizus, and acetic acid bacteria is used for acetic acid bacteria culture medium), culturing at 25-30° C. for 1-2 days, and expanding the culture for 1-2 days (using the corresponding activation culture medium for expansion culture) to obtain the corresponding bacterial liquid.

[0036] In some embodiments of the present invention, when inoculating microorganisms, the microbial cultures are inoculated into a mixture of sea buckthorn juice and sugar at a volume ratio of 1:1 or 1:1:1 or 1:1:1:1, and the inoculation amount of the entire microbial culture is 1 v / v% to 3 v / v% of the sea buckthorn juice.

[0037] In some embodiments of the present invention, the preparation method includes the following steps: mixing the roxburghii juice with sugar, inoculating Lactobacillus plantarum and Pediococcus pentosaceus, and fermenting; inoculating Rhizopus arrhizus CLF001 and acetic acid bacteria; and fermenting again to obtain the roxburghii fermented product.

[0038] In some embodiments of the present invention, the fermentation is carried out at 30-50° C. for 20-30 hours.

[0039] In some embodiments of the present invention, the fermentation is carried out at 30-40° C. for 20-26 hours.

[0040] In some embodiments of the present invention, the secondary fermentation is carried out at 20-40° C., 100-200 r / min, and an aeration rate of 0.2-0.8 vvm for 2-4 days.

[0041] In some embodiments of the present invention, the secondary fermentation is carried out at 25-35° C., 100-200 r / min, and a ventilation volume of 0.2-0.8 vvm for 2-3 days.

[0042] By means of segmented fermentation and dissolved oxygen control, the mixed bacteria fermentation process is optimized, which significantly improves the removal of astringency of the sea buckthorn juice fermentation product, enhances the taste, and is rich in prebiotic polysaccharides.

[0043] In the presence of oxygen, vitamin C is easily oxidized. Fermentation in fermentation tanks uses aeration, so the vitamin C content decreases during the fermentation process, but the content of polyphenols and flavonoids increases significantly. Through process optimization, while the vitamin C content is significantly reduced, the antioxidant activity not only does not decrease, but also slightly increases.

[0044] In some embodiments of the present invention, before inoculating Lactobacillus plantarum and Pediococcus pentosaceus or Rhizopus rhizopus CLF001 and acetic acid bacteria, the above strains are activated to prepare a bacterial solution.

[0045] In some embodiments of the present invention, the bacterial solutions of Lactobacillus plantarum and Pediococcus pentosaceus are mixed in a volume ratio of 1:1 and then inoculated, and the inoculation amount is 1 v / v% to 2 v / v%.

[0046] In some embodiments of the present invention, the inoculation amount of Rhizopus arrhizus CLF001 is 1 v / v% to 2 v / v%, preferably 1 v / v% to 1.5 v / v%.

[0047] In some embodiments of the present invention, the inoculation amount of the acetic acid bacteria is 2 v / v% to 7 v / v%, preferably 2 v / v% to 5 v / v%.

[0048] 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.

[0049] In some embodiments of the present invention, the added amount of the sugar is 1 w / v% to 3 w / v% of the roxburghii juice.

[0050] In some embodiments of the present invention, the preparation method further includes solid-liquid separation and sterilization steps.

[0051] In some embodiments of the present invention, the method for preparing the sea buckthorn juice comprises the following steps: selecting fresh, moderately mature (yellow-green or golden yellow peel), pest-free, and rot-free sea buckthorn fruits, washing with high-pressure water to remove surface impurities, squeezing, filtering and clarifying, and pasteurizing at 80-85° C. for 5-10 minutes to obtain the sea buckthorn juice.

[0052] A fourth aspect of the present invention provides a roxburghii fermented product, which is prepared by the preparation method of the third aspect of the present invention.

[0053] In some embodiments of the present invention, the roxburgh roxburgh fermented product includes a roxburgh ... beverage.

[0054] The beneficial effects of the present invention are:

[0055] The invention provides a microbial agent. When the original roxburgh juice is fermented with the microbial agent, the contents of polysaccharides, polyphenols and brass, etc., can be significantly increased during fermentation for 3 days, and the content of tannins in the original roxburgh juice can be reduced, so that the fermented original roxburgh juice has no obvious astringency, and the bitterness of the original roxburgh juice is greatly improved. That is, the microbial agent is used to ferment the tannins and other polyphenols with strong astringency in the original roxburgh juice and convert them into non-astringent polyphenols, thereby improving the taste of the original juice and increasing the content of active ingredients. For example, the microbial agent can significantly increase the key ingredients with antioxidant activity (such as polyphenols, flavonoids and polysaccharides) in the original roxburgh juice and improve the antioxidant performance of the juice. The invention has important significance in the fermentation and processing of the original roxburgh juice. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The sensory evaluation results of the roxburghii fermented liquid obtained by fermenting the roxburghii juice using the methods of Examples 1 to 13 are shown. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below through specific examples.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] Preparation of bacterial liquid: Take a small amount of bacterial strain and inoculate it into 100 mL of liquid culture medium for activation (MRS broth medium is used for Lactobacillus leucovorus, Bifidobacterium, Pediococcus pentosaceus, Lactobacillus plantarum and Lactobacillus acidophilus; potato dextrose water medium is used for yeast, Ganoderma lucidum, Monascus and Rhizopus arrhizus; acetic acid bacteria medium is used for acetic acid bacteria medium) and culture it at 28°C for 1-2 days. Dip the culture liquid and streak it on the corresponding culture medium solid plate, and culture it at 28°C for 1-3 days. Select a single colony and expand it in the corresponding liquid culture medium for 1-2 days for later use to obtain the corresponding bacterial liquid.

[0062] MRS broth (catalog number HB0384-1) and potato glucose water culture medium (catalog number HB0233-4) were purchased from Haibo Biotechnology Co., Ltd.

[0063] Acetobacter culture medium: slant / plate culture medium: glucose 8 g, yeast powder 10 g, calcium carbonate 5 g, agar 15-20 g, water 1000 mL, pH 5.5, sterilize at 0.1 MPa for 20 min.

[0064] Liquid proliferation medium: 10 g glucose, 10 g yeast, 1000 mL pH 5.5 water, sterilized, and 40 mL of ethanol (food grade).

[0065] The bacterial species information involved in the examples is shown in Table 1.

[0066] Table 1 Fermentation strain information

[0067] name Strain number Purchase Source Pediococcus pentosaceus 1 JYPR-9330 Shandong Zhongke Jiayi Bioengineering Co., Ltd. Pediococcus pentosaceus 2 HH-PP57 Zhengzhou Hehe Bioengineering Technology Co., Ltd. Lactobacillus plantarum 1 JYLP-002 Shandong Zhongke Jiayi Bioengineering Co., Ltd. Lactobacillus plantarum 2 HH-LP56 Zhengzhou Hehe Bioengineering Technology Co., Ltd. Acetobacter ATCC 23768 Guangdong Provincial Microbial Culture Collection Center Yeast 1 Angel-RW yeast for wine and fruit wine Angel Yeast Co., Ltd. Yeast 2 Angel-Wine and Fruit Wine Special Yeast SW Angel Yeast Co., Ltd. Lactobacillus acidophilus JYLA-191 Shandong Zhongke Jiayi Bioengineering Co., Ltd. Rhizopus arrhizus CLF001 Lactobacillus leucoderma LB-Z16 Shandong Zhongke Jiayi Bioengineering Co., Ltd. Bifidobacterium JYBR-390 Shandong Zhongke Jiayi Bioengineering Co., Ltd. Ganoderma lucidum GL0102 Gift from the Fungus Research Center of Fujian Agriculture and Forestry University Monascus GDMCC NO.3.72 Guangdong Provincial Microbial Culture Collection Center

[0068] In the above table, Rhizopus arrhizus CLF001 was independently isolated by the laboratory and has been deposited in the Guangdong Provincial Microbial Culture Collection Center with the deposit number GDMCC No: 66063 and the deposit date is March 27, 2025.

[0069] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0070] Example 1

[0071] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a Lactobacillus leucovorus liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0072] Example 2

[0073] A method for fermenting roxburghii raw juice comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bifidobacterium liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0074] Example 3

[0075] A method for fermenting roxburghii raw juice comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a Lactobacillus acidophilus liquid with an inoculation amount of 1v / v%, and culturing by shaking at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0076] Example 4

[0077] A method for fermenting roxburghii raw juice comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a yeast culture liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a rotation speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0078] Example 5

[0079] A method for fermenting roxburghii raw juice comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a yeast 2 culture liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a rotation speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0080] Example 6

[0081] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial liquid of Pediococcus pentosaceus with an inoculation amount of 1v / v%, and culturing the mixture with shaking at 30°C and a rotation speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0082] Example 7

[0083] A method for fermenting roxburghii raw juice comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a 1v / v% bacterial liquid of Pediococcus pentosaceus, and culturing the mixture under shaking at 30°C and a rotation speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0084] Example 8

[0085] A method for fermenting roxburghii raw juice comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a Lactobacillus plantarum bacterial liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0086] Example 9

[0087] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating 2 bacterial liquids of Lactobacillus plantarum with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0088] Example 10

[0089] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating acetic acid bacteria liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0090] Example 11

[0091] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a Rhizopus arrhizae liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0092] Example 12

[0093] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a Ganoderma lucidum liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0094] Example 13

[0095] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a monascus liquid with an inoculation amount of 1v / v%, and oscillating and culturing at 30°C and a speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0096] Example 14

[0097] The invention discloses a method for fermenting roxburghii raw juice, comprising the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bacterial solution (a mixture of a Rhizopus arrhizae solution and a Pediococcus pentosaceus 1 bacterial solution in a volume ratio of 1:1) with an inoculation amount of 1v / v%, and culturing under shaking at 30°C and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0098] Example 15

[0099] The invention discloses a method for fermenting roxburghii raw juice, comprising the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae and Pediococcus pentosaceus 2 bacterial solutions in a volume ratio of 1:1) with an inoculation amount of 1v / v%, and culturing under shaking at 30°C and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0100] Example 16

[0101] A method for fermenting roxburghii juice comprises the following steps: adding 1 w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae and a Lactobacillus plantarum solution at a volume ratio of 1:1) with an inoculation amount of 1 v / v%, and culturing under shaking at 30° C. and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0102] Example 17

[0103] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae and Lactobacillus plantarum 2 bacterial solution in a volume ratio of 1:1) with an inoculum amount of 1v / v%, and culturing under shaking at 30°C and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0104] Example 18

[0105] A method for fermenting roxburghii juice comprises the following steps: adding 1 w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae and an acetic acid bacteria solution in a volume ratio of 1:1) with an inoculum amount of 1 v / v%, and culturing under shaking at 30° C. and a rotation speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0106] Example 19

[0107] The invention discloses a method for fermenting roxburghii raw juice, comprising the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bacterial solution (a mixture of a Rhizopus arrhizae liquid and a yeast liquid in a volume ratio of 1:1) with an inoculation amount of 1v / v%, and culturing under shaking at 30°C and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0108] Example 20

[0109] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a mixture of a Rhizopus arrhizae solution and a yeast solution in a volume ratio of 1:1) with an inoculation amount of 1v / v%, and culturing under shaking at 30°C and a rotation speed of 150 r / min for 3 days to obtain a roxburghii fermented liquid.

[0110] Example 21

[0111] A method for fermenting roxburghii juice comprises the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae and Lactobacillus acidophilus in a volume ratio of 1:1) with an inoculum amount of 1v / v%, and culturing under shaking at 30°C and a rotation speed of 150 r / min for 3 days to obtain roxburghii fermented liquid.

[0112] Example 22

[0113] A method for fermenting roxburghii juice comprises the following steps: adding 1 w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae, a Pediococcus pentosaceus bacterial solution, and a Lactobacillus plantarum bacterial solution in a volume ratio of 1:1:1) with an inoculation amount of 1 v / v%, and culturing by shaking at 30° C. and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0114] Example 23

[0115] The invention discloses a method for fermenting roxburghii juice, comprising the following steps: adding 1w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a Rhizopus arrhizae solution, a Pediococcus pentosaceus 1 bacterial solution and an acetic acid bacteria solution mixed and inoculated at a volume ratio of 1:1:1) with an inoculation amount of 1v / v%, and carrying out shaking culture at 30°C and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0116] Example 24

[0117] A method for fermenting roxburghii juice comprises the following steps: adding 1 w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae, Lactobacillus plantarum 1 bacterial solution and acetic acid bacteria bacterial solution in a volume ratio of 1:1:1) with an inoculum amount of 1 v / v%, and culturing under shaking at 30° C. and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0118] Example 25

[0119] A method for fermenting roxburghii juice comprises the following steps: adding 1 w / v% glucose to the roxburghii juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae solution, a Pediococcus pentosaceus solution, a Lactobacillus plantarum solution, and an acetic acid bacteria solution in a volume ratio of 1:1:1:1) with an inoculum amount of 1 v / v%, and carrying out shaking culture at 30°C and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0120] Example 26

[0121] The invention discloses a method for fermenting roxburghii raw juice (shake flask fermentation), comprising the following steps: adding 1 w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae liquid, a Pediococcus pentosaceus liquid, a Lactobacillus plantarum liquid and an acetic acid bacteria liquid in a volume ratio of 1:1:1:1) with an inoculum amount of 2 v / v%, and carrying out shaking culture at 30°C and a rotation speed of 150 r / min for 3 days to obtain the roxburghii fermented liquid.

[0122] Example 27

[0123] The invention discloses a method for fermenting roxburghii raw juice (performed in a 2L fermentation tank). The method comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bacterial solution (a mixture of Rhizopus arrhizae solution, a Pediococcus pentosaceus solution, a Lactobacillus plantarum solution and an acetic acid bacteria solution in a volume ratio of 1:1:1:1) with an inoculum amount of 2v / v%, fermenting at 30°C, a rotation speed of 150r / min, a ventilation volume of 0.2vvm, and continuing fermentation for 3 days to obtain the roxburghii fermented liquid.

[0124] Example 28

[0125] The invention discloses a method for fermenting roxburghii raw juice (performed in a 2L fermentation tank). The method comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bacterial solution (a mixture of 1 bacterial solution of Lactobacillus plantarum and 1 bacterial solution of Pediococcus pentosaceus in a volume ratio of 1:1) with an inoculation amount of 1v / v%, and fermenting at 35°C and a rotation speed of 150 r / min for 12 hours; then inoculating a Rhizopus arrhizae liquid (inoculation amount of 1v / v%) and an acetic acid bacteria liquid (inoculation amount of 5v / v%), and fermenting at 30°C and a rotation speed of 150 r / min while passing sterile air with a ventilation volume of 0.4vvm. The fermentation is continued for 2 days to obtain the roxburghii fermented juice.

[0126] Example 29

[0127] The invention discloses a method for fermenting roxburghii raw juice (carried out in a 2L fermentation tank). The method comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bacterial solution (a mixture of 1 bacterial solution of Lactobacillus plantarum and 1 bacterial solution of Pediococcus pentosaceus in a volume ratio of 1:1) with an inoculation amount of 1v / v%, and performing static fermentation at 35°C for 24 hours; then inoculating a Rhizopus arrhizae liquid (inoculation amount of 1.5v / v%) and an acetic acid bacteria liquid (inoculation amount of 3v / v%), and fermenting at 30°C with a rotation speed of 150r / min, passing sterile air with a ventilation volume of 0.5vvm, and continuing the fermentation for 3 days to obtain the roxburghii raw juice.

[0128] Example 30

[0129] The invention discloses a method for fermenting roxburghii raw juice (performed in a 2L fermentation tank). The method comprises the following steps: adding 1w / v% glucose to the roxburghii raw juice (pH value 3.4), inoculating a bacterial solution (a mixture of 1 bacterial solution of Lactobacillus plantarum and 1 bacterial solution of Pediococcus pentosaceus in a volume ratio of 1:1) with an inoculation amount of 1v / v%, and performing static fermentation at 35°C for 24 hours; then inoculating a Rhizopus arrhizae liquid (inoculation amount of 1.5v / v%) and an acetic acid bacteria liquid (inoculation amount of 2v / v%), and continuing the fermentation at 30°C with a rotation speed of 200 r / min, passing sterile air with a ventilation volume of 0.6vvm, and continuing the fermentation for 60 hours to obtain the roxburghii fermented juice.

[0130] Comparative Example 1

[0131] A roxburgh pear juice is prepared by adding 1 w / v % glucose into roxburgh pear original juice (pH value 3.4).

[0132] Effect embodiment

[0133] 1. Single strain fermentation results

[0134] The roxburghii fermented liquor obtained by fermentation in Examples 1 to 13 was subjected to sensory evaluation (the evaluation criteria are shown in Table 3. For the purpose of drawing the graph, the data were converted at a ratio of 10:1, i.e., 100 points were converted to 10 points) and the number of microbial colonies was counted.

[0135] Microbial colony count: Use a sterile pipette to pipette 1 mL of sample into a 9 mL sterile distilled water centrifuge tube. Shake thoroughly for 1-2 minutes to prepare a 1:10 dilution. Follow the above steps to prepare a 10-fold serial dilution of the sample. Use a new 1 mL sterile pipette for each dilution. Select two to three sample dilutions of appropriate dilutions. While performing 10-fold dilutions, pipette 1 mL of each dilution into two sterile plates. Simultaneously, add 1 mL of the sterile dilution to two sterile plates as a blank control. Immediately pour 20-25 mL of potato dextrose agar or MRS medium cooled to 46°C into the plates, swirling to mix thoroughly. Place on a water surface until the medium completely solidifies. Incubate at 30°C for 5 days. Select plates with 10-150 CFU and count colonies based on colony morphology.

[0136] The results showed that 12 microorganisms such as common fermentation bacteria of fruits and vegetables, such as Lactobacillus bulgaricus, Bifidobacterium, Lactobacillus acidophilus, Saccharomyces 1, Saccharomyces 2, Pediococcus pentosaceus 1, Pediococcus pentosaceus 2, Lactobacillus plantarum 1, Lactobacillus plantarum 2, acetic acid bacteria, Ganoderma lucidum, Monascus, and the isolated Rhizopus arrhizae were used to ferment the roxburghii juice. The growth of microorganisms in the roxburghii juice and the improvement of taste were evaluated, as shown in Tables 2 and 3. Figure 1 As shown, Rhizopus arrhizus, yeast, Pediococcus pentosaceus 1, Lactobacillus plantarum 1, and acetic acid bacteria are the dominant strains for fermenting roxburghii juice, and have good fermentation effect.

[0137] Table 2 Evaluation of the results of single-bacteria fermentation of roxburghii juice

[0138]

[0139] 2. Results of mixed fermentation of two bacteria

[0140] The roxburghii fermented liquid of Example 11 and Examples 14 to 21 was taken respectively, and the liquid was collected by centrifugation (8000-10000r / min centrifugation for 10min). After pasteurization of the bacterial liquid, sensory evaluation and polysaccharide content were determined. Among them, the sensory evaluation method was as follows: 5 laboratory personnel were selected to conduct sensory evaluation on the sterilized roxburghii fermented liquid, and the roxburghii fermented product was comprehensively evaluated and scored mainly from the four aspects of color, aroma, taste and appearance. The sensory evaluation standards are shown in Table 3. The polysaccharide content was determined with reference to "T / GZCX019-2022 Determination of polysaccharide content in roxburghii and its products".

[0141] Table 3 Sensory evaluation

[0142]

[0143]

[0144] Saccharomyces, Pediococcus pentosaceus 1, Lactobacillus plantarum 1, acetic acid bacteria are respectively co-fermented with Rhizopus arrhizae roxburghii stoichiometric ratio, result is as shown in Table 4, sensory score and polysaccharide content comprehensive evaluation preferably combine to be Rhizopus arrhizae+Pediococcus pentosaceus 1 (embodiment 14), Rhizopus arrhizae+Pediococcus pentosaceus 2 (embodiment 15), Rhizopus arrhizae+acetic acid bacteria (embodiment 18), Rhizopus arrhizae+Lactobacillus plantarum 1 (embodiment 16), Rhizopus arrhizae+Saccharomyces 2 (embodiment 20), Rhizopus arrhizae++Lactobacillus plantarum 2 (embodiment 17), Rhizopus arrhizae+Saccharomyces 1 (embodiment 19) and Rhizopus arrhizae+Lactobacillus acidophilus (embodiment 21).Illustrate that Pediococcus pentosaceus, acetic acid bacteria and Lactobacillus plantarum are more suitable for cooperative fermentation with Rhizopus arrhizae.Saccharomyces is due to the accumulation of metabolites such as ethanol, and the later stage introduces the bitter taste variation.

[0145] Table 4 Sensory evaluation and exopolysaccharide content of the roxburghii fermentation broth obtained by co-fermentation of the two bacteria

[0146] Experimental groups Sensory evaluation Extracellular polysaccharide content (mg / mL) Example 11 69.8±5.23 1.07±0.15 Example 14 77.8±5.31 1.12±0.21 Example 15 71.2±3.68 1.01±0.07 Example 16 71±3.70 1.20±0.14 Example 17 68.2±5.65 0.93±0.16 Example 18 78.6±5.31 1.09±0.10 Example 19 65.6±5.07 0.92±0.16 Example 20 70.4±5.26 0.84±0.43 Example 21 62.6±4.59 0.79±0.19

[0147] 3. Results of multi-bacteria synergistic fermentation

[0148] The roxburghii fermented liquid obtained in Examples 22 to 25 was taken respectively, centrifuged, and the bacterial liquid was collected. After pasteurization, the bacterial liquid was subjected to sensory evaluation and polysaccharide content determination. The evaluation method and determination method were the same as those in 2.

[0149] The results are shown in Table 5. The best effect was achieved by synergistic fermentation of roxburghii juice with Rhizopus arrhizus + Pediococcus pentosaceus 1 + Lactobacillus plantarum 2 + Acetobacter, with a sensory score of 83.2±5.74 and an exopolysaccharide content of 1.36±0.07 mg / mL.

[0150] Table 5 Sensory evaluation and exopolysaccharide content of the roxburghii fermentation broth obtained by multi-bacteria cooperative fermentation

[0151]

[0152]

[0153] 4. Optimization results of multi-bacteria synergistic fermentation process

[0154] The rosa roxburghii fermented liquid obtained in Examples 26 to 30 and the rosa roxburghii juice of Comparative Example 1 were taken separately, centrifuged, and the supernatant was collected. After pasteurization, the supernatant was subjected to sensory evaluation, and the polysaccharide, total phenol, flavonoid, and VC contents in the supernatant were determined. At the same time, the DPPH antioxidant activity and ABTS antioxidant activity of the supernatant were determined. The sensory evaluation method and the polysaccharide content determination method were the same as 2. Flavonoid content determination: refer to "TGZCX018-2022 Determination of total flavonoid content in rosa roxburghii and its products", total phenol content determination: refer to "T / GZCX 020-2022 Determination of total phenol content in rosa roxburghii and its products", and VC content determination: refer to "GB 5009.86-2016 National Food Safety Standard Determination of ascorbic acid in food".

[0155] DPPH free radical scavenging ability: 1.0 mL of the supernatant diluted 2000 times and different concentrations of VC (0.03, 0.04, 0.05, 0.075, 0.1 mg / mL) were mixed with 1.0 mL of 0.5 mmol / L DPPH solution and 2 mL of 60% ethanol, and the mixture was allowed to stand at room temperature for 30 min. The absorbance A at a wavelength of 517 nm was measured. i VC was used as the positive control. The background absorbance of the sample was determined by replacing the DPPH solution with an equal volume of anhydrous ethanol. j The absorbance A0 of the blank control was determined by replacing the polysaccharide sample solution with an equal volume of distilled water. The DPPH radical scavenging rate was calculated according to formula (1).

[0156]

[0157] ABTS+· scavenging ability: Mix equal volumes of 7.4 mmol / L ABTS and 2.6 mmol / L potassium persulfate solution, incubate in the dark for 12-16 hours, and dilute the solution with 0.2 mol / L pH 7.4 phosphate buffer to a 734 nm absorbance of (0.70 ± 0.02) to prepare an ABTS working solution. 1.0 mL of the 2000-fold diluted fermentation broth solution and 1.0 mL of VC at different concentrations (0.001, 0.002, 0.005, 0.01, and 0.015 mg / mL) were mixed with 6.0 mL of the ABTS working solution, allowed to stand at room temperature for 6 minutes, and the absorbance at 734 nm, Ai, was measured. VC was used as a positive control. An equal volume of distilled water was used to replace the ABTS working solution to determine the sample background absorbance Aj, and an equal volume of distilled water was used to replace the polysaccharide sample solution to determine the blank control absorbance A0. Calculate the ABTS+· scavenging rate according to formula (1).

[0158] The results are shown in Table 6. The polysaccharide, total and total flavonoid contents in Comparative Example 1 were 0.66 ± 0.20 mg / mL, 9.13 ± 0.64 mg / mL and 9.05 ± 0.23 μg / mL, respectively. After Example 26 (shake flask fermentation), the polysaccharide content increased by nearly 1 times, and the flavonoid content increased by about 10%. The total phenol content decreased by about 15%, mainly due to the degradation of tannin polyphenols, and the VC content decreased by about 25%, mainly due to the oxidation of VC in the shaker fermentation process. The polysaccharide and flavonoid contents in the roxburghii fermentation broth of Examples 27 to 30 (fermentation tank) were further improved on the basis of the shake flask, wherein the polysaccharide content increased by 1.62 to 2.50 times, and the flavonoid content increased by 17.92% to 72.20%; however, the VC content decreased significantly, decreasing by 55.38% to 59.96%. It is worth noting that the total phenol content of fermentation tank fermentation also increased by 5.48% to 58.71%. This may be due to the fermentation tank fermentation process converting the astringent tannins and other polyphenols in the sea buckthorn juice into non-astringent polyphenols.

[0159] Table 6 Sensory evaluation and index determination of the roxburghii fermented liquor of Examples 26 to 30

[0160]

[0161] Further comparison was made of the differences in the components of the sea buckthorn juice before and after fermentation using the strains in Example 30. The results are shown in Table 7. After strain screening and process optimization, the sea buckthorn juice was fermented using the method of Example 30. The polysaccharide content in the obtained sea buckthorn fermented liquid was increased by more than 2.5 times compared with the sea buckthorn juice, the total phenol content was increased by 36.89%, and the tannin content (determination method refers to "NY / T 1600-2008 Determination of tannin content in fruits, vegetables and their products") was reduced by 52.36%. Tannin is a type of plant polyphenol with astringency and bitter taste, and is the main substance of the astringency of sea buckthorn. After fermentation, the tannin content was significantly reduced, while the total phenol content was significantly increased, indicating that the fermentation process of this embodiment can convert polyphenols such as tannins with strong astringency in sea buckthorn juice into non-astringent polyphenols. VC, polyphenols, and flavonoids are key components of the strong antioxidant activity of roxburghii juice. VC was used as a positive control in the antioxidant activity evaluation. The antioxidant activity of the juice was equivalent to 32.94±0.72 mg / mL of VC, while the VC content in the juice was 19.43±1.71, accounting for approximately 60% of the total antioxidant activity. In the presence of oxygen, VC is easily oxidized. After aerated fermentation, the VC content decreased by approximately 60%, but the total antioxidant activity was equivalent to 33.16±0.89 mg / mL of VC. This indicates that the content of polysaccharides, polyphenols, and flavonoids increased significantly after fermentation, resulting in a slight increase in antioxidant activity, rather than a decrease. This compensates for the defect that VC is easily oxidized during processing, which leads to a decrease in antioxidant activity.

[0162] Table 7 Differences in the main components of the roxburghii juice before and after fermentation in Example 30

[0163]

[0164]

[0165] In summary, the method of multi-bacteria synergistic fermentation of sea buckthorn juice with Rhizopus arrhizus as the core is adopted. After the sea buckthorn juice is fermented by this method, the polyphenol content and brass content in the fermentation product are significantly increased, and the bitter taste is significantly improved. This shows that the method provided by the present invention can convert polyphenols such as tannins with strong astringency in the sea buckthorn juice into non-astringent polyphenols, improve the taste of the juice and increase the content of active ingredients, which is of great significance in the fermentation and processing of sea buckthorn juice.

[0166] 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 microbial agent comprising Rhizopus arrhizus CLF001 and at least one of Pediococcus pentosaceus, Lactobacillus plantarum, acetic acid bacteria, yeast, and Lactobacillus acidophilus; 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. The microbial agent according to claim 1, characterized in that The microbial agent includes Rhizopus arrhizus CLF001 and Pediococcus pentosaceus; or The microbial agent includes Rhizopus arrhizus CLF001 and Lactobacillus plantarum; or The microbial agent includes Rhizopus arrhizus CLF001 and acetic acid bacteria; or, The microbial agent includes Rhizopus arrhizus CLF001 and Lactobacillus acidophilus; or The microbial agent includes Rhizopus arrhizus CLF001 and yeast.

3. The microbial agent according to claim 1, characterized in that The microbial agent includes Rhizopus arrhizus CLF001, Pediococcus pentosaceus and Lactobacillus plantarum; or The microbial agent includes Rhizopus arrhizus CLF001, Pediococcus pentosaceus and acetic acid bacteria; or, The microbial agent includes Rhizopus arrhizus CLF001, Lactobacillus plantarum and acetic acid bacteria.

4. The microbial agent according to any one of claims 1 to 3, characterized in that The microbial agent includes Rhizopus arrhizus CLF001, Pediococcus pentosaceus, Lactobacillus plantarum and acetic acid bacteria.

5. Use of the microbial agent according to any one of claims 1 to 4 in the preparation of a roxburghii fermented product.

6. A method for preparing a roxburghii fermented product, comprising the step of treating roxburghii juice with the microbial agent according to any one of claims 1 to 4.

7. The preparation method according to claim 6, characterized in that The preparation method comprises the following steps: mixing roxburghii raw juice with sugar, inoculating the microbial agent according to any one of claims 1 to 4, and fermenting to obtain a roxburghii fermented product; Preferably, the fermentation conditions are 20-50° C., 100-200 r / min, and culturing for 2-5 days.

8. The preparation method according to claim 6, characterized in that The preparation method comprises the following steps: mixing roxburghii juice with sugar, inoculating Lactobacillus plantarum and Pediococcus pentosaceus as claimed in claim 4, and fermenting; inoculating Rhizopus arrhizus CLF001 and acetic acid bacteria as claimed in claim 4; and fermenting again to obtain the roxburghii fermented product.

9. The preparation method according to claim 8, characterized in that The fermentation conditions are 30-50° C. for 20-30 hours; and / or the secondary fermentation conditions are 20-40° C., 100-200 r / min, and a ventilation rate of 0.2-0.8 vvm for 2-4 days; Preferably, the added amount of the sugar is 0.1 w / v% to 5 w / v% of the roxburghii juice; Preferably, the pH value of the roxburghii juice is 2-5.

10. A roxburghii fermented product prepared by the preparation method according to any one of claims 6 to 9.

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