Improved medium, improved method and use thereof

Abstract

The application provides an improved culture medium, an improved method and application. The improved culture medium replaces phosphate with glycerol phosphate and replaces phosphate buffer with PIPES buffer on the basis of the original culture medium containing phosphate. The improved culture medium does not produce precipitate in the fermentation process, does not affect the absorption and utilization of phosphorus by microorganisms, and improves the growth stability of the microorganisms in a zinc-containing environment. In addition, all bacteria, fungi and microalgae can be fermented in the improved culture medium to prepare zinc-rich protein with high zinc content, break through the limitation of market zinc-rich single-cell protein to lactic acid bacteria and yeast, and expand the diversity of zinc-rich microbial germplasm and protein products.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and relates to an improved culture medium, an improved method, and its application. Background Technology

[0002] Single-cell protein, also known as microbial protein, is typically formed through fermentation of microorganisms in a culture medium. Many types of microorganisms are used to produce single-cell protein, including bacteria, fungi, and microalgae. Research has shown that single-cell protein can be used as a substitute for dietary protein.

[0003] Zinc is an essential trace element for all organisms, used to improve blood pressure, glucose, and serum low-density lipoprotein cholesterol levels. Among common zinc supplements, inorganic zinc and organically synthesized zinc have low bioavailability and can affect gastrointestinal digestion. Amino acid chelated zinc has high bioavailability, but its price is much higher than that of inorganic zinc supplements. Fermented zinc supplements based on zinc-enriched yeasts achieve zinc absorption and conversion during the growth process by adding inorganic zinc during the strain cultivation stage. Because the form in which zinc exists is more consistent with the natural form of trace elements in organisms, bioavailability is significantly improved, and toxic side effects are greatly reduced.

[0004] Current research on microbial zinc enrichment technology mainly focuses on yeasts such as *Candida tropicalis*, lactic acid bacteria such as *Lactobacillus paracasei* and *Lactobacillus fermentum*, and a few other species such as *Bacillus subtilis*. These species mostly grow under acidic conditions, and the lack of diversity leads to significant homogenization of the produced zinc-enriched protein products. Furthermore, existing technologies generally rely on culture media containing high concentrations of zinc ions, such as 200-500 mg / L, to improve enrichment efficiency. However, this approach not only increases the cost of zinc salt raw materials but also easily leads to the environmental risk of heavy metal residues in fermentation wastewater.

[0005] Currently, most single-cell protein microorganisms are cultured under neutral or alkaline pH conditions, with the culture medium containing a large amount of phosphate. Phosphate serves as both a pH buffer for cell culture and a source of phosphorus for cell growth. However, zinc phosphate has extremely low solubility under neutral and alkaline conditions, making it impossible to increase the concentration of dissolved zinc ions in the culture medium, thus hindering zinc-enriched culture for the production of zinc-enriched single-cell proteins. Summary of the Invention

[0006] The purpose of this invention is to provide an improved culture medium, an improved method, and an application to solve the problem that existing pH-neutral and alkaline culture media cannot be used to cultivate and produce zinc-rich single-cell proteins.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In one aspect, this application provides a modified culture medium comprising a glycerol phosphate and a 1,4-piperazine diethanesulfonic acid (PIPES) buffer solution with a pH greater than 6.5.

[0008] The improved culture medium in this application is an improvement on the original phosphorus-containing culture medium for preparing single-cell proteins. In addition to phosphorus-containing components and phosphate buffer, the original phosphorus-containing culture medium also includes carbon sources and trace elements required for the growth of different microbial strains.

[0009] Specifically, this application modifies the existing phosphorus-containing culture medium for preparing single-cell proteins by replacing phosphorus-containing components, such as phosphate, with glycerophosphate as the sole phosphorus source in the modified culture medium; simultaneously, it uses PIPES buffer as the sole buffer in the modified culture medium. This modified culture medium does not produce precipitation during cell culture and fermentation, does not affect the absorption and utilization of phosphorus by microorganisms, and improves the growth stability of microorganisms in a zinc-containing environment.

[0010] In this application, the final concentrations of glycerophosphate and 1,4-piperazine diethanesulfonic acid buffer in the modified culture medium are 1-300 mmol / L and 1-100 mmol / L, respectively. The glycerophosphate is selected from one or more soluble salts such as sodium glycerophosphate, potassium glycerophosphate, and magnesium glycerophosphate.

[0011] In addition, the modified culture medium in this application also includes a zinc source, and the final concentration of the zinc source in the modified culture medium is ≥5 mg / L. The addition of the zinc source makes the modified culture medium suitable for culturing zinc-rich single-cell proteins.

[0012] In this application, the zinc source includes one or more of soluble inorganic zinc salts, soluble organic zinc salts, chelated zinc, or complexed zinc. Soluble inorganic zinc salts include, but are not limited to, zinc sulfate, zinc chloride, zinc nitrate, zinc bromide, and zinc iodide. Soluble organic zinc salts include, but are not limited to, zinc acetate, zinc lactate, zinc gluconate, zinc citrate, zinc butyrate, and zinc formate. Chelated zinc includes, but is not limited to, ZnEDTA (Ethylenediaminetetraacetic Acid Disodium Zinc Salt), ZnDTPA (Diethylenetriaminepentaacetic acid zinc trisodium), and ZnHEDTA (N-β-hydroxyethylethylenediaminetriacetic acid sodium salt). Complexed zinc includes, but is not limited to, zinc lignin sulfonate, zinc amino acid complexes, and zinc fulvic acid.

[0013] Secondly, this application provides a method for improving a culture medium, the method comprising: replacing the phosphorus-containing component with glycerol phosphate and replacing the phosphate buffer with 1,4-piperazine diethanesulfonic acid buffer, based on a phosphorus-containing original culture medium.

[0014] Thirdly, this application provides an application of an improved culture medium, namely its application in the fermentation preparation of zinc-rich single-cell protein.

[0015] Fourthly, this application provides a method for preparing zinc-rich single-cell protein, the method comprising: inoculating fermenting microorganisms into the modified culture medium in the first aspect at an inoculation ratio of >0.2%, adding a carbon source, and shaking culture at 10-70℃; when fermentation reaches the end of the logarithmic growth phase, the culture is ended, the fermentation broth is centrifuged, and the broth is washed twice with ultrapure water to obtain zinc-rich single-cell protein.

[0016] In this application, the fermenting microorganisms include one or more of bacteria, fungi, and microalgae, and the carbon source is an autotrophic or heterotrophic carbon source.

[0017] Fifthly, this application provides a zinc-rich single-cell protein, which is prepared by the preparation method described in the fourth aspect.

[0018] The present invention has the following beneficial effects: (1) This application is the first to select fermentation bacteria such as methanogenic bacteria and hydroxide bacteria as zinc-enriched carriers, which breaks through the limitations of lactic acid bacteria and yeast and expands the diversity of zinc-enriched strains and protein products.

[0019] (2) This application uses PIPES buffer instead of the traditional phosphate buffer system, and combines glycerol phosphate to optimize the phosphorus source. No precipitation is generated during fermentation, which does not affect the absorption and utilization of phosphorus by microorganisms and improves the growth stability of microorganisms in zinc-containing environment.

[0020] (3) This application can achieve efficient assimilation of zinc and significantly reduce the demand for zinc raw materials. Attached Figure Description

[0021] Figure 1 OD of methanogenic bacteria during the culture process of conventional bacterial protein fermentation medium and modified medium 600 Dynamic change diagram; Figure 2 The dynamic pH changes of methanogenic bacteria during culture in conventional cell protein fermentation medium and modified medium; Figure 3 OD during the growth of methanogenic bacteria under different zinc ion concentrations 600 Dynamic change diagram; Figure 4A comparison of cell dry weight and protein content at the culture endpoint of methanogenic bacteria under different zinc ion concentrations. Figure 5 A comparison of total zinc content in the protein of zinc-rich single-cell proteins obtained from methanogenic bacteria cultured under different zinc ion concentrations; Figure 6 This is a comparison of the growth of hydroxide bacteria under different zinc ion concentrations; where, figure a shows the OD... 600 The dynamic change graph, and graph b is a comparison of dry weight and protein content; Figure 7 This is a comparison chart of the total zinc content in zinc-rich single-cell proteins obtained from bacteria cultured with hydroxide under different zinc ion concentrations. Detailed Implementation

[0022] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0023] Example 1 This application provides an improved culture medium based on a phosphorus-containing original culture medium, in which glycerol phosphate replaces the phosphorus-containing components and 1,4-piperazine diethanesulfonic acid buffer replaces the phosphate buffer. The final concentrations of glycerol phosphate and 1,4-piperazine diethanesulfonic acid buffer in the improved culture medium are 150 mmol / L and 50 mmol / L, respectively, and the pH of the culture medium is 7.0.

[0024] Example 2 This application provides an improved culture medium based on a phosphorus-containing original culture medium. The phosphorus-containing component is replaced by glycerol phosphate and the phosphate buffer is replaced by 1,4-piperazine diethanesulfonic acid buffer. The final concentrations of glycerol phosphate and 1,4-piperazine diethanesulfonic acid buffer in the improved culture medium are 20 mmol / L and 5 mmol / L, respectively. The pH of the culture medium is 6.6.

[0025] Example 3 This application provides an improved culture medium based on a phosphorus-containing original culture medium. The phosphorus-containing components are replaced with glycerol phosphate and the phosphate buffer is replaced with 1,4-piperazine diethanesulfonic acid buffer. The final concentrations of glycerol phosphate and 1,4-piperazine diethanesulfonic acid buffer in the improved culture medium are 300 mmol / L and 100 mmol / L, respectively. The pH of the culture medium is 7.2.

[0026] Example 4 This application provides an improved culture medium based on a phosphorus-containing original culture medium. The phosphorus-containing components are replaced with glycerol phosphate and the phosphate buffer is replaced with 1,4-piperazine diethanesulfonic acid buffer. The final concentrations of glycerol phosphate, zinc source and 1,4-piperazine diethanesulfonic acid buffer in the improved culture medium are 150 mmol / L, 10 mg / L and 50 mmol / L, respectively. The pH of the culture medium is 7.0.

[0027] Example 5 This application provides an improved culture medium based on a phosphorus-containing original culture medium. The phosphorus-containing component is replaced by glycerol phosphate, and the phosphate buffer is replaced by 1,4-piperazine diethanesulfonic acid buffer. The final concentrations of glycerol phosphate, zinc source, and 1,4-piperazine diethanesulfonic acid buffer in the improved culture medium are 20 mmol / L, 5 mg / L, and 5 mmol / L, respectively. The pH of the culture medium is 6.6.

[0028] Example 6 This application provides an improved culture medium based on a phosphorus-containing original culture medium. The phosphorus-containing components are replaced with glycerol phosphate and the phosphate buffer is replaced with 1,4-piperazine diethanesulfonic acid buffer. The final concentrations of glycerol phosphate, zinc source and 1,4-piperazine diethanesulfonic acid buffer in the improved culture medium are 300 mmol / L, 20 mg / L and 100 mmol / L, respectively. The pH of the culture medium is 7.2.

[0029] Example 7 This application provides a zinc-rich single-cell protein, the preparation method of which includes: The methanogenic bacteria were inoculated at a ratio of 2% into 50 mL of the modified culture medium in Example 4. A raw material gas containing methane and oxygen was introduced, with a volume ratio of methane to oxygen of 2:1. The bacteria were cultured at 30°C and a shaking speed of 150 rpm for 11-13 days. When the fermentation reached the end of the logarithmic growth phase, the culture was terminated. The bacterial solution was centrifuged and washed twice with ultrapure water to obtain zinc-rich single-cell protein.

[0030] Example 8 This application provides a zinc-rich single-cell protein, the preparation method of which includes: Hydroxyhydrogen bacteria were inoculated at a ratio of 5% into 50 mL of the modified culture medium in Example 5. Raw material gas containing H2, O2 and CO2 was introduced, with a volume ratio of H2, O2 and CO2 of 7:2:1. The bacteria were cultured at 30°C and a shaking speed of 150 rpm for 9-11 days. When fermentation reached the end of the logarithmic growth phase, the culture was terminated. The bacterial solution was centrifuged and washed twice with ultrapure water to obtain zinc-rich single-cell protein.

[0031] To investigate whether the modified culture medium could promote microbial growth, this application used a conventional bacterial protein fermentation medium containing phosphate as a comparative example and the modified culture medium in Example 4 as an experimental example for comparative culture. The phosphorus content of phosphate was calculated to be the same as the phosphorus content of glycerophosphate in Example 4. During the cultivation of methanogenic bacteria in the comparative example and experimental example according to the method in Example 7, OD was measured daily. 600 Value and pH value, to obtain the attached Figure 1 , 2 From the appendix Figure 1 , 2 It is evident that the zinc-rich single-cell protein provided in this application embodiment has no inhibitory effect on the growth of methanogenic bacteria, indicating that glycerophosphate does not affect the strain's absorption and utilization of phosphorus, thereby improving the strain's growth stability in a zinc-containing environment.

[0032] To verify the effect of zinc sulfate concentration on zinc-rich single-cell protein, the methanogenic bacteria OB3b were cultured in modified culture media containing zinc sulfate at final concentrations of 0 mg / L, 5 mg / L, 10 mg / L, 25 mg / L, and 50 mg / L, respectively, following the method described in Example 7. Each group was divided into three replicates, and OD values ​​were measured daily. 600 Value, obtain attached Figure 3 After the growth experiment, the bacterial culture was collected, centrifuged, washed twice with ultrapure water, and freeze-dried under vacuum. The dry weight and protein content of the zinc-rich single-cell protein were measured. Simultaneously, the zinc content in the zinc-rich single-cell protein was determined using flame atomic absorption spectrometry according to GB5009.14-2017 National Food Safety Standard for the Determination of Zinc in Food. Figure 4 , 5 .

[0033] From the appendix Figure 3 It is evident that the growth of methanogenic bacteria OB3b was significantly inhibited by increasing the zinc ion concentration in the modified culture medium, and the OD... 600 The value decreased significantly, therefore, the growth of methanogenic bacteria OB3b was stagnant on modified culture media containing zinc sulfate at final concentrations of 25 mg / L and 50 mg / L.

[0034] Therefore, the detection of bacterial dry weight, protein content, and zinc content is only applicable to zinc-enriched single-cell protein cultured in modified medium containing zinc sulfate at final concentrations of 0 mg / L, 5 mg / L, and 10 mg / L. (From the attached...) Figure 4 It is evident that, compared to a final zinc ion concentration of 0 mg / L, the dry weight and protein content of zinc-rich single-cell proteins cultured in a modified medium with a final zinc ion concentration of 5 mg / L both decreased. Conversely, the dry weight of zinc-rich single-cell proteins cultured in a modified medium with a final zinc ion concentration of 10 mg / L decreased, but the protein content increased. (See attached...) Figure 5It can be seen that the zinc content in zinc-rich single-cell proteins cultured in modified culture media with final zinc ion concentrations of 5 mg / L and 10 mg / L reached 5.43 mg / g dry weight and 16.93 mg / g dry weight, respectively.

[0035] To verify the effect of zinc sulfate concentration on zinc-rich single-cell protein, in the embodiments of this application, DSM-1083 hydroxide bacteria were cultured in modified culture media containing zinc sulfate at final concentrations of 0 mg / L, 60 mg / L, 90 mg / L, 120 mg / L, 150 mg / L, and 200 mg / L, respectively, according to the method in Example 8. Each group was set up in triplicate, and OD was measured daily. 600 Value. After the growth experiment, the bacterial culture was collected, centrifuged, washed twice with ultrapure water, and freeze-dried under vacuum. The dry weight and protein content of the zinc-rich single-cell protein were measured. Simultaneously, the zinc content in the zinc-rich single-cell protein was determined using flame atomic absorption spectrometry according to GB5009.14-2017 National Food Safety Standard for the Determination of Zinc in Food. The results were obtained. Figure 6 , 7 .

[0036] From the appendix Figure 6 It is evident that, for the same number of culture days, as the zinc ion concentration in the modified culture medium increases, the OD... 600 The values ​​first increased and then decreased. Similarly, as the zinc ion concentration in the modified culture medium increased, the dry weight and protein content of zinc-rich single-cell protein also first increased and then decreased, with the highest dry weight and protein content of zinc-rich single-cell protein observed when the zinc ion concentration was 90 mg / L.

[0037] From the appendix Figure 7 It can be seen that as the zinc ion concentration in the modified culture medium increases, the zinc content in zinc-rich single-cell protein gradually increases, and when the zinc ion concentration in the modified culture medium is 200 mg / L, the zinc content in zinc-rich single-cell protein reaches 11.18 mg.

[0038] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A modified culture medium, characterized in that, It includes glycerol phosphate and 1,4-piperazine diethanesulfonic acid buffer.

2. The improved culture medium according to claim 1, characterized in that, The final concentrations of the glycerol phosphate and the 1,4-piperazine diethanesulfonic acid buffer in the modified culture medium were 1-300 mmol / L and 1-100 mmol / L, respectively.

3. The improved culture medium according to claim 1, characterized in that, The glycerophosphate includes one or more of sodium glycerophosphate, potassium glycerophosphate, and magnesium glycerophosphate.

4. The improved culture medium according to any one of claims 1-3, characterized in that, The modified culture medium also includes a zinc source, wherein the final concentration of the zinc source in the modified culture medium is ≥5 mg / L.

5. The improved culture medium according to claim 4, characterized in that, The zinc source includes one or more of soluble inorganic zinc salts, soluble organic zinc salts, chelated zinc, or complexed zinc.

6. The method for improving the improved culture medium according to any one of claims 1-5, characterized in that, Based on the original phosphorus-containing culture medium, glycerol phosphate was used to replace the phosphorus-containing components, and 1,4-piperazine diethanesulfonic acid buffer was used to replace the phosphate buffer.

7. The use of the improved culture medium according to any one of claims 1-5 in the fermentation preparation of zinc-rich single-cell protein.

8. A method for preparing zinc-rich single-cell protein, characterized in that, include: Fermenting microorganisms are inoculated into the modified culture medium described in claim 4 or 5, and a carbon source is added for cell culture to obtain zinc-rich single-cell protein.

9. The method for preparing zinc-rich single-cell protein according to claim 8, characterized in that, The fermenting microorganisms include one or more of bacteria, fungi, and microalgae, and the carbon source is an autotrophic or heterotrophic carbon source. The inoculation ratio of the fermenting microorganisms is ≥0.2%, and the culture temperature of the fermenting microorganisms is 10-70℃.

10. The zinc-rich single-cell protein prepared by the method for preparing zinc-rich single-cell protein according to claim 8 or 9.

Images