Method for improving yield of Cupriavidus hookworm protein and application of Cupriavidus hookworm protein

Abstract

The invention provides a method for increasing the yield of Cupriavidus hookworm protein and application, and relates to the technical field of microbial fermentation, the method comprises the following steps: step 1, sugar fermentation; step 2, performing gas culture; and step 3, after the set fermentation time is up, collecting and filtering the bacterial liquid. The obtained single-cell protein is used as a zebra fish feed additive, has no side effect on the growth of zebra fish, and can promote the growth of zebra fish and improve the weight gain rate. The single-cell protein disclosed by the invention has a wide application prospect in the fields of foods and feeds.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and in particular relates to a method for improving the yield of copper-coated hookworm protein and its application. Background Art

[0002] Global population growth is driving increasing demand for protein, while traditional protein sources, such as livestock and fish farming, face resource and environmental challenges. New sustainable protein sources are urgently needed. SCP (Sponge Protein) is a bioprotein produced by fungi, bacteria, and microalgae using a variety of raw materials under suitable culture conditions. As an emerging protein source, SCP offers advantages such as high nutritional value and an environmentally friendly production process, reducing reliance on land and water resources and lowering greenhouse gas emissions.

[0003] Cupriella necator is a green, low-carbon, and sustainable source of SCP. It is an inorganic chemolithoautotrophic bacterium that uses H₂ as an energy source and absorbs O₂ and CO₂ for growth. It plays a key role in carbon cycling and hydrogen bioconversion. Due to its high protein content and excellent protein quality, Cupriella necator has been widely studied for SCP production. Cupriella necator growth is independent of organic matter. It not only autotrophically synthesizes SCP but also absorbs CO₂, playing a positive role in reducing carbon emissions and mitigating the greenhouse effect. It is a green, low-carbon, and sustainable source of SCP. As a protein alternative, it offers advantages such as fast growth, low cultivation costs, and protein production that is unaffected by climate and the environment. However, when cultured in gaseous medium, Cupriella necator's growth rate is not as rapid as when fermented in organic matter, and the protein content in organic fermentation is relatively low.

[0004] Therefore, developing a process that first rapidly increases the number of C. necator bacteria by organic matter fermentation and then enhances its protein content by gas culture is of great significance for improving the efficiency of C. necator bacteria in producing SCP. Summary of the Invention

[0005] The purpose of the present invention is to solve the problems of low density of heterotrophic fermentation bacteria of hookworms, insufficient oxygen transfer efficiency in the autotrophic stage and limited increase in protein content in the above-mentioned prior art, and to provide a method and application for improving the protein yield of hookworms. By first using sugar fermentation to accumulate cell density and then switching to a method using gas continuous fermentation, the single-cell protein content and protein yield of hookworms are significantly improved, and its application effect in feed additives is verified.

[0006] The present invention adopts the following technical solutions: A method for improving the yield of hookworm copper-greedy bacteria protein comprises the following steps: step 1. sugar fermentation; step 2. gas culture; step 3. collecting and filtering the bacterial liquid after the set fermentation time is up.

[0007] Wherein, step 1 comprises: S101. inoculating the hookworm copper bacteria into the fermentation tank in an amount of 5% by volume; S102. Add 21 g / L fructose and 4 mol / L ammonia water; S103. Fermentation was carried out at 25-30°C, pH 6.5-7.0, and 200-600 rpm, with filtered air introduced at 5.0-10.0 L / min to maintain the dissolved oxygen value at 30%, and 40 g / L of carbon source was added every 12 h; S104. Ferment and culture until the bacterial density reaches a maximum value.

[0008] Step 2 comprises: S201. transferring the fermentation broth in step 1 to a gas culture fermentation tower; S202. Cultivate at 25-30°C, pH 6.5-7.0.

[0009] Step 1 also includes: before S101, inoculating the hookworm copper-greedy bacteria into a triangular flask containing fermentation medium at a volume percentage of 5%, and expanding the culture in a constant temperature shaker at 30° C. and 180 rpm for 3-6 days.

[0010] Step 2 also includes installing a pipeline connecting the bottom and top of the gas culture fermentation tower for external circulation, and the liquid circulation speed in the pipeline is 300-600L / min.

[0011] Step 2 also includes adding 3 mol / L ammonia water to maintain the pH of the bacterial solution at 6.5-7.0. The ammonia water also serves as a nitrogen source, and supplementing the mixture of H2 and CO2 at a flow rate of 2.5-16.4 SCCM.

[0012] A method for improving the yield of Cupria obliquus protein is disclosed, wherein the protein powder is prepared by using the method.

[0013] The invention discloses an application of hookworm copper-grazing bacteria protein powder as a fish protein feed additive.

[0014] It also includes that the ratio of hookworm copper-greedy bacteria added to the feed is 1:20.

[0015] Beneficial effects of the present invention: The present invention uses fructose as the sole carbon source to carry out sugar fermentation of hookworm copper bacteria, and then introduces H2 and CO2 for continuous gas culture. After the fermentation is completed, the protein content of the bacterial powder of hookworm copper bacteria is increased from 44.98% in the sugar fermentation stage to 74.70% after combined culture, an increase of 39.79%, and the protein yield is increased from 0.1410g / h / L to 0.2418g / h / L, a significant increase of 71.49%. The protein powder of hookworm copper bacteria is used as a feed additive in zebrafish farming, has no negative impact on zebrafish, and can significantly improve its growth performance. It has significant application value in the field of protein products such as protein feed additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flow chart of the steps of the present invention; Figure 2 Schematic diagram of the sugar fermentation to gas culture stage; Figure 3 This is the result graph of CDW determination during sugar fermentation stage; Figure 4 For the gas culture stage, (a) is the Log colony count; (b) is CDW; Figure 5 This is the GC-MS total ion chromatogram of the volatile components of Cupriphora hookworm; Figure 6 Provide a technical roadmap for adding bacterial protein to feed; Figure 7 The effects of different mass percentages of protein addition on the growth performance of zebrafish, (a) shows the effects of no addition, 5% and 10% addition on the growth rate of zebrafish, (b) shows the effects of no addition, 5% and 10% addition on the weight gain rate of zebrafish, and (c) shows the effects of no addition, 5% and 10% addition on the characteristic growth rate of zebrafish.

[0017] 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-pipeline I, 10-pipeline II. DETAILED DESCRIPTION

[0018] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0019] 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.

[0020] 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.

[0021] Single Cell Protein: Single Cell Protein, abbreviated as SCP.

[0022] 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.

[0023] Table 1 Comparison between heterotrophic and autotrophic stages After the above-mentioned culture method converts organic matter into inorganic matter, the culture does not need 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.

[0024] The specific implementation plan is as follows: During the sugar fermentation stage, the strain of Copperbacterium nematophilum was inoculated into a flask containing fermentation medium at a concentration of 5% (v / v). After expansion culture in a constant temperature shaker at 30°C and 180 rpm for 3-6 days, it was inoculated into the fermentation tank at a concentration of 5% (v / v). Valve III3 was opened to introduce 21 g / L of fructose solution. Valves II2 and IV4 were opened to introduce 4 mol / L of ammonia solution. The pH was maintained at 6.5-7.0. The fermentation tank was equipped with a defoamer. The temperature of the fermentation liquid was maintained at 25-30°C through a cooling water circulation pump or a heating base. Valves I1 and V5 were opened to introduce filtered air. The air volume was maintained at 5.0-10.0 L / min. The stirring speed was set at 200-600 rpm. The dissolved oxygen value was maintained at 30%. 40 g / L of carbon source was added every 12 hours. The results of CDW determination during the sugar fermentation stage of Copperbacterium nematophilum are shown in the table. Figure 3 shown.

[0025] When the density of hookworm copper bacteria reaches the maximum in the sugar fermentation stage, the bacterial liquid is transferred to the gas culture fermentation tower through pipeline Ⅰ9, and valves Ⅲ3, Ⅳ4, and Ⅴ5 of the sugar fermentation stage are closed to enter the gas culture stage. 2 mol / L ammonia water is used as a supplementary nitrogen source, while maintaining the pH at 6.5-7.0. CO2 and H2 are used as gas sources, and the gas flow rate is controlled at 2.5-16.4 SCCM by a gas flow meter. Open valve Ⅰ1, valve Ⅵ6, valve Ⅶ7, and valve Ⅷ8, and pass the mixed gas and ammonia water into the gas culture fermentation tower. Efficient continuous fermentation is carried out under the conditions of 25-30℃ and a liquid circulation rate of 200-600L / min. During the fermentation process, the fermentation liquid is circulated through pipeline Ⅱ10. During the 14-day continuous fermentation cycle, 12.95L was harvested, with an average value of 25.17g / L, a total CDW of 326.36g, and an average yield of 23.31g / d for continuous fermentation. Figure 4 shown.

[0026] Example 2 The collected single-cell protein powder was tested for moisture, crude protein, crude ash, crude fat, crude fiber, 17 amino acids, tryptophan, pepsin digestibility, total phosphorus, and calcium. The results are shown in Table 2. The crude protein content far exceeded the required minimum standard, reaching 74.70%, far exceeding traditional plant protein sources. It also has a high digestibility, making it a very high-quality protein with a low water content, a long shelf life, and better storage performance than other commercially available feed grains. Compared to soybean fish meal, single-cell organisms have a very short reproduction cycle and can quickly provide large quantities of high-quality protein. It can replace fish meal to meet the feed industry's demand for protein sources, with greater economic benefits.

[0027] Table 2 Single cell protein content determination results like Figure 5 As shown in the figure, GC-MS analysis of the flavor substances in Copperworm nectar was performed, and the main substances were acids, esters, aldehydes, ketones, and alkanes. The relative content of each substance is shown in Table 3. The experiment showed that Copperworm nectar provided a higher acid flavor and a lower ketone and aldehyde flavor. Among the volatile flavor substances, acid compounds have a higher threshold value and have less impact on the overall flavor. Among them, the relative content of stearic acid is 3.15%, and the relative content of palmitic acid is 1.93%. Esters contribute more to the flavor, generally showing a long-lasting and rich fruity aroma. Among them, the relative content of 9-octadecenoic acid (2-phenyl-1,3-dioxolane-4) methyl ester is 0.77%, and the relative content of phthalic acid diester is 0.77%. Aldehydes have a fatty aroma and are the main source of meat flavor. The relative content of stearaldehyde is 3.84%, and the relative content of octadecane is 0.54%; among the alkane compounds, the relative content of 1-chlorooctane is 0.7%, and the relative content of 1,1-didecodecyloxyhexadecane is 0.54%; most of the ketone compounds have a high threshold value, which may be the oxides of alcohols or the decomposition products of esters, and also play an important role in the formation of meat aroma, among which the relative content of 3-nonen-2-one is 0.63%, and the relative content of nonadecene-3,5-diamine-ketone is 0.62%.

[0028] Table 3 Contents of various aroma substances in Copperobacterium necrotum By using the single cell protein of hookworm copper bacteria as a feed additive, adding it to the feed at 5% and 10% for feeding fish, the effect of this use is determined. The technical route of adding bacterial protein to feed is as follows: Figure 6 Zebrafish were fed a homemade single-cell protein diet with continuous oxygenation. The fish's swimming patterns were observed regularly over a 28-day experimental period. The weight and length of each zebrafish were measured daily and the data were recorded.

[0029] Depend on Figure 7 As shown in the results, the growth rate, weight gain rate and specific growth rate of the experimental group with added bacterial protein were higher than those of the control group without bacterial protein. The growth rate of the 5% group was 12.4%, the weight gain rate was 41.5%, and the specific growth rate was 1.2%. The growth rate of the 10% group was 12.1%, the weight gain rate was 35.5%, and the specific growth rate was 1.0%. The 5% group was better than the 10% group. Based on this experiment, the addition amount of 5% is better. The results show that the bacterial protein of hookworm copper fungus can be added to the feed to replace part of the protein, has no side effects on the growth of zebrafish, and can promote the growth of zebrafish and increase the weight gain rate. In the field of protein products or feed, especially as a feed additive or prepared into protein feed, it has potential application value in the field of fish farming.

[0030] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for increasing the yield of hookworm copper bacteria protein, characterized in that: The method comprises the following steps: step 1. sugar fermentation; step 2. gas culture; step 3. collecting and filtering the bacterial liquid after the set fermentation time is up; Wherein, step 1 includes: S101. The hookworm greedy copper bacteria were inoculated into the fermentation tank in an amount of 5% by volume; S102. Add 21 g / L fructose and 4 mol / L ammonia water; S103. Fermentation was conducted at 25-30°C, pH 6.5-7.0, and 200-600 rpm with filtered air introduced at 5.0-10.0 L / min to maintain a dissolved oxygen concentration of 30%. S104. Fermentation and cultivation until the bacterial cell density reaches a maximum value; Step 2 includes: S201. The fermentation broth in step 1 is transferred to a gas culture fermentation tower; S202. Cultivate at 25-30°C, pH 6.5-7.

0.

2. The method according to claim 1, wherein: Step 1 also includes: before S101, inoculating the hookworm copper-greedy bacteria into a triangular flask containing fermentation medium at a volume percentage of 5%, and expanding the culture in a constant temperature shaker at 30° C. and 180 rpm for 3-6 days.

3. The method according to claim 1, characterized in that Step 2 also includes: installing a pipe connecting the bottom and top of the gas culture fermentation tower for external circulation, so that the bacterial liquid circulation speed in the pipe is 300-600L / min.

4. The method according to claim 1, wherein Step 2 also includes: adding 3 mol / L ammonia water to maintain the pH of the bacterial solution at 6.5-7.0, and the ammonia water also serves as a nitrogen source, while supplementing a mixed gas of H2 and CO2 with a flow rate of 2.5-16.

4.

5. The hookworm greedy copper bacteria protein powder obtained according to the method described in any one of claims 1 to 4.

6. An application of the hookworm copper fungus protein powder as claimed in claim 5 as a fish protein feed additive.

7. The use according to claim 6, characterized in that The ratio of hookworm copper-greedy bacteria added to the feed is 1:20.

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