Kluyveromyces marxianus grx168 and application thereof

Abstract

The invention discloses kluyveromyces marxianus grx168 and application thereof, the kluyveromyces marxianus is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms on June 9, 2025, and the preservation number is CGMCC NO.34816. The kluyveromyces marxianus grx168 can be used for preparing the kluyveromyces marxianus. The strain is screened from Kefir grains in Xinjiang, and a nerve cell cAMP experiment verifies that the opioid activity of a fermentation product of the strain is obviously higher than that of other yeast strains, and the cAMP content of the fermentation product of the strain is obviously lower than that of a blank control. The strain can be used for preparing fermented food or health care products with opioid activity, and a high-quality strain resource is provided for development of functional fermented products.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and relates to a strain of Kluyveromyces marxianus grx168 and application thereof. BACKGROUND

[0002] Opioid active substances are a class of active molecules that can specifically bind to body opioid receptors, and play physiological functions by regulating neurotransmitter release (such as endorphin and dopamine). The importance of the substances in the fields of medicine, food and the like has been widely proved. From the mechanism of action, the substances can significantly affect physiological processes such as pain perception, emotion regulation, immune function and energy metabolism by binding to μ, δ and κ type opioid receptors of the central and peripheral nervous system, and are one of key regulatory factors for maintaining body homeostasis.

[0003] Existing studies show that the opioid active substances have important physiological functions, and the mechanism of action is closely related to opioid receptors. The opioid active substances can bind to opioid receptors on the surface of nerve cells, inhibit the activity of adenylyl cyclase through a G protein-coupled signal pathway, and then affect the synthesis level of intracellular cyclic adenosine monophosphate (cAMP). The change of the cAMP content can be an important detection index of the opioid activity: when the opioid active substances exist, the synthesis of cAMP in nerve cells is inhibited, and the content will present a characteristic change; and when an opioid receptor antagonist (such as naloxone) is added to block the receptor binding, the inhibition is removed, and the content of cAMP will be reversely regulated. Therefore, by detecting the dynamic change of the cAMP level in nerve cells, it can be scientifically and specifically verified whether the substances have opioid activity. Since the method can directly reflect the signal transduction process mediated by opioid receptors, it has become one of the classic methods for verifying the opioid activity in the world.

[0004] At present, there are significant limitations in the natural sources of opioid active substances. In plant sources, poppy is the traditional source, but its components such as thebaine are strictly controlled, and toxic impurities are easily produced in the extraction process; animal sources rely on raw materials such as dairy products and meat, such as beta-casein peptides, which are mainly derived from casein hydrolysis, and are limited by raw material costs and enzymatic efficiency (conversion rate is usually <30%); in terms of microbial sources, existing research focuses on actinomycetes, fungi and the like, such as Penicillium strains that can produce a small amount of opioid metabolites, but most of them are conditional pathogens, and their safety cannot meet the requirements of food-grade applications. Beta-casein peptides produced during the fermentation of dairy products are a typical representative, and studies by Ningya and others have shown that beta-casein peptide-7 and beta-casein peptide-5 in yogurt can be released after digestion and enzymolysis, and can pass through the blood-brain barrier and bind to central opioid receptors to exert calming and soothing (reduce anxiety score by 15%-20%), anti-fatigue (improve exercise endurance by 25%) and other effects, but the cost is high. Whey is a natural by-product of dairy product processing, and is rich in resources and low in cost, which is conducive to industrialization. Developing opioid active peptides from whey is a good strategy for high-value utilization of whey. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a Kluyveromyces marxianus grx168 with opioid activity.

[0006] The technical problem to be solved by the present application is to provide a microbial preparation.

[0007] The technical problem to be solved by the present application is to provide the Kluyveromyces marxianus grx168 and the microbial preparation for producing cAMP.

[0008] The technical problem to be solved by the present application is to provide the Kluyveromyces marxianus grx168 and the microbial preparation for producing cAMP.

[0009] Technical scheme: In order to solve the above technical problems, the present application provides a Kluyveromyces marxianus grx168, the classification and naming of the Kluyveromyces marxianus grx168 is Kluyveromyces marxianus, which was preserved in the China General Microbiological Culture Collection Center on June 9, 2025, and the preservation number is CGMCC NO.34816.

[0010] The OD value of the Kluyveromyces marxianus grx168 is 0.46 at pH 3.0, and the OD value of the strain is 0.73 when the concentration of bile salt is 0.6.

[0011] The present invention also includes a microbial preparation containing the aforementioned Kluyveromyces masculinus grx168.

[0012] The formulation includes single-ingredient or compound formulations, and the formulation type includes liquid formulations or solid formulations.

[0013] This invention also includes the application of the aforementioned Kluyveromyces Marcius grx168 and the aforementioned microbial preparation in the production of opioid-like active fermented products. The fermented products of this invention include fermented foods or health products.

[0014] The fermentation conditions in the preparation of the fermented product are as follows: the Kluyveromyces grx168 is inoculated into whey-based medium and fermented.

[0015] The inoculation amount of the Kluyveromyces grx168 was 3-6%.

[0016] The viable count of the fermented product is 3.7 × 10⁻⁶. 8 CFU / mL, alcohol content 0.88%; lactose utilization rate 79.45%, acetic acid 58.41g / L, lactic acid 22.05g / L, citric acid 24.76g / L.

[0017] The mechanism of this invention: The metabolites (specific active polypeptides) produced by the fermentation of Kluyveromyces martensii grx168 possess opioid-like activity. These fermentation products can specifically bind to opioid receptors on the surface of nerve cells, inhibiting cAMP synthesis through the G protein-coupled signaling pathway, resulting in significantly lower intracellular cAMP levels than the blank control group (verifying its opioid activity). Upon addition of naloxone, naloxone competitively binds to opioid receptors, blocking the binding of the fermentation products to the receptors, thus relieving the inhibition of cAMP synthesis and restoring cAMP levels, further confirming that its activity is specific and mediated by opioid receptors.

[0018] Beneficial Effects: Compared with the prior art, the present invention has the following advantages: The *Kluyveromyces martensii* strain grx168 of the present invention was screened from kefir grains from Xinjiang. Verification by nerve cell cAMP experiments showed that the opioid-like activity of its fermentation products was significantly higher than that of other yeast strains. Only the cAMP content of the *Kluyveromyces martensii* grx168 group was significantly lower than that of the control group, and its opioid-like effect was blocked after the addition of naloxone. The strain of the present invention can be used to prepare fermented foods or health products with opioid-like activity, providing a high-quality strain resource for the development of functional fermented products. Attached Figure Description

[0019] Figure 1 This is a diagram showing the colony morphology of the bacterial strain.

[0020] Figure 2 This is an electrophoretic imaging image of 18S rDNA;

[0021] Figure 3 This is a graph showing the sequencing and identification results;

[0022] Figure 4 Figure showing the results of acid and bile salt resistance;

[0023] Figure 5 Image of a fusion cell (NG108-15) of neuroblastoma cells and rat glioma cells. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the accompanying drawings, embodiments, and experiments, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Those skilled in the art can implement the invention by referring to various commonly used reference books, scientific and technological documents, or related instructions and manuals prior to the filing date of this invention.

[0025] Example 1: Isolation and Identification of Strains

[0026] (1) Sample preparation: Take 1g of Xinjiang kefir grains, add 9mL of sterile physiological saline, shake to mix, and then serially dilute (1000 mg / L). -1 Up to 10 -6 Meanwhile, the purchased KM strain (Kluyveromyces laurierae LAF-4) was used as a positive control;

[0027] (2) Isolation and purification: 100 μL of the diluted solution was spread on YPD medium and incubated at 25°C for 48 h. Single colonies with different morphologies were picked and purified by streak three times to obtain pure culture strains xj01~xj09.

[0028] (3) Tolerance screening: After two generations of activation, the purified strains were inoculated into culture media with different pH values ​​(1.5, 2.0, 2.5, 3.0, 6.5) and different concentrations of bile salts (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 1.0, 1.5, 2.0%). The bacterial culture was incubated at 28℃ for 72 hours, and the OD value of the culture was measured. The results are shown in […]. Figure 4 grx168 exhibits outstanding resistance to acids (2.5, 3.0) and bile salts (0.1%–1.0%).

[0029] (4) Viability screening: Each strain (xj01~xj09, KM strain) was inoculated at 6% onto whey (10% whey powder) substrate (Beijing Tongsheng Youchuang Trading Co., Ltd., item number XI-03556), and fermented at 25℃ for 72h. The fermentation broth was taken, pH adjusted, centrifuged and filtered, and the effect of each strain's fermentation broth on NG108-15 cells was detected. NG108-15 cells were purchased from the China Center for Type Culture Collection (https: / / cctcc.whu.edu.cn / portal / no_center / index.html), CCTCC number GDC0170. The effect of cAMP content was investigated using the following steps: NG108-15 cells were seeded in multiple 6-well plates and cultured for 12–24 h. The blank group (culture medium only), experimental group (culture medium + fermentation broth of each strain), and naloxone group (culture medium + fermentation broth of each strain + naloxone) were simultaneously added to each 6-well plate. The protein concentration in each fermentation sample was adjusted to 2 mg / mL, and the plates were incubated for 15 min. 2 mL of trichloroacetic acid was added to each well to terminate the reaction. Then, 150 μL of lysis buffer was added to each well, cells were collected, centrifuged at 3000 rpm for 10 min, and the supernatant was discarded. The cells were resuspended in 2 mL of 50 mmol / L acetate buffer. Cells were sonicated (300 Hz, 3 min). The cells were centrifuged at 12000 × g for 30 min, and the supernatant was collected in a 15 mL centrifuge tube to precipitate. 2.0 mL of anhydrous ethanol was added, mixed, centrifuged, and the supernatants were combined in a centrifuge tube. Protein concentration was measured, and the cells were dried at 60 °C and stored at 4 °C for later analysis. 50 mmol / L pH... 4.75% acetate buffer was used to dissolve the dried cell contents. The cAMP content of NG108-15 cells was measured using the Cytokine™ cAMP ELISA Kit. The analytical procedures and reagent preparation were performed according to the instructions. 0.1 mL of the solution was added to an analytical tube for double-tube assay. The amount of cAMP produced per milligram of protein was calculated, and a strain (xj04) with a significantly lower cAMP content in its fermentation product than the control was selected as strain grx168.

[0030] (5) Identification: Morphological observation (colonies are milky white and round) Figure 1 ) and 18S rDNA sequencing (sequence 99.78% homology with Kluyveromyces marxianus) Figure 3 The strain was identified as *Kluyveromyces marxianus*, named grx168, and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34816.

[0031] (6) Opioid activity assay:

[0032] The same method as in step (4) was used to detect grx168, KM strain (Max Kluyver LAF4), and xj01~xj03, xj05~xj09. The results showed that the cAMP content of the naloxone-free group of the nine yeast strains was 8.10~8.25 pmol / mg, which was not significantly different from the blank control group. Only the cAMP content of the grx168 group was 6.59 pmol / mg, which was significantly lower than that of the blank group. Moreover, its cAMP content could be reduced after the addition of the antagonist naloxone, which confirmed that it has unique opioid-like activity.

[0033] Table 1 Results of cAMP content determination

[0034]

[0035] Table 1 shows the effect of each bacterial strain on the cAMP content produced by NG108-15 nerve cells. Note: Significance between milk beer and its main components is indicated by lowercase letters, and significance between the groups with and without naloxone is indicated by A to B.

[0036] Example 2: Application of the strain

[0037] Kluyveromyces marxianus grx168 was inoculated at a 6% inoculum into reconstituted whey broth (10% whey powder) (Beijing Tongsheng Youchuang Trading Co., Ltd., XI-03556). The mixture was first cultured at 25°C with shaking for 24 hours (16 rpm), then statically cultured for 72 hours, and finally fermented at 4°C for 4 days to obtain a whey-based fermentation broth, which can be used as a whey-based beverage. The results are shown in Table 2, with a viable count of 3.7 × 10⁻⁶. 8 CFU / mL, alcohol content of 0.88%; lactose utilization rate of 79.45%; organic acids (acetic acid: 58.41 g / L; lactic acid: 22.05 g / L; citric acid: 24.76 g / L); alcohols, acids, and esters are the key volatile flavor compounds.

[0038] Table 2 Volatile flavor compounds and their aroma activity values ​​in milk beer

[0039]

[0040]

[0041]

[0042]

[0043] The whey-based fermentation broth of *Kluyveromyces marxianus* grx168 was adjusted to pH, centrifuged, and filtered. Its effect on cAMP content in NG108-15 cells (China Center for Type Culture Collection, GDC0170) was then examined. The specific steps were as follows: NG108-15 cells were seeded in 6-well plates and cultured for 12-24 h; whey-based fermentation broth was added to the plates to adjust the protein concentration to 2 mg / mL, and incubated for 15 min; 2 mL of trichloroacetic acid was added to each well to terminate the reaction; then... Add 150 μL of lysis buffer to each well, collect cells, centrifuge at 3000 rpm for 10 min, and discard the supernatant. Resuspend cells in 2 mL of 50 mmol / L acetate buffer. Sonicate cells (300 Hz, 3 min). Centrifuge at 12000 × g for 30 min, collect the supernatant in a 15 mL centrifuge tube, and precipitate the cells. Add 2.0 mL of anhydrous ethanol, mix well, centrifuge, combine the supernatants in a centrifuge tube, measure protein concentration, dry at 60 °C, and store at 4 °C until analysis. Dissolve the dried cell contents in 0.5 mL of 50 mmol / L pH 4.75 acetate buffer. Measure the cAMP content of NG108-15 cells using the Cytokine™ cAMP ELISA Kit. The analytical procedures and reagent preparation were performed according to the instructions. 0.1 mL of the solution was added to an analytical tube for double-tube assay. Based on the amount of cAMP generated per milligram of protein, the opioid activity was stable, and the concentration was 6.51 pmol / mg.

Claims

1. A strain of Kluyveromyces Marcius grx168, characterized in that, The strain described as *Kluyveromyces grx168* is classified as *Kluyveromyces grx168*. Kluyveromyces marxianus, It was deposited on June 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34816.

2. The Kluyveromyces Marcius grx168 according to claim 1, characterized in that, The Kluyveromyces grx168 strain had an OD value of 0.46 at pH 3.0 and an OD value of 0.73 at a bile salt concentration of 0.

6.

3. A microbial preparation, characterized in that, It contains the Kluyveromyces magna grx168 as described in claim 1.

4. The microbial preparation according to claim 3, characterized in that, The formulation includes single-ingredient or compound formulations, and the formulation type includes liquid formulations or solid formulations.

5. The use of the Kluyveromyces Marcius grx168 as described in claim 1, or the microbial preparation as described in claim 2 or 3, in the production of fermented products with opioid-like activity.

6. The application according to claim 5, characterized in that, The fermentation conditions in the preparation of the fermented product are as follows: the Kluyveromyces grx168 is inoculated into whey-based medium and fermented.

7. The application according to claim 5, characterized in that, The inoculum size of the Kluyveromyces grx168 was 3-6%.

8. The application according to claim 5, characterized in that, The viable cell count of the fermented product was 3.7 × 10⁻⁶. 8 CFU / mL, alcohol content 0.88%; lactose utilization rate 79.45%, acetic acid 58.41 g / L, lactic acid 22.05 g / L, citric acid 24.76 g / L.

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