Food-sourced acetic acid bacteria capable of producing bacterial cellulose and application of acetic acid bacteria in fermented tea

Abstract

The present invention relates to a foal coconut bacillus, as well as compositions, food products or dietary supplements, cultures and fermentation products comprising the same. The invention further relates to application of the foal coconut bacillus and the composition in preparation of food products or dietary supplements or health care products.

Description

Technical Field

[0001] The present invention relates to a Komagataeibacter cocois, and a composition, food product or dietary supplement, culture and fermentation product comprising the same. The present invention also relates to the use of the Komagataeibacter cocois and the composition in the preparation of a food product or a dietary supplement or a health product. Background Art

[0002] Kombucha, also known as "black tea fungus", "sea treasure", "stomach treasure", etc., originated in the Bohai area of China and is a functional fermented tea beverage that has received much attention in recent years. It is a natural probiotic beverage made by fermenting tea (black tea, green tea, Pu-erh tea or oolong tea) base, sugar, water, bacteria and yeast. The black tea fungus (Symbiotic culture of bacteria and yeast, SCOBY) formed by the symbiotic relationship between acetic acid bacteria and yeast is immersed in tea water added with white sugar (glycolysis) and continuously fermented to obtain. This beverage is rich in components such as probiotics, digestive enzymes, organic acids, vitamin B, antioxidants and trace minerals, and has a series of potential benefits to human health and is deeply loved by people.

[0003] The main strains of kombucha include yeasts and acetic acid bacteria. Yeast components usually include Saccharomyces cerevisiae, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, and Zygosaccharomyces bailii. They are responsible for fermenting the sugars added to the tea base to produce alcohol and provide some special flavors. Acetic acid bacteria are the dominant bacteria during the kombucha fermentation process. They can produce acetic acid using alcohol as a substrate under aerobic conditions, giving kombucha its characteristic sour taste. The acetic acid bacteria that have been found in kombucha include Acetobacteria, Gluconobacter, Gluconacetobaceter, and Komagataeibacter, etc. Among them, Komagataeibacter and Gluconacetobaceter are considered the most characteristic microorganisms in kombucha fermentation. They produce β-1,4-glucan by utilizing the carbon source in the fermentation substrate. β-1,4-glucan can form water-insoluble high-molecular polymers through intramolecular and intermolecular hydrogen bonding, namely bacterial cellulose. Bacterial cellulose has excellent properties such as high crystallinity, high water-holding and water-recovery capacity, high water permeability and air permeability, high mechanical strength, strong wet-state flexibility, as well as biocompatibility, biodegradability, and controllable biosynthesis. It has been commercially applied in many fields such as food, biomedicine, cosmetics, and environmental pollution treatment, and has broad application prospects. Komagataeibacter and Gluconacetobaceter are the main strains that produce bacterial cellulose, and the formation of the kombucha pellicle is largely attributed to the presence of these two strains.

[0004] However, the microbial composition of traditional kombucha is complex, and there are problems such as being difficult to preserve, prone to degradation, and easy to be contaminated by bacteria. At the same time, natural fermentation has problems such as a long fermentation period, low product safety, and poor stability of product taste. Therefore, many scholars have studied the process of producing kombucha by pure mixed bacteria fermentation.

[0005] Therefore, it is necessary to isolate new pure acetic acid bacteria from traditional kombucha to develop their characteristics of producing bacterial cellulose and their potential application in fermented tea. Summary of the Invention

[0006] The inventors of the present application screened a brand-new Komagataeibacter xylinus from kombucha through a large number of experiments. The inventors of the present application confirmed through a large number of experiments that this Komagataeibacter xylinus can form a bacterial cellulose membrane. Further, by fermenting tea sugar water with it, the sensory evaluation results of the tea sugar water were significantly improved, and thus the present invention was completed.

[0007] Komagataeibacter nataicola

[0008] Thus, in a first aspect, the present application provides a Komagataeibacter nataicola having a 16S rDNA sequence with 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95% or 100% identity to SEQ ID NO: 1.

[0009] In certain embodiments, after amplification with primers as shown in SEQ ID NO: 2 and SEQ ID NO: 3, the 16S rDNA yields a fragment of 1400 bp.

[0010] In certain embodiments, the colonies of the Komagataeibacter nataicola are round and opalescent light yellow.

[0011] In certain embodiments, the surface of the colonies of the Komagataeibacter nataicola is moist and slightly wrinkled, and the edge is slightly rough.

[0012] In certain embodiments, the cells of the Komagataeibacter nataicola are short rod-shaped.

[0013] In certain embodiments, under suitable growth conditions (e.g., in the presence of a liquid medium), the Komagataeibacter nataicola is capable of producing a bacterial cellulose membrane.

[0014] In certain embodiments, the moisture content of the wet bacterial cellulose membrane is greater than 85% (e.g., 85% - 90%, 90% - 95%, 95% - 99.9%).

[0015] In certain embodiments, the rehydration rate of the dry bacterial cellulose membrane is greater than 75% (e.g., 75% - 80%, 80% - 85%, 85% - 90%, 90 - 95%).

[0016] In certain embodiments, the cellulose yield of the bacterial cellulose membrane is greater than 0.1 g / L (e.g., 0.1 g / L - 0.3 g / L, 0.3 g / L - 0.5 g / L, 0.5 g / L - 0.7 g / L, 0.7 g / L - 0.9 g / L).

[0017] In certain embodiments, the stretching distance of the bacterial cellulose membrane is greater than 10 mm (e.g., 10 mm - 14 mm, 14 mm - 17 mm, 17 mm - 20 mm).

[0018] In certain embodiments, the tensile strength of the bacterial cellulose membrane is greater than 0.3 Mpa (e.g., 0.3 Mpa - 0.6 Mpa, 0.6 Mpa - 0.9 Mpa, 0.9 Mpa - 1.2 Mpa).

[0019] In certain embodiments, the Komagataeibacter nataicola is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 27966.

[0020] Composition

[0021] In a second aspect, the present application provides a composition comprising the Komagataeibacter nataicola as described above.

[0022] In certain embodiments, the Komagataeibacter nataicola can be used in combination with one or more other species of microorganisms that can have a beneficial effect on the health of the host to which they are administered.

[0023] Thus, in certain embodiments, the composition further comprises a microorganism selected from the following: bacteria, fungi (such as yeast), or any combination thereof.

[0024] In certain embodiments, the microorganism is a probiotic.

[0025] In certain embodiments, the bacteria are selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof.

[0026] In certain embodiments, the yeast is selected from Brettanomyces anomalus, Saccharomyces cerevisiae, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Zygosaccharomyces bailii, or any combination thereof.

[0027] In certain embodiments, the bacteria of the genus Bifidobacterium are selected from: Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium adolescentis, or any combination thereof.

[0028] In certain embodiments, the bacteria of the genus Lactobacillus are selected from: Lactobacillus paracasei, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus jensenii, Lactobacillus iners, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus salivarius, or any combination thereof.

[0029] In certain embodiments, the bacteria of the genus Bacillus are selected from: Bacillus subtilis, Bacillus coagulans, or any combination thereof.

[0030] In certain embodiments, the bacteria of the genus Propionibacterium are selected from: Propionibacterium shermanii, Propionibacterium freudenreichii, Propionibacterium acidipropionici, or any combination thereof.

[0031] In certain embodiments, the bacteria of the genus Streptococcus are selected from: Streptococcus thermophilus, Streptococcus salivarius, or any combination thereof.

[0032] In certain embodiments, the bacteria of the genus Lactococcus is Lactococcus lactis.

[0033] In certain embodiments, the bacteria of the genus Enterococcus are selected from: Enterococcus faecalis, Enterococcus faecium, or any combination thereof.

[0034] In certain embodiments, the composition of the present invention can also be combined with acceptable excipients in foods such as different sweeteners or flavoring agents, colorants, stabilizers, glidants, fillers, etc.

[0035] In certain embodiments, the composition as described above comprises: Komagataeibacter nataicola as described above, yeast, and tea leaves or an extract thereof.

[0036] In certain embodiments, the tea leaves are selected from black tea, green tea, oolong tea, yellow tea, dark tea, white tea, Pu-erh tea, oolong tea, or any combination thereof.

[0037] In certain embodiments, the extract of the tea leaves is obtained by brewing the tea leaves with hot water.

[0038] In certain embodiments, the extract of the tea leaves further comprises sugar (e.g., glucose, granulated sugar, fructose).

[0039] In certain embodiments, the composition further comprises Gluconacetobacter xylinus.

[0040] In certain embodiments, the composition further comprises additional additives.

[0041] In certain embodiments, the additional additive is selected from other nutrients (e.g., dietary fiber, prebiotics, proteins, lipids, plant components), minerals, vitamins, or any combination thereof.

[0042] In certain embodiments, the minerals are selected from iron, zinc, potassium, sodium, calcium, magnesium, and any combination thereof.

[0043] In certain embodiments, the vitamins are selected from vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, and any combination thereof.

[0044] In certain embodiments, the composition comprises Komagataeibacter xylinus, Brettanomyces bruxellensis, Brettanomyces anomalus, and black tea or black tea extract as described above.

[0045] Food product or dietary supplement

[0046] In another aspect, the present application provides a food product or dietary supplement comprising Komagataeibacter xylinus as described above or the composition as described above.

[0047] In the text, the term "food" is used in a broad sense and includes human food and beverages, as well as animal food and beverages (i.e., feed). In certain embodiments, the food product is suitable for and designed for human consumption.

[0048] It will be understood that depending on the use, application method, or administration method, the food product of the present application may be in the form of a liquid, solid, suspension, or powder.

[0049] In certain embodiments, the food product or dietary supplement further comprises excipients acceptable in food (e.g., sweeteners, flavoring agents, coloring substances, stabilizers, glidants, fillers).

[0050] In certain embodiments, the food product is a beverage.

[0051] In certain embodiments, the food product is selected from tea beverages, solid beverages, or fruit juice beverages.

[0052] In certain embodiments, the food product comprises dairy products.

[0053] In certain embodiments, the food product or dietary supplement is formulated for oral administration.

[0054] In certain embodiments, the food product or dietary supplement is in the form of pills, powders, capsules, tablets, granular powders, opercula, orally soluble granules, sachets, dragees, or liquids.

[0055] In certain embodiments, the amount of Komagataeibacter nataicola in the food product or dietary supplement is present in an amount of 10 2 to 10 20 CFU / dose (e.g., 10 4 to 10 18 CFU / dose).

[0056] In certain embodiments, the food product or dietary supplement may further comprise one or any combination of the following substances: probiotics (e.g., probiotic bacteria), dietary fiber, prebiotics, proteins (e.g., enzymes), carbohydrates, lipids (e.g., fats), vitamins, minerals, plant components (e.g., plant extracts), amino acids, immunomodulators, milk substitutes, or metabolites or extracts of Komagataeibacter nataicola or its progeny.

[0057] Culture

[0058] In another aspect, the present application provides a culture comprising Komagataeibacter nataicola as described above or a composition as described above.

[0059] In certain embodiments, the culture further comprises components that provide nutrition (e.g., solid or liquid medium, feeder cell layer).

[0060] In certain embodiments, the components that provide nutrition are selected from proteins, carbohydrates, fats, probiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof.

[0061] In certain embodiments, the culture further comprises derivatives of Komagataeibacter nataicola.

[0062] In certain embodiments, the derivatives are selected from metabolites, enzymes, cell structure components (e.g., cell wall or its components), exopolysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

[0063] Fermentation product

[0064] In another aspect, the present application provides a fermentation product comprising Komagataeibacter nataicola as described above or a composition as described above.

[0065] In certain embodiments, the culture further comprises components that provide nutrition (e.g., solid or liquid medium, feeder cell layer).

[0066] In certain embodiments, the nutrient-providing component is selected from proteins, carbohydrates, fats, probiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof.

[0067] In certain embodiments, the culture further comprises a derivative of Komagataeibacter nataicola.

[0068] In certain embodiments, the derivative is selected from metabolites, enzymes, cell structure components (e.g., cell walls or their components), exopolysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

[0069] Use

[0070] In another aspect, the present application provides the use of Komagataeibacter nataicola as described above or the composition as described above in the preparation of a starter culture for the fermentation of solid foods (e.g., cheese) or beverages (e.g., tea beverages, fruit juice beverages, flavored fermented milk, lactic acid bacteria beverages).

[0071] In another aspect, the present application provides the use of Komagataeibacter nataicola as described above or the composition as described above in the preparation of a food product or a dietary supplement or a health product.

[0072] In certain embodiments, the food product is a beverage.

[0073] In certain embodiments, the food product is selected from tea beverages, solid beverages, or fruit juice beverages.

[0074] In certain embodiments, the food product comprises dairy products (e.g., yogurt, flavored fermented milk, lactic acid bacteria beverages, cheese).

[0075] Term definition

[0076] As used herein, the term "dietary supplement" refers to an edible product that can provide beneficial effects (e.g., nutritional effects, preventive effects, therapeutic effects, or other beneficial effects) to consumers. In this context, dietary supplements cover products such as health products, nutritional products, supplements, etc.

[0077] As used herein, the term "CFU (Colony-Forming Units)" refers to the total number of microbial communities such as bacteria, fungi, yeasts, etc. in a product, and is usually used for calculating the viable cell count.

[0078] As used herein, the term "CFU / dose" refers to the amount of bacteria present in a composition / food product or dietary supplement / drug composition provided to a subject per day or per administration. For example, in certain embodiments, the amount of Komagataeibacter xylinus in the food product or dietary supplement is from 10 6 to 10 12 CFU / dose (e.g., 10 8 to 10 12 CFU / dose). In such an embodiment, if Komagataeibacter xylinus is administered in a food product (e.g., in a solid beverage, yogurt), the food product (e.g., solid beverage, yogurt) provided to the subject per day or per administration may contain about 10 6 to 10 12 CFU of Komagataeibacter xylinus. Of course, alternatively, the amount of this bacterium can be administered in multiple doses, as long as the total amount of Komagataeibacter xylinus received by the subject within any specific time period (e.g., every 24-hour period) is from about 10 6 to about 10 12 CFU of the bacterium, i.e., the amount of Komagataeibacter xylinus in the food product or dietary supplement as described above is from 10 6 to 10 12 CFU / dose (e.g., 10 8 to 10 12 CFU / dose).

[0079] As used herein, the term "Acetobacter" refers to all strains capable of producing acetic acid.

[0080] Advantages of the invention

[0081] The Komagataeibacter xylinus of the present application has good basic properties. For example, it can form a bacterial cellulose membrane, has good water absorption and water retention capacity, certain ductility and good mechanical properties. In addition, it can significantly increase the total acid content of tea sugar water, significantly reduce the pH and sugar content of tea sugar water. Moreover, the tea water fermented by the Komagataeibacter xylinus of the present application has a green apple flavor and a strong sour taste, forming a unique high-quality flavor.

[0082] The embodiments of the present invention will be described in detail below by way of examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and not for limiting the scope of the present invention. According to the following detailed description of the preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art. Brief Description of the Drawings

[0083] Figure 1 It is the surface biofilm formed by the activation of the Kombucha mushroom strain.

[0084] Figure 2 The situation of the purified strain A8-12 in streak plate culture.

[0085] Figure 3 The microscopic result diagram of 1000× of strain A8-12 after methylene blue staining.

[0086] Figure 4 The bacterial cellulose membrane formed on the surface of the culture medium.

[0087] Sequence information

[0088] The information of some sequences involved in the present invention is provided in Table 1 below.

[0089] Table 1: Description of sequences

[0090]

[0091]

[0092] Instructions on the deposit of biological materials

[0093] Komagataeibacter nataicola A8-12 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. It has the deposit number CGMCC No. 27966, and the deposit date is July 21, 2023. Detailed implementation manners

[0094] The present invention will now be described with reference to the following examples, which are intended to illustrate the present invention (but not to limit the present invention).

[0095] Unless otherwise specified, the experiments and methods described in the examples are basically carried out according to the conventional methods well-known in the art and described in various reference documents. For example, for the conventional techniques such as molecular biology, microbiology, cell biology, etc. used in the present invention, reference can be made to Sambrook, Fritsch, and Maniatis, "Molecular Cloning: A Laboratory Manual", 2nd Edition (1989); "Current Protocols in Molecular Biology" (edited by F.M. Ausubel et al., (1987)); "Methods in Enzymology" series (Academic Press): "PCR 2: A Practical Approach" (edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor (1995)).

[0096] In addition, for those without specific conditions indicated in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through commercial purchase. Those skilled in the art know that the examples describe the present invention by way of illustration and are not intended to limit the scope claimed by the present invention. All the published cases and other reference materials mentioned herein are incorporated herein by reference in their entirety.

[0097] Example 1. Obtaining of strains

[0098] The kombucha samples were purchased from Wendeng District, Weihai City, Shandong Province and stored in a 4°C refrigerator.

[0099] 1. Activation of Kombucha starter culture

[0100] Take 10 g of black tea leaves and put them into 1000 mL of ultrapure water, boil for 5 min, extract for 20 min, filter out the tea leaves, add 100 g of granulated sugar, stir until dissolved, then divide and fill into 1 L glass jars, 300 mL per bottle, and cool to room temperature. In a laminar flow hood, inoculate 100 mL of the above-purchased kombucha liquid and a small piece of kombucha film into the tea sugar water, seal it with four layers of sterile gauze, transfer it to a 28°C constant temperature biochemical incubator and statically culture for 7 d until the surface biofilm reaches about 5 mm thick ( Figure 1 ).

[0101] 2. Isolation, purification and culture of strains

[0102] Selective solid medium: Weigh 20 g of glucose, 10 g of yeast powder, and 20 g of agar, add 965 mL of distilled water, sterilize at 121 °C for 20 min. When cooled to 45 °C - 50 °C, add 20 g of calcium carbonate and 35 mL of absolute ethanol, mix well and pour into plates, and let it cool and solidify for standby.

[0103] Cut about 10 g of activated Kombucha biofilm and put it into 90 mL of sterile PBS liquid with glass beads. After fully shaking and breaking, dilute it by 10-fold gradient to 10-1, 10-2, 10-3, 10-4 concentrations. Respectively, pipette 100 μL of each gradient dilution and spread it on the selective plate, and place it in a constant temperature biochemical incubator at 30 °C for 7 d. Pick single colonies with obvious calcium-dissolving zones around the colonies and streak isolate them twice to obtain pure strains (as Figure 2 shown). The single colonies are round, opaque light yellow, with a moist and slightly wrinkled surface, and slightly rough edges, and are stored at -80 °C with 30% glycerol. After staining the A8-12 strain with methylene blue, observe its morphology under a microscope at 1000× and take pictures for recording ( Figure 3 ). The bacteria are short rod-shaped, occurring singly or in pairs.

[0104] 3. Strain identification

[0105] Extract the DNA of the strain by the kit method. Using the bacterial DNA as a template, use the bacterial universal primers 27F (SEQ ID NO:2) and 1492R (SEQ ID NO:3) to perform PCR amplification on the candidate strain, and send the product to Sangon Biotech Co., Ltd. for sequencing. The result is as shown in SEQ ID NO:1, and submit the obtained sequence to GenBank for Blast comparative analysis.

[0106] The Blast alignment result shows that the sequence result of A8-12 has a homology of more than 99% with the 16S rDNA sequence of Komagataeibacter nataicola.

[0107] Based on the above strain identification results, a new strain of Komagataeibacter nataicola was obtained in this application, named Komagataeibacter nataicola A8-12, and was deposited in the China General Microbiological Culture Collection Center (CGMCC) on July 21, 2023, with the deposit number CGMCC NO.27966.

[0108] Example 2. Determination of basic characteristics

[0109] Selective liquid medium: Weigh 20 g of glucose and 10 g of yeast powder, add 965 mL of distilled water, sterilize at 121 °C for 20 min. After cooling, add 35 mL of absolute ethanol, mix well, and dispense for later use. Use a sterile inoculation loop to pick a single colony from the purified plate and inoculate it into 5 mL of liquid medium. Incubate at 30 °C on a shaker at 230 rpm for 5 days.

[0110] Use a pipette to aspirate 3 mL of the bacterial liquid cultured in liquid medium and inoculate it into a sterile glass petri dish containing 350 mL of selective liquid medium. Place it in a constant temperature biochemical incubator at 30 °C and incubate statically for 7 days. A uniform bacterial cellulose membrane can be observed to form on the surface of the medium, as shown. Figure 4

[0111] 1. Basic properties

[0112] Take out the bacterial cellulose membrane from the medium, thoroughly rinse the medium on its surface with distilled water, soak it in distilled water overnight, then soak it in a 0.1 mol / L NaOH solution at 90 °C for 2 h until the membrane becomes milky white and semi-transparent. Rinse it several times with distilled water, then soak it in a 0.5% (v / v) acetic acid solution for 5 min. After taking it out, rinse it several times with distilled water until the surface moisture is neutral as measured by pH test paper. Use kitchen paper to absorb the excess moisture on the surface of the membrane to obtain a wet bacterial cellulose membrane, and measure its wet weight.

[0113] Place the wet bacterial cellulose membrane in a drying oven at 90 °C until it reaches a constant weight to obtain a dry bacterial cellulose membrane, and measure its dry weight.

[0114] Fully soak the dry bacterial cellulose membrane in ultrapure water for 24 h. After taking it out, use kitchen paper to absorb the excess moisture on the surface and measure the mass of the membrane after rehydration.

[0115] Moisture content of wet membrane = (wet membrane mass - dry membrane mass) / wet membrane mass × 100%

[0116] Rehydration rate of dry membrane = (mass of rehydrated membrane - dry membrane mass) / mass of rehydrated membrane × 100%

[0117] Membrane yield = wet membrane mass / culture solution volume

[0118] Bacterial cellulose yield = dry membrane weight / culture solution volume

[0119] Table 2. Basic properties of the bacterial cellulose membrane produced by A8 - 12

[0120]

[0121] 2. Mechanical properties

[0122] ​Take the bacterial cellulose membrane out of the culture medium, rinse the culture medium on its surface thoroughly with distilled water, soak it in distilled water overnight, use a die cutter to apply a certain pressure on the bacterial cellulose membrane slowly and evenly to remove most of the water inside the membrane, press it flat, measure the membrane thickness with a thickness gauge and record it, then use a mold to cut the bacterial cellulose membrane into strips with a size of 150 mm × 10 mm, measure the maximum stretching distance and tearing force, and calculate mechanical properties such as tensile strength, elongation at break, and elastic modulus.

[0123] Tensile strength = Tearing force / (Membrane thickness * Specimen width)

[0124] Elongation at break = Maximum stretching distance / Specimen length * 100%

[0125] Elastic modulus = Tearing force / Elongation at break

[0126] Table 3. Mechanical properties of bacterial fiber membranes produced by Komagataeibacter A8 - 12

[0127]

[0128] The bacterial cellulose membrane produced by Komagataeibacter xylinus A8 - 12 is uniform and flat, and has excellent water absorption and water retention capacity. The moisture content of the wet membrane reaches 98.11%, and the rehydration rate reaches 89.71%; at the same time, it has a certain ductility, the stretching distance reaches 16.48 mm, and the elongation at break is 10.99%; the tensile strength is 0.83 Mpa, and the elastic modulus is as high as 7.57 MPa, both of which are higher than traditional non-woven materials, and it has good mechanical properties, providing data support for its further development and application in the fields of food, medicine, and cosmetics in the future.

[0129] Example 3. Application of Komagataeibacter nataicola in fermented tea

[0130] 1. Comparison of fermented tea with other Komagataeibacter strains screened during the same period

[0131] Table 4. List of Komagataeibacter strains for fermentation

[0132]

[0133] Other Komagataeibacter strains used in this example (specifically as shown in Table 4 above) were all isolated from kombucha by this laboratory during the same period. The specific methods for isolation, purification, cultivation, and strain identification are the same as those in Example 1.

[0134] Preparation of strain liquid medium: Weigh 20.0 g of glucose and 10.0 g of yeast powder, add deionized water to 1000.0 g, fully dissolve it, then aliquot 50 mL into 250 mL baffled conical flasks, and sterilize at 121 °C for 15 min. The liquid medium for yeast uses commercial YPD medium, also aliquot 50 mL into 250 mL baffled conical flasks, and sterilize at 121 °C for 15 min.

[0135] Preparation of tea sugar water: Weigh 13.0 g of black tea into a preheated beaker. According to the tea - water ratio of 1:30, add 390.0 g of hot water at 85 °C. Heat it in a water bath at 85 °C for 15.0 min, stir once every 5 min. After heating, quickly filter out the tea leaves and tea residues with a filter screen, and then put the tea water into an ice - water bath to cool down rapidly. After cooling to room temperature, weigh 32.5 g of glucose (2.0%) and 32.5 g of granulated sugar (2.0%) into a beaker respectively, add the above - mentioned black tea extraction solution, and then add deionized water to 1625.0 g and stir evenly. Weigh 400.0 g of tea sugar water into a conical flask, seal it with double - layer tin foil and sealing film, and perform pasteurization at 70 °C for 15 min. Cool it to room temperature and set aside.

[0136] Activation of strains: Take 1.0 mL of the cryopreserved bacterial liquid of Brettanomyces bruxellensis and inoculate it into 50 mL of YPD medium, and then culture it in a shaker at 28 °C and 120 rpm for 3 days. Take 1.0 mL of the cryopreserved bacterial liquid of Komagataeibacter in Table 4 respectively and inoculate it into 50 mL of the corresponding medium, and then culture it in a shaker at 28 °C and 180 rpm for 3 days.

[0137] Preparation of seed liquid of strains for fermentation: In a laminar flow hood, take 40.0 mL of the cultured yeast liquid into a 50.0 mL centrifuge tube respectively, and then centrifuge it at 6000 rpm for 15 min in a centrifuge. After centrifugation, pour out the supernatant. Add 40.0 mL of sterilized tea soup into the centrifuge tube to resuspend the bacterial liquid, centrifuge it again at 6000 rpm for 15.0 min, and finally pour out the supernatant, and then add 40.0 mL of sterilized tea soup to make yeast seed liquid. The preparation method of Komagataeibacter is the same as that of yeast, but without washing, and directly add tea soup after centrifugation to make Komagataeibacter seed liquid.

[0138] Inoculation and fermentation: Inoculate 3 mL of Brettanomyces bruxellensis seed liquid and 20 mL of Komagataeibacter seed liquid into every 400 g of tea sugar water, and place them in a shaker at 28 °C and oscillate and ferment at 120 rpm for 3 d. After fermentation is completed, take each fermentation liquid and centrifuge it at 6000 rpm for 15 min, take the supernatant and store it in a sterile plastic bottle, refrigerate it at 4 °C for one day and then transfer it to a - 20 °C refrigerator for preservation. Take about 20.0 mL of the centrifuged fermentation liquid and place it in a transparent plastic tasting cup. Three experimental personnel and two R & D experts of Nongfushanquan tea beverages conduct sensory evaluation on the samples, and mainly record the evaluation feelings of the evaluators from the aspects of richness, aroma (fruity aroma, tea aroma) and sour - sweetness.

[0139] After 3 days of fermentation under the same conditions (Table 5), the fermented teas of Kurthia media A8-5, A9-10 and Kurthia gibsonii A9-18 all had off-flavors and were not suitable for further development in the later stage; the fermented tea of Kurthia xylosa A2-1 had a slightly fruity aroma, but the acidity was average; the fermented tea of Kurthia xylosa A5-7 did not have the expected sour taste, and the flavor in the later stage was weak; the fermented tea of Kurthia gibsonii A5-6 had average acidity and a strong astringency; the fermented tea of Kurthia nataicola A8-12 showed significantly better sensory evaluation results. Specifically, the fermented tea of Kurthia nataicola A8-12 had a fruity aroma, high acidity, a bit of umami, and a slightly green radish flavor. The overall fermentation flavor was the best and it had high potential for further development.

[0140] Table 5. Sensory evaluation description of fermented teas by screening Kurthia strains during the same period

[0141]

[0142] 2. Comparison of fermented tea with commercial Acetobacter strains

[0143] Purchase another four commercial strains that also produce acetic acid, ferment fermented teas with Kurthia nataicola A8-12 of this application respectively, and conduct a sensory flavor comparison of the fermented teas.

[0144] Table 6. Commercial acetic acid bacteria strains and A8-12

[0145]

[0146] The methods and operations of medium preparation, tea-sugar water preparation, strain activation, preparation of seed liquid of the strain for fermentation, inoculation and fermentation are the same as those described above. Among them, for Shanghai brewing vinegar koji and Yuyuan vinegar koji, there is no need for strain activation and preparation of seed liquid for fermentation. After inoculating them into the tea-sugar water at a ratio of 1 g / 400 g of tea-sugar water respectively, they can be fermented with 3 mL of Brettanomyces bruxellensis seed liquid.

[0147] After 3 days of fermentation under the same conditions, Shanghai brewing vinegar koji had almost no sour taste, while Yuyuan vinegar koji had an obvious stench after fermentation, and both were not suitable for fermentation in the tea system of this experiment; the acid sense of Zhongke 1.41 was weak after fermentation, and it had a certain bitterness and poor flavor; the fermented tea of Shanghai brewing 1.01 was overall better than the fermentation broth of Zhongke 1.41, but the flavor was relatively flat; the fermented tea of Kurthia nataicola A8-12 had an obvious acid sense, a fruity aroma, and a more abundant and layered overall flavor, and was more suitable for development as a potential strain for fermented tea.

[0148] Table 7. Sensory evaluation description of fermented teas by commercial acetic acid bacteria strains and A8-12

[0149]

[0150] In summary, compared with the six species of Komagataeibacter of the same kind or the other four strains that also produce acetic acid, the Komagataeibacter coco A8-12 of the present application has particularly outstanding sensory evaluation results and is particularly suitable for the preparation of fermented tea.

[0151] 3. Effects of temperature and fermentation duration on the fermentation flavor of Komagataeibacter nataicola A8-12

[0152] The methods and operations of culture medium preparation and tea sugar water preparation are the same as those described above.

[0153] Strain activation: Take 1.0 mL of the frozen stock solutions of Brettanomyces bruxellensis and Brettanomyces anomalus yeasts and inoculate them into 50 mL of YPD medium, and then culture them in a shaker at 28 °C and 120 rpm for 3 days. Take 1.0 mL of the frozen stock solution of A8-12 and inoculate it into 50 mL of the corresponding medium, and then culture it in a shaker at 28 °C and 180 rpm for 3 days.

[0154] Preparation of seed solutions of strains for fermentation: Take 40.0 mL of the cultured yeast solutions in a clean bench and transfer them to 50.0 mL centrifuge tubes, and then centrifuge them at 6000 rpm for 15 min in a centrifuge. After centrifugation, pour out the supernatant, add 40.0 mL of sterilized tea soup to the centrifuge tubes to resuspend the bacterial solution, centrifuge again at 6000 rpm for 15.0 min, finally pour out the supernatant, and add 40.0 mL of sterilized tea soup to make yeast seed solutions. The preparation method of Komagataeibacter coco is the same as that of yeast, but without washing, and directly add tea soup after centrifugation to make Komagataeibacter coco seed solutions.

[0155] Inoculation and fermentation: Inoculate 1.5 mL of Brettanomyces bruxellensis seed solution, 1.5 mL of Brettanomyces anomalus seed solution and 20 mL of A8-12 acetic acid bacteria seed solution into 400 g of tea sugar water, and place them in shakers at 26 °C, 28 °C and 30 °C respectively, and shake and ferment at 120 rpm for 3 d or 4 d. After fermentation is completed, take 45 mL of the sample to measure the sugar content (Brix%), pH and total acid content; centrifuge the remaining fermentation broth at 6000 rpm for 15 min, take the supernatant and store it in a sterile plastic bottle, refrigerate it at 4 °C for one day and then transfer it to a -20 °C refrigerator for storage. Take about 20.0 mL of the centrifuged fermentation broth and place it in a transparent plastic tasting cup, and invite two R & D experts of Nongfu Spring tea beverages to conduct sensory evaluation on the sample, and record the tasting feelings of the tasters mainly from the aspects of richness, aroma (fruity aroma, tea aroma) and sour and sweet taste.

[0156] Fermentation treatment significantly increased the total acid content of the tea sugar water in all groups, significantly decreased the pH, and decreased the sugar content (as shown in Table 8), indicating that yeast and A8-12 grew well in the above tea system. The yeast consumed the sugar in it, and A8-12 fermented using the alcohol produced by the yeast as a substrate to produce substances such as acetic acid, thus significantly increasing the total acid of the tea sugar water and decreasing the pH. At the same fermentation time, the total acid content of the samples increased with the increase in temperature. It is speculated that A8-12 metabolizes faster and produces more acid overall at 30°C. The strength of sour taste in food is closely related to the total acid content, but it does not have an absolute proportional relationship with the pH. At the same fermentation time, the pH of the 30°C sample was the highest, contrary to the total acid result, but the sensory evaluation result (Table 9) showed that the 30°C sample had the strongest sour taste, consistent with the total acid result. The comprehensive sensory evaluation result (Table 9) showed that fermentation at 30°C for 3 days was the better fermentation condition for A8-12 to ferment tea beverages. The fermented tea samples had a high aroma, a green apple flavor, and a strong sour taste, giving them the unique high-quality flavor closest to traditional kombucha. However, with the extension of the fermentation time, the excessive acid production by acetic acid bacteria would cause the fermented tea to have an overly strong vinegar taste and a decrease in the richness of the overall flavor.

[0157] Table 8. Basic Physicochemical Parameters of Fermented Tea

[0158]

[0159] Table 9. Sensory Evaluation Descriptions of Fermented Tea

[0160]

[0161] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that: according to all the teachings that have been published, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A Komagataeibacter nataicola having a 16S rDNA sequence with 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95% or 100% identity to SEQ ID NO:

1.

2. The Komagataeibacter nataicola according to claim 1, wherein, After amplification with primers shown in SEQ ID NO:2 and SEQ ID NO:3, the 16S rDNA generates a 1400bp fragment.

3. The Komagataeibacter nataicola according to claim 1 or 2, having one or more of the following characteristics: (1) The colony is circular and opaque light yellow; (2) The surface of the colony is moist and slightly wrinkled, and the edge is slightly rough; (3) The cells are short rod-shaped; (4) Capable of producing bacterial cellulose membranes under suitable growth conditions (e.g., in the presence of liquid medium); Preferably, the bacterial cellulose membrane has one or more of the following characteristics: (1) The moisture content of the wet membrane is greater than 85% (e.g., 85%-90%, 90%-95%, 95%-99.9%); (2) The rehydration rate of the dry membrane is greater than 75% (e.g., 75%-80%, 80%-85%, 85%-90%, 90-95%); (3) The cellulose yield is greater than 0.1 g / L (e.g., 0.1 g / L - 0.3 g / L, 0.3 g / L - 0.5 g / L, 0.5 g / L - 0.7 g / L, 0.7 g / L - 0.9 g / L); (4) The stretching distance is greater than 10 mm (e.g., 10 mm - 14 mm, 14 mm - 17 mm, 17 mm - 20 mm); (5) The tensile strength is greater than 0.3 Mpa (e.g., 0.3 Mpa - 0.6 Mpa, 0.6 Mpa - 0.9 Mpa, 0.9 Mpa - 1.2 Mpa).

4. The Komagataeibacter nataicola according to any one of claims 1-3, which is deposited in the China General Microbiological Culture Collection Center under the deposit number CGMCC No. 27966.

5. A composition comprising the Komagataeibacter nataicola according to any one of claims 1-4; Preferably, the composition further comprises a microorganism selected from the following: bacteria, fungi (e.g., yeast), or any combination thereof; Preferably, the microorganism is a probiotic; Preferably, the bacteria are selected from the genus Lactobacillus, Bifidobacterium, Bacillus, Propionibacterium, Streptococcus, Lactococcus, Pediococcus, Enterococcus, Staphylococcus, or any combination thereof; Preferably, the yeast is selected from Brettanomyces anomalus, Saccharomyces cerevisiae, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Zygosaccharomyces bailii, or any combination thereof.

6. The composition according to claim 5, comprising: Komagataeibacter xylinus according to any one of claims 1-4, yeast, and tea leaves or an extract thereof; Preferably, the tea leaves are selected from black tea, green tea, oolong tea, yellow tea, dark tea, white tea, Pu-erh tea, oolong tea, or any combination thereof; Preferably, the extract of the tea leaves is obtained by brewing the tea leaves with hot water; Preferably, the extract of the tea leaves further comprises sugar (e.g., glucose, granulated sugar, fructose); Preferably, the composition further comprises Gluconacetobacter xylinus; Preferably, the composition further comprises additional additives; Preferably, the additional additives are selected from other nutrients (e.g., dietary fiber, prebiotics, proteins, lipids, plant components), minerals, vitamins, or any combination thereof; Preferably, the composition comprises Komagataeibacter xylinus according to any one of claims 1-4, Brettanomyces bruxellensis, Brettanomyces anomalus, and black tea or an extract of black tea.

7. A food product or dietary supplement comprising Komagataeibacter xylinus according to any one of claims 1-4 or the composition according to claim 5 or 6; Optionally, the food product or dietary supplement further comprises excipients acceptable in food (e.g., sweeteners, flavoring agents, colorants, stabilizers, glidants, fillers); Preferably, the food product is a beverage; Preferably, the food product is selected from tea beverages, solid beverages, or fruit juice beverages; Preferably, the food product comprises dairy products; Preferably, the food product or dietary supplement is formulated for oral administration; Preferably, the food product or dietary supplement is in the form of pills, powders, capsules, tablets, granular powders, opercula, orally soluble granules, sachets, dragees or liquids; Preferably, in the food product or dietary supplement, the Komagataeibacter cocois is present in an amount of 10 2 to 10 20 CFU / dose (for example, 10 4 to 10 18 CFU / dose).

8. A culture comprising the Komagataeibacter cocois according to any one of claims 1-4 or the composition according to claim 5 or 6; Optionally, the culture further comprises components providing nutrition (for example, solid or liquid medium, feeder cell layer); Preferably, the components providing nutrition are selected from proteins, carbohydrates, fats, probiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof; Preferably, the culture further comprises derivatives of the Komagataeibacter cocois; Preferably, the derivatives are selected from metabolites, enzymes, cell structure components (for example, cell wall or its components), exopolysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

9. A fermentation product comprising the Komagataeibacter cocois according to any one of claims 1-4 or the composition according to claim 5 or 6; Optionally, the culture further comprises components providing nutrition (for example, solid or liquid medium, feeder cell layer); Preferably, the components providing nutrition are selected from proteins, carbohydrates, fats, probiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof; Preferably, the culture further comprises derivatives of the Komagataeibacter cocois; Preferably, the derivatives are selected from metabolites, enzymes, cell structure components (for example, cell wall or its components), exopolysaccharides, bacteriocins, compounds containing immunogenic components, or any combination thereof.

10. Use of the Komagataeibacter cocois according to any one of claims 1-4 or the composition according to claim 5 or 6 in the preparation of a starter culture for the fermentation of solid foods (for example, cheese) or beverages (for example, tea beverages, fruit juice beverages, flavored fermented milk, lactic acid bacteria beverages).

11. Use of the Acetobacter xylinum of any one of claims 1-4 or the composition of claim 5 or 6 in the preparation of a food product or a dietary supplement or a health product or a cosmetic; Preferably, the food product is a beverage; Preferably, the food product is selected from tea beverages, solid beverages or fruit juice beverages; Preferably, the food product contains dairy products (for example, yogurt, flavored fermented milk, lactic acid bacteria beverage, cheese).

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