Use of vitamin d2 in preparing a heat-resistant protective agent for fermentation industry

Abstract

This application discloses the application of vitamin D2 in the preparation of a heat-resistant protective agent for the fermentation industry. This application's research found that VD2 treatment can significantly improve the performance of *Saccharomyces cerevisiae* under heat stress conditions. S. yeast This invention improves the growth status of *Saccharomyces cerevisiae*, significantly increases glutathione peroxidase (GPx) activity, and reduces cell membrane damage. It boasts advantages such as low cost, high safety, simple operation, and wide applicability, and can be widely applied to high-temperature fermentation systems of *Saccharomyces cerevisiae* in the food and pharmaceutical industries. It effectively solves the problem of high-temperature stress in industrial fermentation and has significant industrial application value.

Description

Technical Field

[0001] This application relates to the field of microbial fermentation technology, specifically to the application of vitamin D2 in the preparation of a heat-resistant protective agent for fermentation industry. Background Technology

[0002] brewing yeast ( Saccharomyces cerevisiae Due to its combination of safety and efficient metabolic capabilities, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) is widely used in core industrial fields such as food brewing, bioenergy production, and biopharmaceutical synthesis, making it a core production strain in the industrial biotechnology industry. However, during large-scale fermentation, the intense metabolic heat production of microorganisms and process requirements can easily cause the system temperature to rise above 38°C, creating a high-temperature stress environment. High temperatures damage *Saccharomyces cerevisiae* in multiple ways: disrupting cell membrane fluidity and integrity, leading to the loss of core intracellular substances; disrupting lipid metabolism homeostasis, weakening cell membrane barrier function; inducing the accumulation of reactive oxygen species (ROS), oxidizing and damaging biomolecules and dysregulating the expression of heat shock response genes, ultimately inhibiting cell proliferation. Statistics show that high-temperature stress can reduce *Saccharomyces cerevisiae* biomass by 10%-30% and fermentation product yield by 15%-40%, severely restricting the efficiency and economics of industrial fermentation and becoming a key technological bottleneck for the development of related industries.

[0003] Existing methods for improving yeast's heat tolerance mainly fall into two categories, both with significant drawbacks. One category involves genetically engineered strains that enhance tolerance by optimizing the expression of stress-related genes. However, this approach is complex, costly to develop, and its application in food and pharmaceutical fields is limited due to biosafety controversies surrounding transgenic strains. The other category involves adding exogenous heat protectants, which has become the mainstream industrial practice due to its simplicity and high safety. However, existing protectants have significant shortcomings: peptides require complex extraction and purification processes, are costly, and their protective effect is greatly affected by fermentation conditions; amino acids have limited functions, only alleviating specific heat damage, and their concentration must be strictly controlled; polysaccharides have poor solubility and uneven dispersion, resulting in limited repair effects on cell membrane damage. Furthermore, most existing protectants are complex systems with unclear component interaction mechanisms, which can easily affect the purity of fermentation products, limiting their widespread application.

[0004] Therefore, the development of novel heat protection agents with simple composition, low cost, high safety, and stable protective effect has become an urgent industrial need. Summary of the Invention

[0005] To address the shortcomings of existing heat protectants, such as inhibited growth and low fermentation efficiency caused by high temperature stress during high-temperature fermentation of brewing yeast, and the complex composition, high cost, and single function of existing heat protectants, this application provides an application of vitamin D2 in the preparation of heat protectants for fermentation industry. The application of vitamin D2 as a heat protectant for brewing yeast can effectively improve the high temperature tolerance of brewing yeast and ensure fermentation efficiency and stability.

[0006] Vitamin D2 (VD2), as a natural active substance, is widely available and inexpensive to prepare, far lower than existing peptide and polysaccharide heat protectants. It is listed as a food additive, has a well-established biosafety profile, and is suitable for various demanding fermentation environments. Furthermore, it shares structural homology with sterols, a core component of the Saccharomyces cerevisiae cell membrane, and theoretically possesses the function of regulating membrane stability. Currently, VD2 applications are concentrated in food fortification and pharmaceuticals; there are no reports or patents disclosing its use as a heat protectant for Saccharomyces cerevisiae, nor are there reports on its role and mechanism in enhancing yeast's high-temperature tolerance. This application develops a new application for VD2, which can overcome the deficiencies of existing technologies, providing a new path to solve the problem of high-temperature stress in industrial fermentation, while expanding the industrial value of VD2, and has significant theoretical and industrial application prospects.

[0007] Based on this, this application provides an application of vitamin D2 in the preparation of a heat-resistant protective agent for the fermentation industry.

[0008] Optionally, the fermentation industry is a fermentation industry using brewer's yeast.

[0009] Furthermore, the brewing yeast is Saccharomyces cerevisiae CEN.PK2-1C strain.

[0010] This application also provides the application of vitamin D2 as a heat-resistant protectant in the fermentation industry using brewer's yeast as a starter culture.

[0011] Optionally, the application includes adding VD2 to the fermentation material at the initial stage of fermentation.

[0012] Optionally, the initial fermentation stage refers to the period from 0 to 96 hours after the start of fermentation.

[0013] Optionally, VD2 can be added to the culture medium for expanding the culture of Saccharomyces cerevisiae, and then added together with Saccharomyces cerevisiae to the material to be fermented.

[0014] Optionally, the amount of vitamin D2 added is calculated based on its concentration in the material to be fermented being 10-100 mg / L. Further, the amount of vitamin D2 added is calculated based on its concentration in the material to be fermented being 10 mg / L or 100 mg / L. Even further, the amount of vitamin D2 added is calculated based on its concentration in the material to be fermented being 100 mg / L.

[0015] This application also provides the use of vitamin D2 in the preparation of a heat-resistant protectant for brewer's yeast.

[0016] This application discovers that adding VD2 to the fermentation medium of Saccharomyces cerevisiae at a final concentration of 10-100 mg / L (preferably 100 mg / L) can significantly enhance the fermentation performance of Saccharomyces cerevisiae (preferably). Saccharomyces cerevisiaeThe tolerance of CEN.PK2-1C strain to high temperatures of 40°C was investigated. The core mechanism involved VD2 regulating yeast lipid metabolism (promoting an increase in GL and inhibiting a decrease in GP), enhancing glutathione peroxidase activity, and protecting cell membrane integrity.

[0017] This application also provides a method for fermenting and culturing Saccharomyces cerevisiae under high temperature stress, including: After activating the Saccharomyces cerevisiae strain, single colonies were picked, inoculated into fresh liquid fermentation medium, and cultured. Cells were then collected by centrifugation. The collected cells were resuspended in fresh liquid fermentation medium, and vitamin D2 was added to the liquid fermentation medium at a final concentration of 10-100 mg / L. Fermentation was carried out at 38-45℃.

[0018] Optionally, the fermentation process can be carried out at 150–250 r·min. -1 Fluctuation.

[0019] Optionally, fermentation culture can be carried out at 40°C.

[0020] Compared with the prior art, this application has at least one of the following beneficial effects: (1) It was innovatively discovered that VD2 has significant heat protection activity for Saccharomyces cerevisiae: VD2 can significantly increase the growth rate of Saccharomyces cerevisiae under high temperature stress (OD600 value is significantly higher than that of the high temperature group), enhance the activity of glutathione peroxidase (GPx), reduce cell membrane damage, effectively solve the problem of high temperature inhibiting the growth of Saccharomyces cerevisiae, and has more advantages than existing single-function heat protectants.

[0021] (2) The mechanism of action is clear and efficient: VD2 can regulate lipid metabolism, which is manifested by promoting the increase of GL (glycerides) and the decrease of GP (glycerophospholipids), thereby improving the high temperature tolerance of Saccharomyces cerevisiae and providing stable and reliable heat protection.

[0022] (3) Low cost and high safety: VD2 has a wide range of sources and its preparation cost is lower than that of polypeptide complex regulators. It has been proven to have good biological safety and can be widely used in the fermentation of brewing yeast in food, medicine and other fields with high safety requirements. It is easy to promote industrialization.

[0023] (4) Simple operation and wide applicability: There is no need to genetically modify the brewing yeast. Just add an appropriate amount of single component VD2 to the fermentation medium to achieve heat protection of the brewing yeast. The operation process is simple and can be adapted to brewing yeast high-temperature fermentation systems of different scales. It is more practical than compound protectants. Attached Figure Description

[0024] Figure 1 This is a growth curve diagram of different groups of brewer's yeast in Example 2.

[0025] Figure 2 This is a comparison of SEM morphology of different groups of Saccharomyces cerevisiae cells in Example 3.

[0026] Figure 3 The bar chart shows the activity of different groups of Saccharomyces cerevisiae GPx in Example 4.

[0027] Figure 4 This is a comparison diagram of the lipid composition of different groups of Saccharomyces cerevisiae in Example 5.

[0028] Figure 5 VD2 and yeast in Example 6 IP3 Image showing the docking results of the encoded protein molecule. Detailed Implementation

[0029] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0031] In the following embodiments: The Saccharomyces cerevisiae strain (CEN.PK2-1C) was purchased from Wuhan Ruizhi Magic Cube Biotechnology Co., Ltd.

[0032] YPD solid culture medium, consisting of 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone and 20 g / L agar, was purchased from Nanjing Jiancheng Biotechnology Institute.

[0033] Except for the absence of agar, the components of YPD liquid medium are the same as those of YPD solid medium.

[0034] VD2 was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0035] Example 1 (1) Activation of strain: The Saccharomyces cerevisiae strain was revived from the glycerol inoculum at -80℃, inoculated into YPD solid medium, and cultured at 28℃ for 4-5 days; (2) Pre-culture: Pick a single colony and inoculate it into YPD liquid medium, incubate at 28℃ and 200 r·min -1After oscillating to the logarithmic growth phase, at 8000 r·min -1 Cells were collected by centrifugation; (3) Group fermentation: Resuspend cells in YPD liquid medium and adjust initial OD 600 The value was 0.2, and the cells were divided into 4 groups, each with a 50 mL culture system. The specific groupings are as follows: Blank control group: No additives were added, and the mixture was placed at 28℃ and 200 r·min. -1 Shaking culture; High-temperature group: No additives, placed at 40℃ and 200 r·min -1 Shaking culture; HT+VD2 low concentration treatment group (VD2-10): Vitamin D2 was added to a final concentration of 10 mg / L and placed at 40℃ with an incubator at 200 r·min. -1 Shaking culture; HT+VD2 high concentration treatment group (VD2-100): Vitamin D2 was added to a final concentration of 100 mg / L and placed at 40℃ at 200 r·min -1 Shaking culture.

[0036] (4) Monitoring and collection: After culturing for 96 hours, samples were taken every 12 hours and the OD was measured using a UV spectrophotometer. 600 Value, collect cells after fermentation.

[0037] Example 2: Verification of the promoting effect of VD2 on the growth of Saccharomyces cerevisiae under high temperature stress This example was used to verify the promoting effect of vitamin D2 (VD2) on the growth of Saccharomyces cerevisiae under high temperature stress. The experimental procedure and grouping were the same as in Example 1. During the culture process, the optical density (OD) at 600 nm was measured using a UV spectrophotometer at 0, 12, 24, 36, 48, 60, 72, 84, and 96 hours of culture. 600 ), plot growth curves and analyze yeast growth rates.

[0038] The effect of VD2 on yeast growth under high temperature stress is as follows: Figure 1 As shown in the figure, the results indicate that the yeast in the blank control group (control) showed better growth, with an OD of 60 hours. 600 The value reached 11.16, and the OD600 value stabilized at 10.19 after 96 hours; the growth of the high-temperature group (HT) was severely inhibited, with the OD600 value decreasing after 60 hours. 600 The value was only 8.24; the 48-hour OD of the HT+VD2 (10 mg / L) group (VD2-10) was... 600 The value reached 11.16, with a 96-hour OD. 600The 48-hour OD600 value of the HT+VD2 (100 mg / L) group (VD2-100) reached 12.89, and the 96-hour OD600 value reached 14.36; 600 The value reached 12.76, which was significantly higher than that of the high-temperature group during the same period in both groups.

[0039] The above experimental results show that VD2 can significantly promote the growth of Saccharomyces cerevisiae under high temperature stress, and this promoting effect can last up to 96 hours; among them, the 100 mg / L concentration group has a better promoting effect within 84 hours, and the 10 mg / L concentration group has a better promoting effect at 96 hours.

[0040] Example 3: Verification of the protective effect of VD2 on the cell membrane structure of Saccharomyces cerevisiae under high temperature stress This embodiment was used to investigate the protective effect of VD2 on the cell membrane structure of Saccharomyces cerevisiae under high temperature stress. The experimental procedure and grouping were the same as in Example 1. The cells were cultured for 48 hours, and sample preparation was performed after the culture was completed. The specific steps are as follows: Measure 2 mL of bacterial culture from each of the following groups: blank control group, high-temperature group, HT+VD2 (10 mg / L) group, and HT+VD2 (100 mg / L) group, and place them into centrifuge tubes. Centrifuge the tubes at 10,000 rpm for 5 minutes at 4°C. After centrifugation, discard the supernatant from each tube. Add 200 μL of 2.5% glutaraldehyde to the bacterial cells and fix at 4°C for 12 hours. After fixation, carefully remove the glutaraldehyde using a pipette. Elute each group of bacteria twice with ultrapure water, centrifuging at 10,000 rpm for 15 minutes after each elution. Then, sequentially elute with 200 μL of 2.5% glutaraldehyde at concentrations of 30%, 50%, 70%, 90%, and 100%. Gradient elution of each group of bacteria was performed using μL of ethanol. After each elution, the cells were centrifuged at 10,000 rpm and 4°C for 15 minutes. The remaining liquid in the bacterial solution was removed and discarded. The bacterial cells were then removed from the centrifuge tubes and placed in an oven to dry at 30°C. The dried samples were fixed on the sample stage, sputtered with gold, and then observed and photographed under a scanning electron microscope (SEM) (accelerating voltage 10 kV) to analyze the integrity and morphological differences of the cell membranes in each group.

[0041] The results are as follows Figure 2 As shown, Figure 2 The differences in yeast cell morphology and cell membrane integrity among the groups were shown, demonstrating the protective effect of VD2 on yeast cell membrane structure. In the figure, a is the blank control group, b is the high temperature group, c is the HT+VD2 (10 mg / L) group, and d is the HT+VD2 (100 mg / L) group.

[0042] Depend on Figure 2The results showed that in the blank control group (a), the yeast cells had regular morphology, smooth surface, and intact cell membrane without damage; in the high temperature group (b), the yeast cells showed obvious shrinkage and deformation, and the cell membrane surface was rough with multiple breaks and depressions; in the HT+VD2 (10 mg / L) group (c), the yeast cells showed reduced shrinkage and fewer cell membrane breaks; and in the HT+VD2 (100 mg / L) group (d), the yeast cells had basically regular morphology, relatively smooth surface, good cell membrane integrity, and only a few slight depressions.

[0043] The above experimental results show that VD2 can effectively protect the cell membrane structure integrity of Saccharomyces cerevisiae under high temperature stress and reduce cell membrane damage caused by high temperature.

[0044] Example 4: Verification of the effect of VD2 on the GPx activity of Saccharomyces cerevisiae under high temperature stress This embodiment was used to detect the effect of VD2 on the activity of glutathione peroxidase (GPx) in Saccharomyces cerevisiae cells under high temperature stress. The experimental materials were based on those in Example 1, with the addition of a GPx activity detection kit.

[0045] The experimental procedure and grouping were the same as in Example 1. The culture was carried out for 48 hours. After the culture was completed, sample preparation was performed, and the specific steps were as follows: After culture, cells from each group were collected, washed three times with sterile physiological saline, and lysed in an ice bath for 30 minutes with lysis buffer. The lysis buffer was then centrifuged at 10,000 rpm and 4°C for 15 minutes, and the supernatant was collected as the crude cell extract. The GPx activity of each group of crude cell extracts was measured according to the instructions of the GPx activity assay kit. The results are expressed as nmol / min / mL (nmol / min is the number of nanomoles of substrate converted per minute, and mL is the volume of the sample being tested).

[0046] The results are as follows Figure 3 The results show the effect of VD2 on the antioxidant capacity of yeast. The GPx activity in the blank control group (control) was 1749.25 nmol / min / mL; the GPx activity in the high-temperature (HT) group of *Saccharomyces cerevisiae* was only 1342.96 nmol / min / mL; the GPx activity in the HT+VD2 (10 mg / L) group reached 1623.58 nmol / min / mL; and the GPx activity in the HT+VD2 (100 mg / L) group reached 1802.41 nmol / min / mL. Figure 3 The VD2 group shown in the figure is the VD2-100 group. Both groups were significantly higher than the high temperature group, indicating that VD2 can significantly enhance the GPx activity of Saccharomyces cerevisiae under high temperature stress and enhance the antioxidant capacity of cells.

[0047] Example 5: Verification of the effect of VD2 on the lipid composition of Saccharomyces cerevisiae under high temperature stress This example investigates the regulatory effect of vitamin D2 on the lipid composition of Saccharomyces cerevisiae under high-temperature stress, focusing on the changes in GL (glycerol esters) and GP (glycerophospholipids) under the influence of vitamin D2. In addition to the materials used in Example 1, lipidomics analysis reagents and a liquid chromatography-mass spectrometry (LC-MS / MS) instrument were added. The experimental procedure and grouping were the same as in Example 1. The culture was carried out for 48 hours, and sample preparation was performed after the culture period. The specific steps are as follows: After culture, cells from each group were collected, freeze-dried, and then extracted with extraction buffer (chloroform:methanol = 2:1, v / v) by sonication. The supernatant was collected by centrifugation, dried under nitrogen, and reconstituted with methanol. Lipidomics analysis was performed by LC-MS / MS to detect changes in the content of GL (glycerol esters) and GP (glycerophospholipids).

[0048] The results are as follows Figure 4 As shown, the horizontal axis represents the experimental groups, and the vertical axis represents the relative lipid content (%), demonstrating the regulatory effect of VD2 on yeast lipid metabolism. The results showed that compared with the high-temperature group (HT), the HT+VD2 (100 mg / L) group had a significantly increased GL content and a significantly decreased GP content; the HT+VD2 (10 mg / L) group also showed a similar regulatory trend, but the effect was weaker than that of the 100 mg / L concentration group. Figure 4 The VD2 group shown is the VD2-100 group.

[0049] The above results indicate that VD2 can enhance heat tolerance by regulating the lipid composition of Saccharomyces cerevisiae, thereby promoting an increase in GL, a decrease in GP, ​​and improving cell membrane stability.

[0050] Example 6: Investigation of potential targets and mechanisms of VD2 action in Saccharomyces cerevisiae (molecular docking verification) This embodiment uses molecular docking technology to predict potential target proteins of VD2 acting on Saccharomyces cerevisiae, and preliminarily elucidates the molecular mechanism of its thermoprotective effect. The three-dimensional structure of VD2 used in the experiment was obtained using KingDraw_v5.0 software; the candidate target protein of Saccharomyces cerevisiae was selected as... IP3 The encoded protein, whose three-dimensional structure was downloaded from the PDB database, underwent molecular docking preprocessing using AutoDockTools software, specifically to remove... IP3 The water molecule and ligand encoding the protein are modified by adding polar hydrogen and charge; VD2 is used as a ligand, and... IP3 The active pocket encoding the protein was docked, the docking parameters for covering the active pocket with a grid box were set, and the docking program was run; the docking results were visualized using PyMOL software, and the interaction between VD2 and the active pocket was analyzed. IP3 The binding modes of encoded proteins (such as hydrogen bonds and hydrophobic interactions) are determined, and the binding energy is calculated (the lower the binding energy, the stronger the binding ability) to verify the binding stability.

[0051] The results are as follows Figure 5 As shown in the figure, a is a PyMOL visualization 3D diagram (showing the spatial binding state of the two), and b is a 2D planar schematic diagram (showing the binding sites and interaction types), together illustrating VD2 and IP3 Stable binding relationships of encoded proteins.

[0052] The results showed that VD2 could interact with brewer's yeast. IP3 The encoded protein forms a stable binding bond with a binding energy below -5.0 kcal / mol (a binding energy ≤ -5.0 kcal / mol is considered a stable binding bond), primarily through hydrogen bonds and hydrophobic interactions.

[0053] The above results indicate that VD2 may be able to [achieve this through interaction with...] IP3 The encoded protein binds to and regulates its activity, thereby modulating the SAM (S-adenosylmethionine) / SAH (S-adenosylhomocysteine) metabolic balance. Ultimately, it exerts a thermoprotective effect on Saccharomyces cerevisiae through this metabolic regulatory pathway, providing molecular-level evidence for its mechanism of action.

[0054] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. Application of Vitamin D2 in the preparation of heat-resistant protective agents for fermentation industry.

2. The application according to claim 1, characterized in that, The fermentation industry mentioned is a fermentation industry that uses brewing yeast.

3. The application according to claim 2, characterized in that, The brewing yeast is Saccharomyces cerevisiae CEN.PK2-1C strain.

4. Application of Vitamin D2 as a heat-resistant protectant in the fermentation industry using Saccharomyces cerevisiae as a starter culture.

5. The application according to claim 4, characterized in that, The application includes adding vitamin D2 to the fermentation material during the initial stage of fermentation.

6. The application according to claim 5, characterized in that, The initial fermentation period refers to the period from 0 to 96 hours after the start of fermentation.

7. The application according to claim 4, characterized in that, Vitamin D2 was added to the culture medium for the expansion culture of Saccharomyces cerevisiae, and then added together with Saccharomyces cerevisiae to the material to be fermented.

8. The application according to claim 7, characterized in that, The amount of vitamin D2 added is calculated based on its concentration in the material to be fermented, which is 10~100 mg / L.

9. Application of Vitamin D2 in the preparation of heat-resistant protectants for Saccharomyces cerevisiae.

10. A method for fermenting and culturing *Saccharomyces cerevisiae* under high-temperature stress, characterized in that, include: After activating the Saccharomyces cerevisiae strain, single colonies were picked, inoculated into fresh liquid seed culture medium, and the cells were collected by centrifugation. The collected cells were resuspended in fresh liquid fermentation medium, and vitamin D2 was added to the liquid fermentation medium at a final concentration of 10-100 mg / L. Fermentation was carried out at 38-45℃.

Images