Acetobacter culture medium prepared from plant waste and application thereof

Abstract

The invention discloses an acetic bacteria culture medium prepared from plant waste and application of the acetic bacteria culture medium, and belongs to the technical field of acetic bacteria culture, the acetic bacteria culture medium comprises bean curd yellow serofluid and a stabilizer, and the stabilizer is sodium alginate-polyamino acid gel particles. The acetic bacteria culture medium is prepared by the following steps: boiling bean curd yellow serofluid for 20-30 minutes, cooling to 40-45 DEG C, adding a stabilizer, stirring and mixing, and cooling to room temperature to obtain the acetic bacteria culture medium. According to the application of the acetic bacteria culture medium prepared from the plant waste materials in preparation of bacterial cellulose, the stabilizer is added into the culture medium, the concentration of calcium ions in a culture medium system is stabilized, the fermentation efficiency of acetic bacteria is improved, and in the later period of fermentation, as a large amount of reducing sugar is consumed in the fermentation process, the concentration of H < + > in the fermentation system is increased; the calcium ions adsorbed in the stabilizer are desorbed, the stability of the calcium ions in the system is maintained, and the cellulose is continuously and stably produced.

Description

Technical Field

[0001] The invention belongs to the technical field of acetic acid bacteria culture, and particularly relates to an acetic acid bacteria culture medium prepared from plant waste and application thereof. Background Art

[0002] The cells of acetic acid bacteria are oval to rod-shaped; single, paired or chain; terminal hairs or peritrichous; motile or immotile; Gram-negative; obligate aerobic bacteria, oxidizing various organic substances into organic acids and other oxidants. Generally, they grow vigorously on ethanol or other oxidizable yeast decoction or yeast digestion medium.

[0003] The important feature of Acetobacter is that it can oxidize ethanol to acetic acid and oxidize acetic acid and lactic acid to CO. 2 and H 2 O. Some strains are able to synthesize cellulose, which forms the matrix outside the cell wall, and the bacteria are embedded in the tangled sheets of cellulose microfilaments. When these species of bacteria grow in a static liquid medium, they will form a biofilm on the surface. Acetobacteria in the biofilm state have a higher tolerance to antibiotics than planktonic acetic acid bacteria. When acetic acid bacteria encounter harmful bacteria, they can form a biofilm to deal with them together, and even inhibit the growth of harmful bacteria.

[0004] Patent application with publication number CN114774329A discloses an acetic acid bacteria culture medium and a method for cultivating acetic acid bacteria. In the application, the acetic acid bacteria culture medium is prepared with yellow pulp water (a large amount of organic wastewater and waste residue will be generated during the processing of soy products) and genistein as raw materials, which not only solves the problem of waste utilization, but also promotes the growth of acetic acid bacteria. However, the quality of yellow pulp water of tofu is unstable, and the use of halide as a coagulant will lead to excessive metal ions in the yellow pulp water. Among them, too high or too low calcium ion concentration will affect the formation of acetic acid bacteria biofilm, which is not conducive to the growth of acetic acid bacteria. Summary of the invention

[0005] The object of the present invention is to provide an acetic acid bacteria culture medium prepared from plant waste and application thereof, so as to solve the problem of unstable growth of acetic acid bacteria.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] An acetic acid bacteria culture medium prepared from plant waste comprises tofu yellow pulp water and a stabilizer, wherein the stabilizer is sodium alginate-polyamino acid gel particles; the stabilizer is used for stabilizing the calcium ion content in the culture medium.

[0008] Furthermore, the acetic acid bacteria culture medium is prepared by the following steps:

[0009] The tofu yellow pulp water is boiled for 20-30 minutes and then cooled to 40-45°C, a stabilizer is added, stirred and mixed, and cooled to room temperature to obtain an acetic acid bacteria culture medium. The stabilizer is added when the water is cooled to 40-45°C after boiling. 40-45°C is a relatively high temperature (relative to room temperature), which is conducive to the adsorption process.

[0010] Furthermore, the stabilizer is prepared by the following steps:

[0011] Sodium alginate and polyamino acid are added to water in a mass ratio of 2:1-1.2, toluene is added after stirring and dispersing, and then dripped into a 5% calcium chloride solution in stirring, filtered and washed with water to obtain a stabilizer; the dosage ratio of sodium alginate to water is 1g:50mL; the dosage ratio of sodium alginate to toluene is 1g:1.2mL. The stabilizer uses sodium alginate and polyamino acid as raw materials, wherein sodium alginate can be used as a carbon source and polyamino acid can be used as a nitrogen source, and can also be used as raw materials after recycling to save resources.

[0012] Furthermore, the polyamino acid is one of polyglutamic acid and polyaspartic acid, both of which are acidic amino acids, participate in the chelation and adsorption of calcium ions, and are in a stable state.

[0013] The relative molecular mass of the polyamino acid is 500KD.

[0014] Furthermore, the tofu yellow slurry has a protein content of 6-7 mg / g, a total sugar content of 9.5-10 mg / g, a raffinose content of 0.6-0.8 mg / g, a stachyose content of 2.5-3 / g, an isoflavone content of 0.1-0.3 mg / g, a saponin content of 0.4-0.5 mg / g, a fat content of 3.5-4 mg / g, and an initial pH of 6-6.5. Under the condition of an initial pH of 6-6.5, it is easy for the stabilizer to adsorb calcium ions, but the growth rate of acetic acid bacteria under this condition is low, so the production efficiency in the early stage of fermentation is low.

[0015] Furthermore, calcium sulfate is added to the tofu yellow slurry water to adjust the calcium ion concentration to 1.5 g / L; and 8-10 g of stabilizer is added to each liter of tofu yellow slurry water.

[0016] The invention discloses an application of an acetic acid bacteria culture medium prepared from plant waste in the preparation of bacterial cellulose.

[0017] Furthermore, the acetic acid bacteria is one of Gluconacetobacter, Acetobacter pasteurianus, Acetobacter orlanii, Acetobacter xueri, Acetobacter putida and Acetobacter climbing membrane.

[0018] Furthermore, the concentration of the acetic acid bacteria solution was 1×10 6 cfu / mL-1×10 8cfu / mL; the inoculation amount of the acetic acid bacteria liquid is 4% to 8%; the fermentation temperature for preparing bacterial cellulose is 28-32°C.

[0019] Furthermore, the fermentation time for preparing bacterial cellulose is 7-10 days.

[0020] Beneficial effects of the present invention:

[0021] The present invention prepares an acetic acid bacteria culture medium prepared from plant waste, wherein a stabilizer is added to the culture medium to stabilize the calcium ion concentration in the culture medium system and improve the fermentation efficiency of the acetic acid bacteria. In the late fermentation period, as a large amount of reducing sugar is consumed during the fermentation process, H + As the concentration increases, the calcium ions adsorbed in the stabilizer are desorbed, maintaining the calcium ion stability of the system, thereby continuously and stably producing cellulose.

[0022] The acetic acid bacteria culture medium prepared from plant waste in the present invention uses sodium alginate-polyamino acid gel particles as a stabilizer. The stabilizer uses sodium alginate and polyamino acid as raw materials, wherein sodium alginate can be used as a carbon source and polyamino acid can be used as a nitrogen source. It is used as a raw material in the culture medium system and can be reused after being recovered. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Preparation Example 1

[0025] Preparation of stabilizer:

[0026] According to the proportion, 5g of sodium alginate and 2.5g of polyglutamic acid are added to 250mL of water, and 3mL of toluene is added after stirring and dispersing. After stirring and dispersing, the mixture is dropped into a stirred calcium chloride solution with a mass fraction of 5%. Cross-linked particles will occur under the action of calcium ions. The toluene and residual ions are removed by filtering and washing to obtain a stabilizer. The relative molecular mass of polyglutamic acid is 500KD.

[0027] Preparation Example 2

[0028] Preparation of stabilizer:

[0029] According to the proportion, 5g of sodium alginate and 3g of polyglutamic acid are added to 250mL of water, and 3mL of toluene is added after stirring and dispersing. After stirring and dispersing, the mixture is dropped into a stirred calcium chloride solution with a mass fraction of 5%. Cross-linked particles will occur under the action of calcium ions. The toluene and residual ions are removed by filtering and washing to obtain a stabilizer. The relative molecular mass of polyglutamic acid is 500KD.

[0030] The tofu yellow slurry in the embodiments and comparative examples is fresh material provided by the tofu factory. The protein content of the tofu yellow slurry is 6.5 mg / g; the total sugar content is 9.9 mg / g; the raffinose content is 0.7 mg / g; the stachyose content is 2.8 mg / g; the isoflavone content is 0.2 mg / g; the saponin content is 0.3 mg / g; the fat content is 3.6 mg / g, and the initial pH is 6.

[0031] Example 1

[0032] The steps for preparing acetic acid bacteria culture medium prepared from plant waste are as follows:

[0033] The calcium ion concentration in the tofu yellow slurry was adjusted to 1.5 g / L by adding calcium sulfate; the tofu yellow slurry was boiled for 20 minutes and then cooled to 45° C., 10 g of stabilizer was added to each liter of the tofu yellow slurry, stirred and mixed, and cooled to room temperature to obtain an acetic acid bacteria culture medium.

[0034] Example 2

[0035] Compared with Example 1, 9 g of stabilizer is added to each liter of tofu yellow slurry and stirred and mixed. The remaining raw materials and preparation process are the same as those in Example 1.

[0036] Example 3

[0037] Compared with Example 1, in this example, 8.5 g of stabilizer is added to each liter of tofu yellow slurry and stirred and mixed. The remaining raw materials and preparation process are the same as those in Example 1.

[0038] Example 4

[0039] Compared with Example 1, 8 g of stabilizer is added to each liter of tofu yellow slurry and stirred and mixed. The remaining raw materials and preparation process are the same as those in Example 1.

[0040] Comparative Example 1

[0041] Compared with Example 4, the calcium ion concentration in the tofu yellow slurry in this comparative example is 1 g / L, and no stabilizer is added. The remaining raw materials and preparation process are the same as those in Example 4.

[0042] Comparative Example 2

[0043] After testing, before inoculation, the concentration of calcium ions in the culture medium in Example 4 was stabilized at about 600 mg / L. Compared with Example 4, the calcium ion concentration in the tofu yellow slurry water was adjusted to 600 mg / L in this comparative example, and no stabilizer was added. The remaining raw materials and preparation process were the same as those in Example 4.

[0044] Comparative Example 3

[0045] Compared with Comparative Example 2, acetic acid was added to adjust the pH value of the acetic acid bacteria culture medium to 4, and the other raw materials and preparation process remained the same as those of Comparative Example 2.

[0046] Application Examples

[0047] In the application example, Gluconacetobacter was selected as the test strain. The Gluconacetobacter strain was activated before use. The steps were as follows: a culture medium containing 20 g / L glucose, 5 g / L yeast extract, 5 g / L peptone, and 2.7 g / L disodium hydrogen phosphate was prepared, and the pH value was adjusted to 6.0 with acetic acid. After sterilization at 121°C, 20 mL / L of anhydrous ethanol was added to obtain an activated culture medium. After secondary activation (Gluconacetobacter was aspirated from the glycerol tube and added to the activated culture medium, and cultured at 28°C for 20 h, the cultured bacterial solution was heated to a concentration of 1×10 6 cfu / mL, inoculation amount of 10%, inoculated into activation medium at 28°C constant temperature culture for 20h) to obtain activated Gluconacetobacter bacterial liquid, which was stored at 4°C for later use.

[0048] Application Example 1

[0049] In this application example, the acetic acid bacteria culture medium prepared in Example 1 was used as the substrate for fermentation, and the concentration of the Gluconacetobacter broth was 1×10 6 cfu / mL, the inoculation amount of Gluconacetobacter was 5%, and the fermentation temperature was 30°C.

[0050] Application Example 2

[0051] Compared with Application Example 1, the acetic acid bacteria culture in this application example is replaced with the acetic acid bacteria culture medium prepared in Example 2, and the other raw materials and preparation process remain the same as those in Application Example 1.

[0052] Application Example 3

[0053] Compared with Application Example 1, the acetic acid bacteria culture in this application example is replaced with the acetic acid bacteria culture medium prepared in Example 3, and the other raw materials and preparation process remain the same as those in Application Example 1.

[0054] Application Example 4

[0055] Compared with Application Example 1, the acetic acid bacteria culture in this application example is replaced with the acetic acid bacteria culture medium prepared in Example 4, and the other raw materials and preparation process remain the same as those in Application Example 1.

[0056] Comparative application example 1

[0057] Compared with Application Example 1, the acetic acid bacteria culture in this application example is replaced by the acetic acid bacteria culture medium prepared in Comparative Example 1, and the other raw materials and preparation process remain the same as those in Application Example 1.

[0058] Comparative Application Example 2

[0059] Compared with Application Example 1, the acetic acid bacteria culture in this application example is replaced by the acetic acid bacteria culture medium prepared in Comparative Example 2, and the other raw materials and preparation process remain the same as those in Application Example 1.

[0060] Comparative Application Example 3

[0061] Compared with Application Example 1, the acetic acid bacteria culture in this application example is replaced by the acetic acid bacteria culture medium prepared in Comparative Example 3, and the other raw materials and preparation process remain the same as those in Application Example 1.

[0062] The cellulose yields of Application Examples 1 to 4 and Comparative Example 1 to 3 were recorded, and the fermentation time was 6 to 10 days. The results are shown in Table 1 below:

[0063] Table 1

[0064]

[0065] From the comparison of the results of Application Examples 1 to 4 and Comparative Application Examples 1 to 3 in Table 1, it can be seen that the data of Application Examples 1 to 4 do not increase significantly from the ninth day as the fermentation time increases, and the yield of the prepared acetic acid bacteria culture medium is greater than 2 g / L. By adding a stabilizer to the acetic acid bacteria culture medium, the calcium ion concentration in the culture medium system is stabilized, and the fermentation efficiency of acetic acid bacteria is improved. In the late fermentation period, as a large amount of reducing sugar is consumed during the fermentation process, H + As the concentration increases, the calcium ions adsorbed in the stabilizer are desorbed, maintaining the calcium ion stability of the system, thereby continuously and stably producing cellulose.

[0066] The corresponding yields of Comparative Example Application 1 to Comparative Example Application 3 are all less than 1.7 g / L, wherein the calcium ion content in the acetic acid bacteria culture medium used in Comparative Example Application 1 is too high, which inhibits the growth of acetic acid bacteria, hinders the activity of microorganisms, and reduces production efficiency. The concentration of calcium ions in Comparative Example Application 2 is appropriate, which helps the extracellular polysaccharides to connect with each other, thereby making the bacterial membrane structure more solid, and calcium ions will be consumed in the promotion process. During the production process, calcium ions will promote acetic acid bacteria to resist the pressure of osmotic pressure changes in the external environment and promote the growth of acetic acid bacteria. Therefore, as the fermentation time increases, the calcium ion concentration of Comparative Example Application 2 decreases, and a bacterial cellulose film is formed at the gas-liquid interface of the culture medium. As the film continues to increase, the growth of microorganisms is restricted, and the cellulose yield is difficult to continue to rise. In comparative example 3, the pH value at the beginning of fermentation is adjusted to 4, which is suitable for the growth of acetic acid bacteria. In addition, the acetic acid used to adjust the pH value of the system can also provide the energy required for the growth of acetic acid bacteria, promote bacterial growth and metabolism, and the growth rate of acetic acid bacteria is fast in the early stage, which improves the efficiency of fermentation, shortens the fermentation cycle, and the fermentation system is not easily contaminated by miscellaneous bacteria. Therefore, compared with application examples 1 to application examples 3 and comparative application examples 1 to comparative application examples 2, a higher yield can be achieved in the early stage of fermentation, but in the later stage, with the consumption of calcium ions and the consumption of nutrients in the culture medium system, the total production volume cannot reach a high level.

[0067] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0068] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An acetic acid bacteria culture medium prepared from plant waste, characterized in that: The method comprises tofu yellow pulp water and a stabilizer, wherein the stabilizer is sodium alginate-polyamino acid gel particles.

2. The acetic acid bacteria culture medium prepared from plant waste according to claim 1, characterized in that: The acetic acid bacteria culture medium is prepared by the following steps: The tofu yellow slurry water is boiled for 20-30 minutes and then cooled to 40-45° C., a stabilizer is added, stirred and mixed, and cooled to room temperature to obtain an acetic acid bacteria culture medium.

3. The acetic acid bacteria culture medium prepared from plant waste according to claim 1, characterized in that: The stabilizer is prepared by the following steps: Sodium alginate and polyamino acid are added into water in a mass ratio of 2:1-1.2, toluene is added after stirring and dispersing, and then the mixture is dropped into a 5% by mass calcium chloride solution after stirring and dispersing, filtered, and washed with water to obtain a stabilizer.

4. The acetic acid bacteria culture medium prepared from plant waste according to claim 3, characterized in that: The polyamino acid is one of polyglutamic acid and polyaspartic acid; The relative molecular mass of the polyamino acid is 500KD.

5. The acetic acid bacteria culture medium prepared from plant waste according to claim 1, characterized in that: The tofu yellow slurry has a protein content of 6-7 mg / g, a total sugar content of 9.5-10 mg / g, a raffinose content of 0.6-0.8 mg / g, a stachyose content of 2.5-3 / g, an isoflavone content of 0.1-0.3 mg / g, a saponin content of 0.4-0.5 mg / g, a fat content of 3.5-4 mg / g, and an initial pH of 6-6.

5.

6. The acetic acid bacteria culture medium prepared from plant waste according to claim 1, characterized in that: Calcium sulfate is added to the tofu yellow slurry to adjust the calcium ion concentration to 1.5 g / L; 8-10 g of stabilizer is added per liter of tofu yellow slurry.

7. Use of the acetic acid bacteria culture medium prepared from plant waste as claimed in any one of claims 1 to 6 in the preparation of bacterial cellulose.

8. The use of an acetic acid bacteria culture medium prepared from plant waste according to claim 7 in the preparation of bacterial cellulose, characterized in that: The acetic acid bacteria is one of Gluconacetobacter, Acetobacter pasteurianus, Acetobacter orlanis, Acetobacter xueri, Acetobacter putida and Acetobacter climbing membrane.

9. The use of an acetic acid bacteria culture medium prepared from plant waste in the preparation of bacterial cellulose according to claim 7, characterized in that: The concentration of acetic acid bacteria solution is 1×10 6 cfu / mL-1×10 8 cfu / mL; the inoculation amount of the acetic acid bacteria liquid is 4% to 8%; the fermentation temperature for preparing bacterial cellulose is 28-32°C.

10. The use of an acetic acid bacteria culture medium prepared from plant waste in the preparation of bacterial cellulose according to claim 7, characterized in that: The fermentation time for preparing bacterial cellulose is 7-10 days.

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