Process for the production of high selenium content selenomethionine product from saccharomyces marxianus

Abstract

The application provides a method for preparing a high-selenium selenomethionine product by fermenting a Kluyveromyces marxianus, which comprises the following steps: inoculating a Kluyveromyces marxianus strain domesticated by inorganic selenium, placing the strain in a substrate medium with a specific composition to carry out primary fermentation culture, using a feed solution with a specific composition to carry out primary feed culture, and then adding a specific amount of glutathione to carry out secondary feed culture. The fermentation technical route can obtain a high-selenium selenomethionine product liquid and an organic selenium product.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing high-selenomethionine products by fermentation of Kluyveromyces martensii. Background Technology

[0002] Organic selenium generally exists in the form of selenomethionine, which is metabolized along the methionine metabolic pathway and participates in protein synthesis. It is easily stored and absorbed in tissues and can be rapidly utilized by the human body, effectively improving selenium deficiency. Compared to inorganic selenium, consuming organic selenium foods is safer and more effective, and easier for the body to absorb. As a feed additive for poultry and livestock, selenomethionine is characterized by improving the quality of poultry and livestock products, enhancing animal reproductive capacity, improving immunity, high absorption rate, and strong biological activity.

[0003] Currently, microbial fermentation is a method for preparing yeast-derived organic selenium products containing selenomethionine, but the selenomethionine content in the products obtained by current fermentation methods is relatively low. Summary of the Invention

[0004] Therefore, it is necessary to provide a method for preparing high-selenomethionine products by fermentation of *Kluyveromyces martensii*.

[0005] The present invention adopts the following technical solution:

[0006] This invention discloses a method for preparing a high-selenomethionine product by fermentation of *Kluyveromyces martensii*, comprising the following steps: *Kluyveromyces martensii* strain domesticated with inorganic selenium is inoculated into a substrate culture medium containing: 1–5 wt% glucose, 0.5–2 wt% magnesium phosphate, 1.5–2.5 wt‰ calcium chloride, 4–6 wt‰ potassium sulfate, 6–10 wt‰ ammonium phosphate, 0.1 vol‰–0.5 vol‰ trace elements, 1–3 wt‰ sodium selenite, 3–6 mL defoaming oil, and the remainder water, for primary aerobic fermentation; a first-stage fed-batch culture is then performed, wherein the first-stage feed solution is a glucose solution; a second-stage fed-batch culture is then performed, wherein the second-stage feed is a fed-batch operation, and the fed-batch component includes glutathione, and the amount of glutathione added to the fermentation broth must be controlled to 0.5–5 wt‰; fermentation is then terminated to obtain a product liquid containing selenomethionine.

[0007] In some embodiments, the primary aerobic fermentation process conditions are: temperature 30°C, pH 5.0, and dissolved oxygen controlled at 5%–35%.

[0008] Preferably, the substrate culture medium comprises: 3 wt% glucose, 1 wt% magnesium phosphate, 2 wt‰ calcium chloride, 5 wt‰ potassium sulfate, 8 wt‰ ammonium phosphate, 0.3 vol% trace elements, 1-3 wt‰ sodium selenite, 5 mL defoaming oil, and the remainder water.

[0009] Preferably, the primary feed solution contains an aqueous solution of 50 wt% glucose and 0.1 wt% biotin.

[0010] Once the glucose in the substrate culture medium is depleted and dissolved oxygen rebounds, glucose solution is continuously added for feeding until the secondary feeding culture is completed.

[0011] Preferably, the amount of glutathione added in the secondary fed-batch culture is controlled to be 2.5 wt‰ of the total fermentation broth. The feeding time of glutathione during the secondary fed-batch culture is controlled to be 1–11 h.

[0012] More preferably, the feeding time of glutathione during the secondary fed culture process is controlled to be 10 hours.

[0013] In some embodiments, the method for preparing high-selenomethionine products by fermentation of *Kluyveromyces maculae* further includes a step of sulfidation and granulation of the selenomethionine-containing product liquid.

[0014] The present invention can also provide a product of high selenomethionine content obtained by the above method.

[0015] Compared with the prior art, the core advantage of this invention is:

[0016] The preparation method of this invention uses a strain of Kluyveromyces roxburghii that has been domesticated with inorganic selenium as an inoculator, which is placed in a substrate culture medium with a specific composition for primary fermentation culture, followed by a fed-batch culture with a specific composition of feed solution, and then a secondary fed-batch culture with glutathione added. Overall, a product liquid with high selenomethionine content and an organic selenium product can be obtained. Detailed Implementation

[0017] The technical concept of this invention lies in providing a method for preparing a high-selenomethionine product using Kluyveromyces martensii fermentation, comprising the following steps:

[0018] 1) Strain selection: Select the strain of Kluyveromyces roxburghii that has been domesticated with inorganic selenium, and culture it in shake flasks using YPD medium. After growing for 24 hours, it is inoculated into a fermenter.

[0019] 2) Primary fermentation culture:

[0020] Primary fermentation was carried out using a substrate culture medium, with the fermentation temperature controlled at 30℃ and the fermentation pH controlled at 5.0. In the early stage of fermentation, the fan speed was increased based on dissolved oxygen, with a maximum speed of 500 rpm / min and a maximum air volume of 3.5 m³ / h.

[0021] The preferred substrate culture medium comprises: 1-5 wt% glucose, 0.5-2 wt% magnesium phosphate, 1.5-2.5 wt‰ calcium chloride, 4-6 wt‰ potassium sulfate, 6-10 wt‰ ammonium phosphate, 0.1 vol‰-0.5 vol‰ trace elements, 1-3 wt‰ sodium selenite, 3-6 mL defoaming oil, and the remainder water.

[0022] 3) Primary fed culture:

[0023] Under full ventilation and rotation conditions, after 14-16 hours of primary fermentation, when dissolved oxygen drops to its lowest point and begins to rebound, glucose feed solution is added continuously until the subsequent secondary feed culture is completed.

[0024] Preferably, the glucose feed solution is composed of 50 wt% glucose and 0.1 wt% D-biotin.

[0025] 4) Secondary fed culture:

[0026] Once the Kluyveromyces maculae is in the exponential growth phase, a secondary fed-batch culture is carried out using fed-batch glutathione. The amount of glutathione added to the fermentation broth is controlled to be 0.5-5 wt‰, and the secondary fed-batch culture time is controlled to be about 1-11 hours. Fermentation is then completed.

[0027] 5) Detect the content of selenomethionine in the product.

[0028] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0029] The method for obtaining the domesticated strain of *Kluyveromyces martensii* is as follows:

[0030] Kluyveromyces martensii (strain deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 10621) was inoculated onto culture plates containing 1 wt% sodium selenite. Three culture plates were selected, each containing 1 ml of sodium selenite solution, and incubated at 30°C for 24 h. The strain from the culture plate with better cell size and morphology was selected for subculturing. Each time, three culture plates containing 1 wt% sodium selenite were selected, and subculturing was carried out sequentially for 7 days. Finally, the best one of the three plates was selected as the domesticated selenium yeast strain as the Kluyveromyces martensii domesticated strain for subsequent experiments.

[0031] Example 1

[0032] This embodiment provides a method for preparing selenomethionine products using Kluyveromyces martensii fermentation, including the following steps:

[0033] S1, the domesticated strain of *Kluyveromyces roxburghii* was cultured in shake flasks using YPD medium and grown for 24 hours before being inoculated into a fermenter.

[0034] S2 uses a base culture medium for primary fermentation. The fermentation temperature is controlled at 30℃, the fermentation pH is controlled at 5.0, and the fan and turbine start when the dissolved oxygen level is below 30%. The maximum speed is 500 rpm / min and the maximum air volume is 3.5 m³ / h.

[0035] In this embodiment, the composition of the substrate culture medium includes: 3wt% glucose, 1wt% magnesium phosphate, 2wt‰ calcium chloride, 5wt‰ potassium sulfate, 8wt‰ ammonium phosphate, 0.3vol‰ trace elements (including manganese chloride, cobalt chloride, copper sulfate, boric acid, and potassium iodide), 2wt‰ sodium selenite, 5ml defoaming oil, and the remainder water.

[0036] S3, primary fed culture:

[0037] Under full ventilation and full rotation, after primary fermentation for 14-16 hours, when dissolved oxygen drops to its lowest point and begins to rebound, glucose feed solution is added to control dissolved oxygen between 5% and 35% for primary fed culture, and glucose is added continuously until the subsequent secondary fed culture is completed.

[0038] In this embodiment, the glucose feed solution consists of 50 wt% glucose, 0.1 wt% D-biotin, and the remainder water.

[0039] S4, secondary fed culture:

[0040] Fermentation was carried out for approximately 25 hours. When the wet weight of the fermentation broth reached 155 g / kg and the *Kluyveromyces martensii* was in the exponential growth phase, glutathione was added for 10 hours, with the amount added controlled to 0.5‰. After approximately 35 hours of fermentation, when the wet weight of the fermentation broth stopped increasing, secondary fed-batch fermentation was stopped. Fermentation was then completed, yielding the fermentation product broth.

[0041] S5. After the fermentation product liquid is filtered through a plate and frame filter, it is granulated by fluidized bed to obtain organic selenium product.

[0042] S6, according to the method specified in Appendix A of the People's Republic of China Supply and Marketing Cooperative Industry Standard GH / T 1135-2017, the content of selenomethionine in its organic selenium product was found to be 5616 ppm.

[0043] Example 2

[0044] This embodiment provides a method for preparing selenomethionine products using Kluyveromyces martensii fermentation. The preparation process steps are basically the same as those in Example 1, with the only difference being:

[0045] In the secondary fed-batch culture of step S4: after fermentation for approximately 25 hours, when the wet weight of the fermentation broth reaches 160 g / kg and the *Kluyveromyces martensii* is in the exponential growth phase, glutathione is added for 10 hours, controlling the amount of glutathione added to the fermentation broth to 2.5‰. After fermentation for approximately 35 hours, when the wet weight of the fermentation broth no longer increases, the secondary fed-batch fermentation is stopped.

[0046] In step S6, the content of selenomethionine in its organic selenium product was measured to be 7164 ppm.

[0047] Example 3

[0048] This embodiment provides a method for preparing selenomethionine products using Kluyveromyces martensii fermentation. The preparation process steps are basically the same as those in Example 1, with the only difference being:

[0049] In the secondary fed-batch culture of step S4: after fermentation for approximately 25 hours, when the wet weight of the fermentation broth reaches 162 g / kg and the *Kluyveromyces martensii* is in the exponential growth phase, glutathione is added for 10 hours, controlling the amount of glutathione added to the fermentation broth to 5 wt‰. After fermentation for approximately 35 hours, when the wet weight of the fermentation broth no longer increases, the secondary fed-batch fermentation is stopped.

[0050] In step S6, the content of selenomethionine in its organic selenium product was measured to be 6282 ppm.

[0051] Example 4

[0052] This embodiment provides a method for preparing selenomethionine products using Kluyveromyces martensii fermentation. The preparation process steps are basically the same as those in Example 2, with the only difference being:

[0053] In the secondary fed culture of step S4: the amount of glutathione added to the fermentation broth was controlled at 2.5 wt‰, and fed for 2 hours.

[0054] In step S6, the content of selenomethionine in its organic selenium product was measured to be 7128 ppm.

[0055] Example 5

[0056] This embodiment provides a method for preparing selenomethionine products using *Kluyveromyces martensii* fermentation. The preparation process steps are basically the same as in Example 3, with the only difference being:

[0057] In the secondary fed culture in step S4: the amount of glutathione added to the fermentation broth was controlled at 5 wt‰, and fed for 2 hours;

[0058] In step S6, the content of selenomethionine in its organic selenium product was measured to be 6444 ppm.

[0059] Comparative Example 1

[0060] This comparative example provides a method for preparing selenomethionine products using *Kluyveromyces martensii* fermentation. The preparation process steps are basically the same as in Example 1, with the only difference being:

[0061] The S4 step of adding glutathione in the secondary fed culture is cancelled, and fermentation is stopped directly when the wet weight of the fermentation broth no longer increases.

[0062] In step S6, the content of selenomethionine in its organic selenium product was measured to be 2074 ppm.

[0063] Comparative Example 2

[0064] This comparative example provides a method for preparing selenomethionine products using *Kluyveromyces martensii* fermentation. The preparation process steps are basically the same as in Example 1, with the only difference being:

[0065] In the secondary fed culture in step S4: when the wet weight of the fermentation broth reaches 155 g / kg, the amount of glutathione added to the fermentation broth is controlled to 0.25‰, and fed for 10 h.

[0066] In step S6, the content of selenomethionine in its organic selenium product was measured to be 3653 ppm.

[0067] Comparative Example 3

[0068] This comparative example provides a method for preparing selenomethionine products using *Kluyveromyces martensii* fermentation. The preparation process steps are basically the same as in Example 1, with the only difference being:

[0069] In the secondary fed culture in step S4: when the wet weight of the fermentation broth reaches 155 g / kg, the amount of glutathione added to the fermentation broth is controlled to 10 wt‰, and fed for 10 h.

[0070] In step S6, the content of selenomethionine in its organic selenium product was measured to be 3125 ppm.

[0071] Comparative Example 4

[0072] This comparative example provides a method for preparing selenomethionine products using *Kluyveromyces martensii* fermentation. The preparation process steps are basically the same as in Example 2, with the only difference being:

[0073] In the secondary fed culture of step S4: when the wet weight of the fermentation broth reaches 155 g / kg, control the amount of glutathione added to the fermentation broth to 2.5 wt‰ and add glutathione all at once.

[0074] In step S6, the content of selenomethionine in its organic selenium product was measured to be 2065 ppm.

[0075] The following is a summary list of key parameters and selenomethionine in organic selenium products:

[0076] Example 1 0.5wt‰ 10 5616 Example 2 2.5wt‰ 10 7164 Example 3 5wt‰ 10 6282 Example 4 2.5wt‰ 2 7128 Example 5 5wt‰ 2 6444 Comparative Example 1 0wt‰ 0 2074 Comparative Example 2 0.25wt‰ 10 3653 Comparative Example 3 10wt‰ 10 3125 Comparative Example 4 2.5wt‰ 0 2065

[0077] As can be seen from the table above, the feeding method of glutathione and its proportion in the fermentation broth during the secondary fed culture in the above experiment have a significant impact on the selenomethionine in the final organic selenium product.

[0078] In particular, when the amount of glutathione added in the secondary feed is controlled to 2.5‰, and the feeding time is controlled to 2-10 hours, the selenomethionine content in the organic selenium product can be guaranteed to reach more than 7000ppm.

[0079] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a high-selenomethionine product by fermentation of *Kluyveromyces martensii*, characterized in that, Includes the following steps: A strain of *Kluyveromyces martensii* domesticated with inorganic selenium was inoculated into a substrate culture medium containing: 1-5 wt% glucose, 0.5-2 wt% magnesium phosphate, 1.5-2.5 wt‰ calcium chloride, 4-6 wt‰ potassium sulfate, 6-10 wt‰ ammonium phosphate, 0.1 vol‰-0.5 vol‰ trace elements, 1-3 wt‰ sodium selenite, 3-6 mL defoaming oil, and the remainder water, for primary aerobic fermentation. Alternatively, the substrate culture medium could contain: 3 wt% glucose, 1 wt% magnesium phosphate, 2 wt‰ calcium chloride, 5 wt‰ potassium sulfate, 8 wt‰ ammonium phosphate, 1-3 wt‰ sodium selenite, 5 mL defoaming oil, and the remainder water. Primary fed culture was carried out, wherein the primary fed solution was an aqueous solution containing 50 wt% glucose and 0.1 wt‰ biotin; Then, a secondary fed-batch culture is carried out. The secondary fed-batch culture adopts a fed-batch operation, and the fed-batch component includes glutathione. The amount of glutathione added in the fermentation broth needs to be controlled to 0.5~5wt‰. The feeding time of glutathione during the secondary fed-batch culture is controlled to be 2~10h. After fermentation is completed, a product liquid containing selenomethionine is obtained. The method for obtaining the domesticated strain of *Kluyveromyces martensii* is as follows: Kluyveromyces martensii, deposited at the China General Microbiological Culture Collection Center (CGMCC No. 10621), was inoculated onto culture plates containing 1 wt% sodium selenite. Three culture plates were selected, each containing 1 ml of sodium selenite solution, and incubated at 30°C for 24 hours. The strain from the culture plate with the best size and morphology was selected for subculturing. Each time, three culture plates containing 1 wt% sodium selenite were selected, and subculturing was carried out sequentially for 7 days. Finally, the best one of the three plates was selected as the domesticated selenium yeast strain as the domesticated Kluyveromyces martensii strain.

2. The method for preparing high-selenomethionine products by fermentation of *Kluyveromyces martensii* according to claim 1, characterized in that, The process conditions for the primary aerobic fermentation are: temperature 30℃, pH value 5, and dissolved oxygen controlled at 5%~35%.

3. The method for preparing high-selenomethionine products by fermentation of *Kluyveromyces martensii* according to claim 1, characterized in that, Once the glucose in the substrate culture medium is depleted and dissolved oxygen rebounds, glucose solution is continuously added for feeding until the secondary feeding culture is completed.

4. The method for preparing high-selenomethionine products by fermentation of *Kluyveromyces martensii* according to claim 1, characterized in that, In the secondary fed culture, the amount of glutathione added is controlled to be 2.5~5wt‰ of the total fermentation broth.

5. The method for preparing high-selenomethionine products by fermentation of *Kluyveromyces martensii* according to claim 1, characterized in that, The feeding time of glutathione during the secondary fed culture process was controlled to be 10 hours.

6. The method for preparing high-selenomethionine products by fermentation of *Kluyveromyces martensii* according to claim 1 or 2, characterized in that, It also includes the step of sulfiding and granulating the product containing selenomethionine.