Method for improving the yield of cupriavidus necator protein and application

Abstract

The application provides a method for improving the yield of Cupriavidus necator protein and application, and relates to the technical field of microbial fermentation, and the method comprises the following steps: step 1, sugar fermentation; step 2, gas culture; and step 3, collecting and filtering the bacterial liquid after a set fermentation time. The obtained single-cell protein is used as a zebrafish feed additive, and it is found that the single-cell protein has no side effect on the growth of zebrafish, and can promote the growth of zebrafish and improve the weight gain rate. The single-cell protein has a wide application prospect in the fields of food and feed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation, and particularly relates to a method for improving protein yield of Cupriavidus necator and application thereof. BACKGROUND

[0002] The global population growth leads to the increase of protein demand, and traditional protein sources such as livestock and fishery farming processing face resource and environmental problems, and new sustainable protein sources are urgently needed. SCP is the cell protein produced by fungi, bacteria and microalgae using various raw materials under suitable culture conditions. SCP, as a new protein source, has the advantages of rich nutrition, environmentally friendly production process, etc., and can reduce the dependence on land and water resources, reduce greenhouse gas emissions, etc.

[0003] Cupriavidus necator is a green and low-carbon sustainable SCP source. It is an inorganic chemoautotrophic bacterium that uses H2 as an energy source, absorbs O2 and CO2 for growth, and plays an important role in carbon cycle and hydrogen bioconversion. Cupriavidus necator is widely studied for SCP production due to its high protein content and good protein quality. Cupriavidus necator grows independently of organic matter, not only can autotrophically synthesize SCP, but also can absorb CO2, thereby playing a positive role in reducing carbon emissions and mitigating the greenhouse effect, and is a green and low-carbon sustainable SCP source. As a substitute protein, it has the advantages of fast growth rate, low cultivation cost and protein production not affected by climate and environment, etc., but the growth rate of Cupriavidus necator in gas culture is not as fast as that in organic matter fermentation, and the protein content of Cupriavidus necator in organic matter fermentation is low.

[0004] Therefore, it is of great significance to develop a process that first rapidly increases the number of Cupriavidus necator cells by organic matter fermentation and then strengthens the protein content by gas culture to improve the efficiency of SCP production by Cupriavidus necator. SUMMARY

[0005] The purpose of the present application is to solve the problems of low cell density in heterotrophic fermentation of Cupriavidus necator, insufficient oxygen transfer efficiency in autotrophic stage and limited protein content improvement in the prior art, and to provide a method for improving protein yield of Cupriavidus necator and application thereof. By using the method of first accumulating cell density by sugar fermentation and then switching to continuous fermentation by gas, the protein content and protein yield of Cupriavidus necator are significantly improved, and the application effect in feed additives is verified.

[0006] The present application adopts the following technical solutions:

[0007] A method for improving protein yield of Cupriavidus necator, comprising the following steps: step 1. sugar fermentation; step 2. gas culture; and step 3. collecting and filtering the bacterial liquid after a set fermentation time.

[0008] Wherein, step 1 comprises: S101. inoculating the Cupriavidus necator in an amount of 5% of volume percentage into a fermenter;

[0009] S102. adding 21 g / L fructose and 4 mol / L ammonia water;

[0010] S103. fermenting under the conditions of 25-30 DEG C, pH 6.5-7.0 and 200-600 rpm, with filtered air being blown in at 5.0-10.0 L / min to maintain the dissolved oxygen value at 30%, and supplementing the carbon source at 40 g / L every 12 h;

[0011] S104. fermenting the culture to the maximum cell density.

[0012] Step 2 comprises: S201. transferring the fermentation broth in step 1 into a gas culture fermentation tower;

[0013] S202. culturing at 25-30 DEG C and pH 6.5-7.0.

[0014] Step 1 further comprises: inoculating the Cupriavidus necator strain in an amount of 5% of volume percentage into a flask containing the fermentation broth medium, and carrying out the expansion culture in a constant-temperature shaker at 30 DEG C and 180 rpm for 3-6 days.

[0015] Step 2 further comprises installing a pipeline for external circulation between the bottom and the top of the gas culture fermentation tower, and the liquid circulation speed in the pipeline is 300-600 L / min.

[0016] Step 2 further comprises adding 3 mol / L ammonia water to maintain the pH of the broth at 6.5-7.0, and the ammonia water also serves as a nitrogen source, while supplementing the mixed gas of H2 and CO2 at a flow rate of 2.5-16.4 SCCM.

[0017] A Cupriavidus necator protein powder prepared by the method for improving the protein yield of Cupriavidus necator.

[0018] An application of the Cupriavidus necator protein powder as a fish protein feed additive.

[0019] Further comprising that the proportion of the Cupriavidus necator added in the feed is 1:20.

[0020] The beneficial effects of the present application are:

[0021] The present application uses fructose as the sole carbon source to carry out sugar fermentation of Cupriavidus necator, and then H2 and CO2 are introduced for continuous gas culture. After fermentation, the protein content of Cupriavidus necator powder is increased from 44.98% in the sugar fermentation stage to 74.70% after combined culture, increased by 39.79%, and the protein yield is increased from 0.1410 g / h / L to 0.2418 g / h / L, increased by 71.49%. Cupriavidus necator cell protein powder as a feed additive applied to zebrafish breeding has no negative effect on zebrafish, and can significantly improve the growth performance, and has significant application value in the field of protein products such as protein feed additives. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a step flow chart of the present application;

[0023] Figure 2 It is a schematic diagram from sugar fermentation to gas culture stage;

[0024] Figure 3 It is a result graph of CDW determination in the sugar fermentation stage;

[0025] Figure 4 In the gas culture stage, (a) is the Log colony count; (b) is the CDW;

[0026] Figure 5 It is a total ion current chromatogram of GC-MS of volatile components of Cupriavidus necator;

[0027] Figure 6 It is a technical route diagram of adding cell protein in feed;

[0028] Figure 7 It is the influence of different mass percentages of protein addition on the growth performance of zebrafish, (a) is the influence of no addition, 5% and 10% addition on the growth rate of zebrafish, (b) is the influence of no addition, 5% and 10% addition on the weight gain rate of zebrafish, (c) is the influence of no addition, 5% and 10% addition on the specific growth rate of zebrafish.

[0029] In the figure, 1-valve I, 2-valve II, 3-valve III, 4-valve IV, 5-valve V, 6-valve VI, 7-valve VII, 8-valve VIII, 9-pipe I, 10-pipe II. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

[0031] The experimental methods in the examples, unless otherwise specified, are conventional methods and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] Cupriavidus necator (C. necator) was purchased from the General Microbiology Center of China Culture Collection Administration, with the strain number 1.7092, and was cultured on nutrient broth agar.

[0033] Single Cell Protein: Single Cell Protein, abbreviated as SCP.

[0034] like Figure 1 、 Figure 2 As shown, the protein yield of hookworm copper bacteria is improved by first fermenting sugar and then culturing with gas. The sugar fermentation stage adopts the organic matter feeding method, and it is necessary to maintain a sterile environment to prevent contamination by other bacteria. The cell number increases rapidly in this stage, but the cell protein content is not high. After switching to gas culture, the cell number will increase further and the cell protein content will increase significantly. During this period, no sterile environment is required, and continuous feeding and discharging can be achieved, thereby improving the protein production efficiency of the single equipment and reducing production costs. According to the automatic Kjeldahl nitrogen analyzer, the protein content in the sugar fermentation stage is 44.98%, and the protein content in the gas culture stage is 74.70%. The calculated protein yields are 0.1410g / h / L and 0.2418g / h / L, respectively. After two fermentations, the protein yield is increased by 71.49%, as shown in Table 1.

[0035] Table 1 Comparison between heterotrophic and autotrophic stages

[0036]

[0037] After the above-mentioned culture method converts organic matter into inorganic matter, the culture can be cultivated without a sterile system, which can further improve the production efficiency of the equipment. At the same time, bacteria require more enzyme systems for carbon assimilation, and the protein content is also increased, thereby increasing the protein yield.

[0038] The specific implementation plan is as follows:

[0039] The sugar fermentation stage, Hookworm Cupriavidus bacteria species according to 5% (v / v) amount of inoculation into the fermentation broth medium in a triangular flask, in a constant temperature shaker at 30℃, 180 rpm conditions for 3-6 d, according to 5% (v / v) amount of inoculation into the fermenter, open valve III 3 into the fructose solution 21 g / L, open valve II 2 and valve IV 4 into the ammonia solution 4 mol / L, maintain pH at 6.5-7.0, the fermenter is equipped with defoaming agent, through the cooling water circulating pump or heating base to maintain the temperature of the fermentation broth at 25-30℃, open valve I 1 and valve V 5 into the filtered air, the gas amount is maintained at 5.0-10.0 L / min, set the stirring speed at 200-600 rpm, maintain the dissolved oxygen value 30%, every 12 h supplement 40 g / L of carbon source. The results of measuring CDW of Hookworm Cupriavidus bacteria in sugar fermentation stage are shown in Figure 3

[0040] In the sugar fermentation stage, when the density of Hookworm Cupriavidus bacteria reaches the maximum, the bacteria solution is transferred into the gas culture fermentation tower through pipeline I 9, and the valves III 3, IV 4 and V 5 of the sugar fermentation stage are closed, entering the gas culture stage. 2 mol / L ammonia water is used as a supplementary nitrogen source, and the pH is maintained at 6.5-7.0. CO2 and H2 are used as gas sources, and the gas flow is controlled by a gas flow meter to be 2.5-16.4 SCCM. Valves I 1, VI 6, VII 7 and VIII 8 are opened, and the mixed gas and ammonia water are introduced into the gas culture fermentation tower. High-efficiency continuous fermentation is carried out at 25-30℃ and a liquid circulation speed of 200-600 L / min. During the fermentation process, the fermentation broth is circulated through pipeline II 10. In a 14 d continuous fermentation cycle, 12.95 L of product is harvested, with an average value of 25.17 g / L, a total CDW of 326.36 g, and an average yield of 23.31 g / d in continuous fermentation, as shown in Figure 4

[0041] Example 2

[0042] The collected single-cell protein powder is tested for moisture content, crude protein, crude ash, crude fat, crude fiber, 17 amino acids, tryptophan, pepsin digestibility, total phosphorus and calcium content. The test results are shown in Table 2. The crude protein content far exceeds the minimum standard, reaching 74.70%, far exceeding traditional plant protein sources, and has a high digestibility, making it a very high-quality protein. It also has a lower water content, a longer shelf life, and better storage performance than other feed grains on the market. Compared with soybean and fish meal, single-cell organisms have a short reproductive cycle and can quickly provide large amounts of high-quality protein. They can replace fish meal to meet the demand for protein sources in the feed industry, resulting in stronger economic benefits.

[0043] Table 2: Single-cell protein component content determination results ​​

[0044]

[0045] As Figure 5 shown, by GC-MS analysis of flavor substances in Leptospirillum hook, mainly for acid, ester, aldehyde, ketone and alkyl, the relative content of each type of material is shown in Table 3, the test shows that Leptospirillum hook provides higher acid flavor and lower ketone and aldehyde flavor. Among the volatile flavor substances, the threshold of acid compounds is higher, and the influence on the overall flavor is smaller, among which the relative content of stearic acid is 3.15%, and the relative content of palmitic acid is 1.93%; ester substances contribute greatly to the flavor, generally showing a lasting and rich fruit aroma, among which the relative content of 9-octadecene oleic acid (2-phenyl-1,3-dioxolane-4) methyl ester is 0.77%, and the relative content of phthalic acid diester is 0.77%; aldehyde substances have a fatty aroma, which is the main source of meat flavor, the relative content of stearic aldehyde is 3.84%, and the relative content of octadecanal is 0.54%; among the alkyl compounds, the relative content of 1-chloro octane is 0.7%, and the relative content of 1,1-bis dodecyl oxyhexadecane is 0.54%; most of the ketone compounds have a high threshold, which may be the oxidation product of alcohol or the decomposition product of ester, and also play an important role in the formation of meat flavor, among which the relative content of 3-nonen-2-ketone is 0.63%, and the relative content of 19-alkene-3,5-diamine-ketone is 0.62%.

[0046] Table 3 Content of each type of aroma substance in Leptospirillum hook

[0047]

[0048] By adding Leptospirillum hook single-cell protein as a feed additive to the feed at 5% and 10% for feeding fish, the effect of this use is determined, and the technical route of adding bacterial protein to the feed is shown in Figure 6 Zebra fish are fed with self-made single-cell protein feed, and oxygen is continuously supplied during the feeding process. The swimming condition of zebra fish is observed regularly every day, the experimental period is 28 days, the body weight and body length of each zebra fish are measured every day, and the data is recorded.

[0049] As Figure 7As shown, the growth rate, weight gain rate and specific growth rate of the test group with added bacterial protein are higher than those of the control group without added bacterial protein, the growth rate of the 5% group is 12.4%, the weight gain rate is 41.5%, and the specific growth rate is 1.2%, the growth rate of the 10% group is 12.1%, the weight gain rate is 35.5%, and the specific growth rate is 1.0%, the 5% group is better than the 10% group, based on the experiment, the addition amount of 5% is better, the results show that the bacterial protein of Cupriavidus necator can be added in feed to replace part of the protein, and has no side effect on the growth of zebrafish, and can promote the growth of zebrafish and improve the weight gain rate, in the field of protein products or feed, especially as a feed additive or prepared into protein feed, has potential application value in the field of fish farming.

[0050] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of increasing the yield of Cupriavidus necator (formerly Ralstonia eutropha) protein, characterized by, Cupriavidus necator ) culturing the bacterium in the presence of a compound that increases the yield of the protein. The method comprises the following steps: step 1, sugar fermentation; step 2, gas culture; and step 3, collecting and filtering the bacterial liquid after setting a fermentation time. Before step 1, the Cupriavidus necator is inoculated into a triangular flask containing a fermentation liquid medium at a volume percentage of 5%, and expanded culture is carried out in a constant temperature shaker at 30 DEG C and 180 rpm for 3-6 days. Step 1 comprises the following steps: S101. Inoculating the Cupriavidus necator into a fermentation tank at a volume percentage of 5%; S102. Adding 21 g / L fructose and 4 mol / L ammonia water; S103. Fermenting at 25-30 DEG C, pH 6.5-7.0 and 200-600 rpm, and passing filtered air at 5.0-10.0 L / min to maintain the dissolved oxygen value at 30%; S104. Fermenting the culture to the maximum bacterial density, and maintaining a sterile environment during the process; Step 2 comprises the following steps: S201. Transferring the fermentation bacterial liquid in step 1 into a gas culture fermentation tower; S202. Culturing at 25-30 DEG C and pH 6.5-7.0; Step 2 further comprises the following steps: adding 3 mol / L ammonia water to maintain the pH of the bacterial liquid at 6.5-7.0, the ammonia water also serving as a nitrogen source, simultaneously supplementing H2 and CO2 mixed gas at a flow rate of 2.5-16.4 SCCM, and installing a pipeline in the gas culture fermentation tower to realize external circulation between the bottom and the top, so that the circulation speed of the bacterial liquid in the pipeline is 300-600 L / min.

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