Method for producing bacterial cellulose by using rice wine vinasse stock solution and bacterial cellulose culture medium

Abstract

The invention discloses a method for producing bacterial cellulose by using rice wine vinasse stock solution and a bacterial cellulose culture medium, and belongs to the technical field of bacterial cellulose preparation. The bacterial cellulose culture medium comprises the following components in percentage by mass: 0-10% of a carbon source and a clear liquid of a rice wine vinasse stock solution. The invention also provides a method for preparing a bacterial cellulose membrane by using the bacterial cellulose culture medium, and the method comprises the following steps: heating and sterilizing the bacterial cellulose culture medium to obtain bacterial cellulose fermentation liquor, and inoculating a target strain into the bacterial cellulose fermentation liquor for fermentation to obtain the bacterial cellulose membrane. And washing and soda boiling the bacterial cellulose membrane to obtain a bacterial cellulose membrane finished product. According to the method provided by the invention, the production cost of the bacterial cellulose can be greatly reduced, the bacterial cellulose membrane with high solid content can be obtained, and a new technology is provided for efficient and large-scale production of the bacterial cellulose.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial cellulose preparation, in particular to a method for producing bacterial cellulose by utilizing rice wine lees stock solution and a bacterial cellulose culture medium. Background Art

[0002] Wine lees, also known as sweet lees, red lees and fermented lees, are the residues left after brewing sorghum, barley, rice and other wines. Wine lees are rich in nutrients, such as protein, B vitamins, starch and lipids, and have high utilization value and huge resource potential. Compared with beer lees and white wine lees, rice wine lees are a mixture of solid matter and water left after the raw materials for brewing rice wine are processed by brewing. A typical example is the wine lees produced during the brewing process of Sanhua Wine in Guilin, Guangxi, with a water content of up to 90%. However, the recycling rate of this high-water content wine lees has always been low. Improper treatment will not only seriously pollute the ecological environment, but also lead to a huge waste of resources. With increasingly stringent environmental policies and the continuous and in-depth construction of national ecological civilization, the reduction, harmlessness and resource utilization of wine lees are imperative.

[0003] Bacterial cellulose is a high-purity cellulose product formed by the fermentation of cellulose-producing bacteria. Due to its excellent biodegradability, high degree of polymerization and high crystallinity, it can be used as an environmentally friendly biomaterial with broad application prospects in new energy, medicine, bioengineering and food packaging.

[0004] Currently, the raw materials used to produce bacterial cellulose are mainly sucrose, glucose, coconut water, kiwi fruit, grapes, watermelon juice and maltose. The schemes for preparing bacterial cellulose using the above method are as follows: (1) preparing bacterial cellulose fermentation liquid: taking 2.5g of yeast extract, 10g of glucose, 2.5g of soy peptone, 1.35g of sodium hydrogen phosphate dodecahydrate and 0.575g of citric acid monohydrate; (2) preparing strain seed liquid: adding 100g of sucrose and 6.5g of tea leaves into a glass jar containing 1000mL of boiling water, stirring to dissolve the sugar, filtering the tea leaves after cooling to obtain sugar tea water, pouring the black tea strain into the cooled sugar tea water, sealing it with gauze, and fermenting it for 7 days. The obtained bacterial liquid can be used as bacterial seed liquid; (3) fermenting to prepare bacterial cellulose: inoculating the bacterial seed liquid in step (2) into the fermentation liquid prepared in step (1) at a volume ratio of 8%, fermenting it at 25°C for 7 days, and collecting the bacterial cellulose film generated on the surface of the liquid. However, using sucrose and glucose as raw materials has the problem of high production cost and is difficult to mass produce. Although other natural raw materials are low-cost, they are affected by production seasons and preservation issues, and have problems such as unstable production capacity and inconvenient storage.

[0005] In addition, the prior art also uses rice koji juice and distiller's grains mixed fermentation as a new special culture medium for bacterial cellulose (CN110894478A), specifically: a culture medium for efficiently producing bacterial cellulose, comprising: preparing a culture medium base liquid: adding wet distiller's grains to rice koji juice for soaking, centrifuging, and filtering to obtain a rice koji juice and distiller's grains culture medium base liquid; adding water to the rice koji juice and distiller's grains culture medium base liquid to adjust the sugar content, and then adding a supplementary nitrogen source, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, citric acid and glycerol, and adjusting the pH value to 5.0-7.0 to obtain a rice koji juice and distiller's grains fermentation liquid, which is a culture medium for producing bacterial cellulose. The prior art discloses a method for preparing bacterial cellulose using white wine lees (CN116837049A), comprising: crushing and diluting the lees and then steaming to obtain a slurry, adjusting the pH value of the slurry after cooling, adding cellulase, hemicellulase and xylanase, heating and inactivating the enzymes, and filtering to obtain a lees separation liquid; adding a carbon source, an inorganic salt and a growth additive to the lees separation liquid to obtain a bacterial cellulose fermentation liquid; inoculating the target strain into the bacterial cellulose fermentation liquid for fermentation, and collecting the bacterial cellulose film after the fermentation is completed. Among them, the added weight of the cellulase, the hemicellulase and the xylanase is 0.2-0.5%, 0.12-0.22% and 0.04-0.09% of the weight of the slurry, respectively. It can be seen that there is no disclosure of using the waste rice wine lees liquid as a culture medium for bacterial cellulose in the prior art. How to directly utilize the waste rice wine vat liquid generated during the rice wine production process as a natural culture medium for bacterial cellulose has important environmental, economic and social benefits for the large-scale production of bacterial cellulose. Summary of the invention

[0006] The purpose of the present invention is to provide a method for producing bacterial cellulose using rice wine lees and a bacterial cellulose culture medium to solve the problems existing in the above-mentioned prior art. The solid content of the bacterial cellulose film obtained by the method of the present invention can be as high as 2.9%.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] Technical solution 1: A bacterial cellulose culture medium, the components of which include a carbon source and a clarified liquid of rice wine lees stock solution, wherein the carbon source is 0 to 10% by mass, and preferably 8% by mass.

[0009] Furthermore, the carbon source is one of sucrose, glucose, fructose and maltose, or a mixture of the four.

[0010] When there are four carbon sources, the addition ratio of each component is 2:2:1:1. Preferably, the carbon source is sucrose.

[0011] Furthermore, the rice wine lees stock solution is a mixture of solid matter and water remaining after the brewing of the raw materials for brewing rice wine is completed, and the water content of the mixture is 70-90%.

[0012] Furthermore, the clarified liquid is obtained by the following method: directly centrifuging the rice wine lees stock liquid to obtain the rice wine lees clarified liquid.

[0013] Preferably, the centrifugal parameter is 8500r / min for 5min.

[0014] Technical Solution 2: A method for preparing a bacterial cellulose film using the bacterial cellulose culture medium, comprising the following steps: heating and sterilizing the bacterial cellulose culture medium to obtain a bacterial cellulose fermentation broth, inoculating a target strain into the bacterial cellulose fermentation broth for fermentation to obtain the bacterial cellulose film.

[0015] Furthermore, the temperature is raised to 100°C for sterilization.

[0016] When the carbon source content is 0%, the bacterial cellulose fermentation liquid is obtained by heating and sterilizing, specifically comprising: heating the rice wine lees clarified liquid to 100°C and boiling it in a sealed container for 0.5 to 1 hour, and then cooling it to 25 to 30°C to obtain the sterilized bacterial cellulose fermentation liquid, preferably boiling it for 1 hour and cooling it to 25°C.

[0017] Furthermore, the target strain is Kombucha.

[0018] The target strain is activated to obtain a seed solution containing the target strain, comprising: inoculating the bacterial solution containing the target strain into rice wine lees clarified liquid at a volume ratio of 5-18%, preferably 8%, and activating the solution at 25-32° C. or shaking for 2-4 days to obtain the seed solution containing the target strain. Preferably, the solution is activated at 28° C. for 2 days.

[0019] The preparation of the bacterial solution containing the target strain is carried out as follows: 100g of sucrose and 6.5g of tea leaves are added to a glass jar containing 1000mL of boiling water, stirred to dissolve the sugar, and after cooling, the tea leaves are filtered to obtain sugar tea water, and the black tea strain is poured into the cooled sugar tea water, sealed with gauze, and fermented for 7 days to obtain a bacterial solution containing the target strain.

[0020] Technical solution three: The bacterial cellulose membrane prepared by the method described above.

[0021] Technical solution three: A bacterial cellulose membrane finished product, the bacterial cellulose membrane is washed and alkali boiled to obtain the bacterial cellulose membrane finished product.

[0022] Furthermore, the washing is performed with deionized water; and the alkali boiling is performed with a sodium hydroxide solution.

[0023] The present invention discloses the following technical effects:

[0024] The present invention uses discarded rice wine lees to prepare bacterial cellulose, and uses the obtained rice wine lees clarified liquid directly as bacterial cellulose fermentation liquid, which simplifies the process and greatly reduces the production cost of bacterial cellulose from raw materials to processes. The solid content of the obtained bacterial cellulose film can be as high as 2.9%, which improves the production efficiency and utilization rate of bacterial cellulose. The present invention also provides a bacterial cellulose culture medium, which can completely use rice wine lees as raw materials, reduce the pollution of lees waste in the rice wine production process, reduce the production cost of bacterial cellulose, and improve the utilization rate of resources. The culture medium can choose to add a carbon source or not to add any carbon source, and the preparation process is simple. The present invention expands the preparation method and process of bacterial cellulose, and is suitable for the promotion of industrial production technology of bacterial nanocellulose products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a photo of bacterial cellulose produced in Example 1;

[0027] Figure 2 The figure is a comparison chart of the solid content and bacterial cellulose yield of the bacterial cellulose films produced in the example and the comparative example. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The centrifugal parameters were 8500r / min for 5min. Sanhua wine lees stock solution was from Guilin Sanhua Co., Ltd. Vacuum freeze dryer (model YTLG-10B).

[0034] Example 1

[0035] The steps for preparing bacterial cellulose using rice wine lees are as follows:

[0036] (1) preparing rice wine lees clarified liquid: directly centrifuging Sanhua wine lees stock liquid (water content 85%) to obtain Sanhua wine lees clarified liquid;

[0037] (2) preparing bacterial cellulose fermentation liquid: taking the Sanhua wine lees clarified liquid obtained in step (1), heating it to 100° C., sealing it and boiling it for 0.5 hours, and then cooling it to 30° C. for later use;

[0038] (3) Preparation of target strain seed solution: Add 100 g of sucrose and 6.5 g of tea leaves into a glass jar containing 1000 mL of boiling water, stir to dissolve the sugar, let it cool, filter the tea leaves to obtain sugar tea water, pour the black tea strain into the cooled sugar tea water, seal it with gauze, and ferment for 7 days. The resulting bacterial solution can be used as bacterial seed solution;

[0039] (4) Fermentation to prepare bacterial cellulose: The seed solution of the strain in step (3) was inoculated into the fermentation solution in step (2) at a volume ratio of 18%, and the solution was shaken at 32° C. for 4 days to obtain a seed solution containing the target strain. The solution was fermented at 25° C. for 7 days, and the bacterial cellulose film formed on the surface of the solution was collected, washed with deionized water for 3 times, boiled with 1% sodium hydroxide solution for 1 hour, and finally repeatedly washed with deionized water until the pH value was 7.0. After wiping off excess water on the surface, the finished bacterial cellulose film was obtained;

[0040] (5) Drying of bacterial cellulose: The bacterial cellulose membrane obtained in step (4) was freeze-dried using a vacuum freeze dryer, and the bacterial cellulose membrane before and after freeze-drying was weighed and calculated to obtain a solid content of 2.10% (see Figure 1 ), the yield of bacterial cellulose was 7.27 g / L.

[0041] Example 2

[0042] The steps for preparing bacterial cellulose using rice wine lees are as follows:

[0043] (1) preparing rice wine lees clarified liquid: directly centrifuging Sanhua wine lees stock liquid (water content 90%) to obtain Sanhua wine lees clarified liquid;

[0044] (2) preparing bacterial cellulose fermentation liquid: taking 92% of the Sanhua wine lees clarified liquid obtained in step (1) and 8% of sucrose, by weight percentage, heating to 100° C., sealing and boiling for 1 hour, and then cooling to 25° C. for later use;

[0045] (3) Preparation of target strain seed solution: Add 100 g of sucrose and 6.5 g of tea leaves into a glass jar containing 1000 mL of boiling water, stir to dissolve the sugar, let it cool, filter the tea leaves to obtain sugar tea water, pour the black tea strain into the cooled sugar tea water, seal it with gauze, and ferment for 7 days. The resulting bacterial solution can be used as bacterial seed solution;

[0046] (4) Fermentation to prepare bacterial cellulose: inoculate the bacterial seed solution prepared in step (3) into the fermentation solution prepared in step (2) at a volume ratio of 8%, let it stand at 28° C. for 2 days to obtain a seed solution containing the target strain, ferment it at 25° C. for 7 days, collect the bacterial cellulose film formed on the surface of the liquid, wash it three times with deionized water, boil it with 1% sodium hydroxide solution for 1 hour, and finally wash it repeatedly with deionized water until the pH value is 7.0. After wiping off excess water on the surface, the finished bacterial cellulose film is obtained;

[0047] (5) Bacterial cellulose drying: The bacterial cellulose membrane obtained in step (4) was freeze-dried using a vacuum freeze dryer, and the bacterial cellulose membrane before and after freeze-drying was weighed and calculated to obtain: the solid content of the bacterial cellulose membrane was 2.47%, and the bacterial cellulose yield was 7.138 g / L.

[0048] Example 3

[0049] The steps for preparing bacterial cellulose using rice wine lees are as follows:

[0050] (1) preparing rice wine lees clarified liquid: directly centrifuging Sanhua wine lees stock liquid (water content 70%) to obtain Sanhua wine lees clarified liquid;

[0051] (2) preparing bacterial cellulose fermentation liquid: taking 95% of the Sanhua wine lees clarified liquid obtained in step (1) and 5% of a carbon source (sucrose, glucose, fructose, maltose in a mass ratio of 2:2:1:1), mixing the mixture, heating the mixture to 100° C., sealing the mixture and boiling the mixture for 0.6 hours, and then cooling the mixture to 27° C. for later use;

[0052] (3) Preparation of target strain seed solution: Add 100 g of sucrose and 6.5 g of tea leaves into a glass jar containing 1000 mL of boiling water, stir to dissolve the sugar, let it cool, filter the tea leaves to obtain sugar tea water, pour the black tea strain into the cooled sugar tea water, seal it with gauze, and ferment for 7 days. The resulting bacterial solution can be used as bacterial seed solution;

[0053] (4) Fermentation to prepare bacterial cellulose: The bacterial seed solution prepared in step (3) was inoculated into the fermentation solution prepared in step (2) at a volume ratio of 8%, and the solution was allowed to stand at 28° C. for 3 days to obtain a seed solution containing the target strain. The solution was fermented at 25° C. for 7 days, and the bacterial cellulose film formed on the surface of the solution was collected. The solution was washed three times with deionized water, boiled with 1% sodium hydroxide solution for 1 hour, and repeatedly washed with deionized water until the pH value was 7.0. The excess water on the surface was wiped off to obtain a finished bacterial cellulose film.

[0054] (5) Drying of bacterial cellulose: The bacterial cellulose membrane obtained in step (4) was freeze-dried using a vacuum freeze dryer, and the bacterial cellulose membrane before and after freeze-drying was weighed and calculated to obtain: the solid content of the bacterial cellulose membrane was 2.64%, and the bacterial cellulose yield was 6.53 g / L.

[0055] Comparative Example 1

[0056] Preparation of bacterial cellulose

[0057] (1) Preparation of bacterial cellulose fermentation liquid: 2.5 g yeast extract, 10 g glucose, 2.5 g soy peptone, 1.35 g disodium hydrogen phosphate dodecahydrate, and 0.575 g citric acid monohydrate;

[0058] (2) Preparation of strain seed solution: Add 100 g of sucrose and 6.5 g of tea leaves into a glass jar containing 1000 mL of boiling water, stir to dissolve the sugar, let it cool, filter the tea leaves to obtain sugar tea water, pour the black tea strain into the cooled sugar tea water, seal it with gauze, and ferment for 7 days. The resulting bacterial solution can be used as the bacterial seed solution;

[0059] (3) Fermentation to prepare bacterial cellulose: inoculate the bacterial seed solution prepared in step (2) into the fermentation solution prepared in step (1) at a volume ratio of 8%, ferment at 25° C. for 7 days, collect the bacterial cellulose film formed on the surface of the liquid, wash it three times with deionized water, boil it with 1% sodium hydroxide solution for 1 hour, and finally wash it repeatedly with deionized water until the pH value is 7.0. After wiping off excess water on the surface, the finished bacterial cellulose film is obtained;

[0060] (4) Bacterial cellulose drying: The bacterial cellulose membrane obtained in step (4) was freeze-dried using a vacuum freeze-drying machine, and the bacterial cellulose membrane before and after freeze-drying was weighed and calculated to obtain: the solid content of the bacterial cellulose membrane was 0.86%, and the bacterial cellulose yield was 2.03 g / L.

[0061] Comparative Example 2

[0062] The difference between this comparative example and Example 2 is that in step (2) of this comparative example, when preparing the bacterial cellulose fermentation liquid, 88% of the Sanhua wine lees clarified liquid obtained in step (1) and 12% of sucrose are mixed according to mass percentage, heated to 100°C and sealed and boiled for 0.6 hours, and then cooled to 27°C for standby use; the remaining steps are the same as those in Example 2. The solid content of the bacterial cellulose film in this comparative example is 0.68%, and the yield of bacterial cellulose is 4.51 g / L.

[0063] Figure 2 This is a comparison chart of the solid content and bacterial cellulose yield of the bacterial cellulose films produced in Example 1-3 and Comparative Example 1-2.

[0064] The embodiments described are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A bacterial cellulose culture medium, characterized in that The components include a carbon source and a clarified liquid of a rice wine lees stock solution, and the carbon source accounts for 0 to 10% by mass.

2. A bacterial cellulose culture medium according to claim 1, characterized in that The carbon source is one of sucrose, glucose, fructose and maltose or a mixture of the four.

3. A bacterial cellulose culture medium according to claim 1, characterized in that: The rice wine lees stock solution is a mixture of solid matter and water remaining after the brewing of the raw materials for brewing rice wine is completed, and the water content of the mixture is 70-90%.

4. The culture medium according to claim 1, characterized in that The clarified liquid is obtained by the following method: directly centrifuging the rice wine lees stock liquid to obtain the rice wine lees clarified liquid.

5. A method for preparing a bacterial cellulose film using the bacterial cellulose culture medium according to claim 1, characterized in that: The method comprises the following steps: heating and sterilizing the bacterial cellulose culture medium according to any one of claims 1 to 4 to obtain a bacterial cellulose fermentation broth, inoculating a target strain into the bacterial cellulose fermentation broth for fermentation to obtain the bacterial cellulose film.

6. The method according to claim 5, characterized in that The temperature-raising sterilization is to raise the temperature to 100°C.

7. The method according to claim 5, characterized in that The target strain is kombucha.

8. The bacterial cellulose film prepared by the method according to any one of claims 5 to 7.

9. A bacterial cellulose membrane product, characterized in that: The bacterial cellulose membrane of claim 8 is washed and alkali boiled to obtain the finished bacterial cellulose membrane.

10. The bacterial cellulose membrane product according to claim 9, characterized in that: The washing is performed with deionized water; and the alkali boiling is performed with a sodium hydroxide solution.

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