Hansenula debayii with ability of degrading ochratoxin a and application thereof

Abstract

The application discloses a Hansenula dubuquensis strain with the ability of degrading ochratoxin A and an application thereof, relates to the technical field of microorganisms, and has the preservation number CCTCC NO: M 2026936. The strain is screened from a Panxian ham, and an active substance for degrading OTA is intracellular metalloproteinase, which can play a degrading role in the range of pH 6-8. The strain is inoculated into fermented sausages, and the initial concentration of OTA of 15.83 μg / kg can be reduced to 7.01 μg / kg after 21 days of fermentation, and the degradation rate is 55.73%. The strain can inhibit the growth of bacteria in the early fermentation stage, effectively inhibit the growth of molds in the whole fermentation cycle, and promote the protease activity in the early fermentation stage and continuously improve the lipase activity. The strain can also significantly improve the flavor quality of the fermented sausages, reduce the content of bad flavor substances such as methyl and sulfides, and improve the level of aromatic compounds. When coexisting with high-concentration OTA, the strain can reduce the intensity of sour taste, bitter taste, astringent taste and aftertaste, improve the richness of taste, and has the dual functions of biological detoxification and quality improvement.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Hansenula barley yeast with the ability to degrade ochratoxin A and its applications. Background Technology

[0002] Fermented sausages are a traditional fermented meat product with a long history, their production process dating back over 2,000 years to the Mediterranean region. With the continuous development of fermentation technology, fermented sausages have become one of the largest-scale fermented meat products in terms of production volume. However, sausages are rich in protein and fat, making them highly susceptible to mold contamination during fermentation, leading to the accumulation of mycotoxins in the final product and posing a potential food safety risk. Among various mycotoxins, ochratoxin A is one of the most serious contaminants in fermented meat products, mainly produced by filamentous fungi such as Aspergillus and Penicillium that colonize the product surface through secondary metabolism. Ochratoxin A is teratogenic, carcinogenic, hepatotoxic, and nephrotoxic, and has been classified as a Group 2B carcinogen by the International Agency for Research on Cancer. Furthermore, the unique molecular structure of ochratoxin A exhibits high thermodynamic stability during food processing, making it difficult to remove from food once contaminated using conventional methods. Therefore, effectively controlling ochratoxin A contamination in fermented meat products has become a crucial issue for ensuring meat safety and promoting the sustainable development of the industry.

[0003] The formation of mycotoxins is regulated by multiple factors, including the genetic factors of the toxin-producing fungi, environmental factors, and the characteristics of the food matrix. Physical and chemical methods have long dominated the control of ochratoxin A. However, physical methods such as high-temperature degradation, radiation induction, and activated carbon adsorption often have limitations, including low efficiency, significant environmental constraints, and potential damage to food nutrients. Meanwhile, chemical antimicrobial agents such as potassium sorbate, propionic acid, and sorbic acid are increasingly causing consumer concern due to their potential environmental residues and health risks. Against this backdrop, biological control methods have received widespread attention due to their environmental friendliness, ease of operation, and minimal impact on product nutritional quality. The biological control of mycotoxins mainly consists of two strategies: first, inhibiting the growth of toxin-producing fungi and their toxin synthesis through microbial antagonism; and second, utilizing the enzyme systems produced by microbial metabolism or the adsorption of cell walls to transform or adsorb existing ochratoxin A. While microbial adsorption is largely a physical process, it can effectively reduce the biological exposure of ochratoxin A in the gastrointestinal tract, thereby reducing its harm to human health. In terms of biodegradation, some microorganisms can produce carboxypeptidase or amidase during metabolism, which can convert ochratoxin A into ochratoxin α and L-β-phenylalanine, which have significantly lower toxicity, thus achieving detoxification. Furthermore, the application of microorganisms can improve food flavor, extend shelf life, and enhance nutritional value. However, existing detoxification strains are mostly isolated from non-food environments such as soil and feces, or primarily used in substrates such as grape juice and grains; their application in fermented meat products is relatively limited. Therefore, screening native microbial strains with both fermentation potential and ochratoxin A degradation capabilities from traditional fermented meat products and applying them to the production process of fermented sausages has significant practical importance and application value in solving the problem of ochratoxin A contamination in fermented meat products. Summary of the Invention

[0004] The technical problem this invention aims to solve is to provide a *Hansøe du Barry* strain capable of degrading ochratoxin A (OTA), a microbial preparation containing this strain, and the application of this strain or microbial preparation in the degradation of ochratoxin A and the preparation of fermented meat products. This invention also provides a method for preparing fermented sausages using this strain, thereby addressing the food safety issue of the difficulty in effectively controlling OTA contamination in fermented meat products, while simultaneously improving the flavor and quality of the product.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A strain of Hansenbali yeast ( Debaryomyces hanseniiStrain 9F is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2026936. This strain was screened from Panxian ham fermented for 1-2 years in Panzhou City, Guizhou Province, China. It was identified as *Hansøe du Barry* yeast by morphological and molecular biological methods. Its cells are round and reproduce by budding. On YPD medium, it forms round, raised, smooth, dull, milky white, opaque colonies.

[0006] The present invention also provides a microbial preparation comprising the above-mentioned Hansenula d'Barry yeast 9F.

[0007] This invention also provides the application of the *Hansophila barley* 9F strain or the aforementioned microbial preparation in the degradation of ochratoxin A. Specifically, the active substance for degrading ochratoxin A is a metalloproteinase within the *Hansophila barley* 9F strain, which exerts its degradation effect under pH 6-8 conditions. Experiments have demonstrated that the cell lysate of this strain exhibits significant OTA degradation activity. However, treatment of the cell lysate with high temperature, proteinase K, SDS, and EDTA significantly inhibits or completely eliminates its degradation activity, thus proving that the active substance for OTA degradation is a thermosensitive metalloproteinase. Further research has found that Zn... 2+ Cu 2+ Ca 2+ Mg 2+ and K + It can significantly inhibit the activity of this enzyme, while Al 3+ Na + Fe 2+ There was no significant effect, further verifying the metalloprotein nature of the enzyme. Furthermore, the optimal pH range for this active enzyme is 6–8, and its activity significantly decreases under acidic or strongly alkaline conditions.

[0008] This invention also provides the application of the *Hansenula polymorpha* 9F or the above-mentioned microbial preparation in the preparation of fermented meat products. Preferably, the fermented meat product is a fermented sausage.

[0009] This invention also provides a method for preparing fermented sausage, comprising inoculating minced meat with the above-mentioned Hansenula d'Barry yeast 9F or the above-mentioned microbial preparation, and then fermenting it. Preferably, the inoculation amount of Hansenula d'Barry yeast 9F is 3% of the weight of the minced meat, and the concentration of the inoculated bacterial suspension is 10. 8 CFU / mL. The preferred fermentation conditions are: 28℃ and 85%~90% relative humidity for 3 days, followed by 15℃ and 75%~85% relative humidity for 18 days.

[0010] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention marks the first time that a *Hansenula d'Barry* strain capable of degrading ochratoxin A (OTA) has been screened from traditional fermented meat products, filling a gap in strain resources for OTA biodegradation. This strain exerts its degradation effect through intracellular metalloproteinases, with a clearly defined degradation mechanism. Furthermore, the enzyme exhibits optimal activity under neutral to slightly acidic conditions, demonstrating good controllability in its application. This strain not only possesses OTA degradation capabilities but also exhibits excellent fermentation potential, tolerating conditions such as temperature, salinity, and nitrite levels during the processing of fermented meat products.

[0011] In practical applications of fermented sausages, this strain exhibited a significant OTA degradation effect. With an initial OTA content of 15.83 μg / kg, after 21 days of fermentation, the inoculated group reduced OTA to 7.01 μg / kg, achieving a degradation rate of 55.73%, far exceeding the 26.73% natural degradation rate of the control group. In the early stages of fermentation, this strain showed some inhibitory effect on bacterial growth, and throughout the entire fermentation cycle, it effectively inhibited mold growth, reducing the risk of secondary contamination by mold and toxins. Simultaneously, this strain promoted the enhancement of protease activity in the early stages of fermentation and continuously enhanced lipase activity throughout the fermentation process, which is beneficial for the hydrolysis of proteins and fats and the formation of flavor precursors. Regarding physicochemical properties, this strain had no significant effect on the pH, salt content, and moisture content of the fermented sausage, but it moderately increased the water activity of the product, thereby reducing sausage firmness and improving the product's texture.

[0012] More notably, this strain significantly improves the flavor characteristics of fermented sausages. Electronic nose analysis shows that inoculation with this strain can substantially reduce the content of undesirable flavor compounds such as methyl compounds, inorganic sulfides, and organic sulfides, while significantly increasing the content of benzene and ammonia aromatic compounds, resulting in a purer and more harmonious overall flavor in the sausage. Electronic tongue analysis further shows that when the strain coexists with high concentrations of OTA, it can significantly improve the flavor profile of the sausage, reduce the intensity of sourness, bitterness, astringency, and their aftertaste, and enhance the richness of the flavor, achieving the dual effects of biological detoxification and quality improvement. In conclusion, this Hansenula d'Barry yeast 9F strain possesses both OTA degradation capabilities and fermentation characteristics, and has important application value in the safe production and quality improvement of fermented meat products. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Picture 1Figure 1 shows the screening and identification results of OTA-degrading strains. A: Tolerance of yeast strains to OTA; B: Degradation ability of yeast strains to OTA; C: Phylogenetic tree of strain 9F; D: Colony morphology of strain 9F on culture medium; E: Microscopic morphology of strain 9F.

[0015] Picture 2 strain D. hansenii Growth characteristics of strain 9F. Where: A: Growth curve of strain; B: Optimal temperature of strain; C: Optimal pH of strain; D: Sodium tolerance of strain; E: Nitrite tolerance of strain; F: Safety evaluation of strain; G: Antimicrobial properties of strain.

[0016] Picture 3 strain D. hansenii Figure 9 shows the characteristics of OTA degradation by 9F strains. A: OTA degradation ability of the strains; B: OTA degradation ability of different strain components; C: Effect of the metal chelating agent EDTA on the active ingredient; D: Effects of proteinase K, SDS, and high temperature on the active ingredient; E: Effects of metal ions on the active ingredient; F: Effect of pH on the active ingredient.

[0017] Picture 4 This graph shows the changes in the appearance and OTA content of fermented sausages. A: Appearance of fermented sausages; B: Changes in OTA content during fermentation.

[0018] Picture 5 This graph shows the changes in microbial colony counts during fermentation. A represents the change in total yeast count; B represents the change in total mold count; and C represents the change in total bacterial count.

[0019] Picture 6 This diagram shows the changes in enzyme activity during fermentation. In the diagram, A represents changes in protease activity, and B represents changes in lipase activity.

[0020] Picture 7 This is a graph showing the changes in the physicochemical properties of fermented sausage. A: Changes in water activity; B: Changes in water-holding capacity; C: Changes in moisture content; D: Changes in NaCl content; E: Changes in pH.

[0021] Picture 8 This is a graph for electronic sensory evaluation analysis. A: Principal component analysis of electronic nose response; B: Electronic nose response radar graph; C: Principal component analysis of electronic tongue response; D: Electronic tongue response radar graph. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0024] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0025] In this invention, OTA standard (purity >99%) was purchased from TMRM (Beijing, China), dissolved in methanol (1.00 mg / mL) to prepare a standard stock solution, and further diluted with methanol to 0.1 mg / mL and 0.01 mg / mL for subsequent studies. The ochratoxin A ELSEA kit was purchased from Pribolab (Shandong, China). Methanol was chromatographic grade and purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). All other chemical reagents were analytical and chemically pure.

[0026] Example 1: Screening, isolation and identification of ochratoxin A-degrading strains 1. Strain source and activation The experimental strains were derived from 76 yeast strains isolated and purified from Panxian ham fermented for 1 to 2 years in Panzhou City, Guizhou Province, China. Through a comprehensive evaluation of the fermentation potential of these strains, including protease degradation, lipase degradation, and alcohol production, 12 strains with fermentation potential were initially screened. Eight of these strains were isolated from ham samples (fermented for 1 year) from Yanglao Dairy Food Co., Ltd. in Panzhou City, Guizhou Province, and four were isolated from ham samples (fermented for 2 years) from Panfu Ham Co., Ltd. in Panzhou City, Guizhou Province. The strains, stored at -80℃, were slowly thawed in 37℃ warm water. The thawed bacterial culture was streaked onto YPD agar plates using a sterile inoculation loop and incubated at 28℃ for 3 days. Single colonies with full growth and good condition were selected and inoculated into YPD liquid medium, and cultured at 28℃ and 150 rpm for 48 hours in a shaker to obtain the seed culture. The seed culture was centrifuged at 4℃ and 5000 rpm for 10 minutes, the supernatant was discarded, and the bacterial sludge was collected. After resuspending the bacterial sludge in sterile saline and washing it twice, the cells were collected by centrifugation again. The cells were counted using a hemocytometer, and the concentration of the bacterial suspension was adjusted to 10⁷ CFU / mL with sterile saline for later use.

[0027] 2. Screening of strains with ochratoxin A degradation ability The bacterial suspensions of the above 12 yeast strains were inoculated at a volume ratio of 1% into YPD liquid medium containing different concentrations of ochratoxin A (OTA) (0 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL). The cultures were incubated at 28°C and 150 rpm for 24 hours. The absorbance (OD600) of each culture was measured at 600 nm using a microplate reader to assess the tolerance of each strain to OTA.

[0028] The results are as follows Picture 1 As shown in Figure A, most strains exhibited growth promotion at low OTA concentrations (20 ng / mL), while their growth activity decreased slightly at high concentrations (100 ng / mL), but the OD600 value remained above 1.4. Among them, strains 9F and 9C showed almost no effect from OTA concentration, with their OD600 values ​​consistently remaining above 1.6, demonstrating excellent OTA tolerance.

[0029] The OTA degradation capacity was then determined. Specifically, 200 μL of yeast culture at a concentration of 107 CFU / mL was inoculated into 5 mL of YPD liquid medium containing 100 ng / mL OTA, and cultured for 48 hours in a constant temperature shaker at 28℃ and 150 rpm. After the culture was completed, 200 μL of the culture medium was taken, the residual concentration of OTA was measured, and the degradation rate was calculated. The results are as follows: Picture 1 As shown in Figure B, there were significant differences in the ability of different yeast strains to degrade OTA (P < 0.05). Nine strains showed OTA degradation ability, while three strains did not. Strains 9F and 9C exhibited the highest OTA degradation rates, reaching 26.57% and 24.42%, respectively, followed by strains 9G, 4-5, and 9B, all with degradation rates exceeding 15%. Considering both the fermentation potential and OTA removal capacity of the strains, strain 9F was ultimately selected as the target strain for subsequent studies.

[0030] 3. Identification of strain 9F The selected target strain 9F was purified and cultured on YPD agar medium using the streak plate method. After incubation at 28°C for 3 days, single colonies with good growth were selected for observation, and their color, morphology, and other characteristics were recorded. Picture 1 As shown in Figure D, after culturing strain 9F on YPD medium for 3 days, it formed round, raised, smooth, dull, milky-white, opaque colonies. Under a light microscope, its cell morphology was round, and it reproduced asexually via budding. Picture 1 E).

[0031] The target strain 9F was sent to Shanghai Sangon Biotech Co., Ltd. for bidirectional sequencing, and molecular biological identification of the strain was performed according to the method of Chen et al. The sequencing results were assembled using the BLAST algorithm and compared with the NCBI database to screen known strain sequences with high similarity to the target sequence. A phylogenetic tree was constructed using the Neighbor-Joining method, and evolutionary analysis was performed using MEGA12 software.

[0032] like Picture 1 As shown in C, strain 9F and *Hansøe d'Bary yeast* ( Debaryomyces hansenii The strain BRRL Y-7426 is highly similar and belongs to the same family. Debaryomyces hansenii The phylogenetic branch of the fungus. Therefore, the taxonomic classification of strain 9F is Ascomycota, Saccharomycetes, Saccharomycetales, and Saccharomycetaceae. Based on morphological and molecular biological identification results, strain 9F was identified as *Hansøe bale* and named... Debaryomyces hansenii 9F (hereinafter referred to as 9F) D. hansenii (9F). The sequence data of this strain has been uploaded to the NCBI database and obtained the GenBank accession number: SUB15713003.

[0033] Furthermore, the Hansenula baiji yeast obtained by screening in this invention ( Debaryomyces hansenii Strain 9F was deposited on May 12, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2026936, and classified as follows: Debaryomyces hansenii 9F.

[0034] 4. Growth characteristics of the strain The strains screened in this example D. hansenii Growth characteristics of strain 9F were studied. Results showed that this strain exhibited strong growth activity, growing well within a temperature range of 18–28℃ and a pH range of 3–11. It also tolerated 0–100 g / kg NaCl and 0–750 mg / kg NaNO2, fully meeting the processing requirements for fermented meat products. Safety evaluation showed that this strain had no hemolytic activity. In both ordinary culture medium and ham broth agar medium, this strain significantly antagonized ochratoxin A-producing Aspergillus niger (…). Aspergillus niger Growth of strain A-8. Results related to strain growth characteristics are as follows: Picture 2 As shown.

[0035] Example 2: Strains D. hanseniiCharacteristic study of 9F degradation of ochratoxin A 1. Determination of the ability of different cellular components to degrade OTA To investigate the localization of the active components in the strain that degrade OTA, a method similar to that of Yang et al. was used to prepare... D. hansenii Cell-free supernatant, live cells, heat-inactivated cells, and cell lysate from 9F were analyzed, and their degradation abilities were compared. Specifically, 200 μL of a 10% concentration was used. 7 CFU / mL D. hansenii Strain 9F was inoculated into 100 mL of YPD liquid medium and cultured at 28°C and 150 rpm for 2 days. 100 mL of the cultured bacterial solution was centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain a cell-free supernatant. The centrifuged bacterial sludge was washed twice with sterile PBS buffer (pH 7.4, 0.1 mol / L), and then resuspended in 100 mL of the same PBS buffer to obtain a live yeast cell suspension. 25 mL of the live yeast cell suspension was autoclaved at 121°C for 30 minutes to obtain a heat-inactivated yeast cell suspension. Another 25 mL of the live yeast cell suspension was placed in an ice-water bath and cell disruption was performed using a cell disruptor at 300 W for 3 seconds, followed by a 5-second interval, until the cell solution became clear and transparent. The lysate was centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm microporous membrane to obtain the cell lysate.

[0036] Take 2 mL each of the above cell-free supernatant, live cell suspension, heat-inactivated cell suspension, and cell lysis buffer, and mix them with OTA standard to achieve a final OTA concentration of 100 ng / mL in the reaction system. After reacting each mixture at 28°C and 150 rpm for 48 hours, determine the residual OTA content in the liquid. Results are as follows: Picture 3 As shown in B, D. hansenii The degradation capacity of different cell components of 9F cells for OTA showed significant differences (P < 0.05). Specifically, the degradation rates of 100 ng / mL OTA by heat-inactivated cells, live cells, and cell-free supernatant within 48 hours showed no significant difference (P > 0.05), being 17.42%, 15.16%, and 13.18%, respectively, all significantly lower than the degradation rate of cell lysate (40.18%). This result indicates that... D. hansenii 9F primarily degrades OTA in the environment through its intracellular metabolites, and the intracellular active substances do not significantly permeate extracellularly under normal culture conditions. Therefore, it is preliminarily speculated that... D. hansenii 9F may adsorb and take in OTA through the cell wall into the cell, where it is then degraded by intracellular active substances.

[0037] 2. Effects of high temperature, proteinase K, SDS, and EDTA on bioactive substances Given that cell lysates exhibited the strongest OTA degradation activity, subsequent experiments used cell lysates as the research object to further analyze the properties of the active substances. To investigate the effects of different treatments on degradation activity, the following experiments were conducted: High temperature treatment: 900 μL D. hansenii Mix 9F cell lysis buffer with 100 μL PBS buffer and heat in a 100°C water bath for 10 minutes.

[0038] Proteinase K and SDS treatment: 900 μL of each protein was used. D. hansenii 9F cell lysis buffer was mixed with (1) 50 μL 20 mg / mL proteinase K and 50 μL PBS buffer, (2) 50 μL 20% SDS and 50 μL PBS buffer, and (3) 50 μL 20% SDS and 50 μL 20 mg / mL proteinase K and incubated at 37°C for 3 hours.

[0039] EDTA treatment: Mix cell lysate with EDTA solution to achieve final EDTA concentrations of 1 mM, 5 mM, and 10 mM, and incubate at 37°C for 1 hour.

[0040] Each of the treated liquids was mixed with 1 mL of 100 ng / mL OTA standard solution to achieve a final OTA concentration of 100 ng / mL in the reaction system. The mixture was incubated at 28°C and 150 rpm for 48 hours. Cell lysis buffer with an equal volume of PBS buffer was used as a control group. After the reaction, the residual OTA concentration in each group was measured.

[0041] The results are as follows Picture 3 C and Picture 3 As shown in Figure D, the control group cell lysate with only PBS buffer was able to degrade most of the OTA. After treatment with proteinase K, the activity of the cell lysate decreased rapidly, with an OTA degradation rate of only 12.7%. Furthermore, the cell lysate treated with SDS and high temperature completely lost its ability to degrade OTA. This indicates that the active substance degrading OTA is most likely a heat-sensitive protein enzyme.

[0042] Meanwhile, EDTA, a widely used metal chelating agent, exhibited a significant inhibitory effect on intracellular enzyme activity (P < 0.05). After treatment with 1 mg / mL and 5 mg / mL EDTA solutions, the degradation rates of OTA after 48 hours were only 5% and 4.16%, respectively; when the EDTA concentration reached 10 mg / mL, intracellular enzyme activity was completely inhibited, and the ability to degrade OTA was completely lost. Many known enzymes capable of degrading OTA, such as carboxypeptidase A and amidases, are classified as metalloproteins. Therefore, based on the above results, it can be inferred that… D. hansenii The active substance in 9F that degrades OTA intracellularly is a metalloproteinase.

[0043] 3. The effects of different metal ions on active substances Following the method of Xu et al. with slight modifications, this study investigated the effects of different metal ions on intracellular OTA detoxification active substances. This was achieved by adding Al-containing metal ions to the cell lysis buffer. 3+ Zn 2+ Na + Fe 2+ Cu 2+ K + Ca 2+ Mg 2+ Metal salt solutions (Al2(SO4)3, ZnSO4, NaCl, FeSO4, CuSO4, KCl, CaCl2, and MgCl2, respectively) were prepared to a final concentration of 10 mM. After incubating the treated solution at 37°C for 1 hour, OTA standard solution was added to bring the final OTA concentration in the system to 100 ng / mL, and the system was incubated at 28°C and 150 rpm for 48 hours. An equal volume of PBS buffer was added as a control group. The results are as follows: Picture 3 E shown, Al 3+ Na + Fe 2+ It had no significant effect on the degradation of OTA by intracellular enzymes (P>0.05), with degradation rates reaching 41.33%, 45.04%, and 41.89%, respectively; while Zn 2+ Cu 2 + Ca 2+ Mg 2+ It significantly inhibited the degradation of OTA by intracellular enzymes (P < 0.05), with degradation rates of only 27.00%, 26.21%, 32.23%, and 31.35% after 48 hours; K + This completely inhibited the activity of the intracellular enzyme in degrading OTA (P < 0.05). This result further supports the inference that the intracellular enzyme that degrades OTA is a metalloproteinase.

[0044] 4. The effect of pH value on active substances Following the method of Yang et al. with slight modifications, the effect of different pH values ​​on the detoxification activity of the substance was investigated. Cell lysates were extracted using buffers with different pH values: working solutions at pH 4–6 were prepared using 20 mmol / L sodium citrate buffer; working solutions at pH 7–8 were prepared using 20 mmol / L PBS buffer; and working solutions at pH 9–10 were prepared using 20 mmol / L glycine-NaOH buffer. One mL of each pH buffer was taken, and OTA standard solution was added to achieve a final OTA concentration of 100 ng / mL. After incubation at 28°C and 150 rpm for 48 hours, the OTA concentration in the solution was measured.

[0045] The results are as follows Picture 3 As shown in F, D. hansenii The optimal pH range for OTA degradation by the intracellular enzyme 9F is 6–8. The highest degradation rate (40.57%) was observed at pH 6, followed by pH 7 and pH 8, with degradation rates reaching 37.70% and 38.53%, respectively. Although the strain… D. hansenii 9F itself can tolerate a wide pH range, but its degradation activity decreased significantly in acidic (pH 4–5) and alkaline (pH 9–10) environments (P < 0.05). This indicates that the enzyme exhibits optimal activity under neutral to slightly acidic conditions.

[0046] Example 3: Strains D. hansenii The effects of 9F on the degradation of ochratoxin A in fermented sausages and on product quality 1. Preparation and experimental grouping of fermented sausage Following and slightly modifying the method of Zheng et al., fermented sausages were produced. Pork tenderloin and backfat that passed hygiene inspection were selected, trimmed, and the tendons and membranes removed. The meat was then washed thoroughly with clean water, drained, and minced into granules using a meat grinder. The mixture was added according to a formula ratio of 70% pork tenderloin, 30% pork backfat, 3% salt, 3% glucose, 0.015% NaNO2, and 0.005% D-isoascorbic acid sodium salt, and mixed evenly. The mixture was then chilled and marinated at 4°C for 4 hours. To simulate toxin contamination during meat processing, OTA standard solution was added to a portion of the minced meat and mixed thoroughly, resulting in a final concentration of approximately 20 ng / g. The control group was treated with an equal volume of PBS buffer (pH 7.4, 0.1 mol / L). To investigate... D. hansenii The ability of 9F to degrade OTA during sausage fermentation and its effect on flavor quality were investigated by adding 9F at an inoculum concentration of 10⁸ CFU / mL at a rate of 3% (by weight) in the corresponding experimental groups. D. hansenii9F bacterial suspension was used. The control group was replaced with an equal volume of sterile saline. The prepared minced meat was stuffed into 25 mm diameter protein casings, each approximately 20 cm long (about 150 g), and both ends were tied tightly with cotton thread. Appropriate holes were pricked in the surface of the sausage with a needle to facilitate air release. The stuffed sausages were then suspended in a constant temperature and humidity incubator for fermentation. The fermentation conditions were set as follows: Stage 1: 28℃, 85%–90% relative humidity (RH) for 3 days; Stage 2: 15℃, 75%–85% RH for 18 days, for a total fermentation period of 21 days.

[0047] The experiment was set up with 4 treatment groups, as follows: (1) CK group: PBS buffer and physiological saline were added; (2) CO group: OTA and physiological saline were added; (3) FK group: PBS buffer and bacterial strain were added. D. hansenii 9F; (4) FO group: Add OTA and strain D. hansenii 9F.

[0048] Samples were taken on day 0 (raw meat), day 3 (early fermentation), day 15 (mid-fermentation), and day 21 (mature fermentation) of fermentation. The samples were stored in a -20°C refrigerator until the various indicators were analyzed.

[0049] 2. Changes in ochratoxin A content during fermentation Changes in OTA content in fermented sausages from different treatment groups at different fermentation stages, as shown below Picture 4 As shown. At the start of fermentation (day 0), the background OTA content in the CK and FK groups was 1.093 μg / kg. As fermentation progressed, the OTA content in the FK group gradually decreased, and was significantly lower than that in the CK group at all stages (P < 0.05). To simulate the situation of sausages being contaminated with high concentrations of mycotoxins during fermentation, OTA was additionally added to the CO and FO groups. Initial testing showed that the OTA content after addition reached 15.83 μg / kg. Picture 4 As shown, the OTA content in both the CO and FO groups gradually decreased with increasing fermentation time. At the fermentation endpoint (day 21), the contents were 11.56 μg / kg and 7.01 μg / kg, respectively, corresponding to OTA degradation rates of 26.73% and 55.73%. Notably, throughout the entire fermentation process, the OTA content in the FO group was significantly lower than that in the CO group (P < 0.05). This indicates that even under conditions of high OTA contamination, the inoculated strain... D. hansenii 9F can effectively degrade most OTA, keeping it at a low level.

[0050] 3. Changes in the microbial community during fermentation. Following the method of Gong et al., microbial colony counting was performed on fermented sausages at different fermentation stages. Specifically, in an aseptic workbench, sausage meat samples (after removing the casing) were chopped, and 5 g of the meat sample was accurately weighed and placed in a sterile homogenization bag. 25 mL of sterile physiological saline was added, and the mixture was homogenized for 5 minutes using a beater-type sterile homogenizer. 1 mL of the homogenate was then serially diluted 10-fold to 10⁻⁶. -7 100 μL of the appropriate dilution solution was spread onto YPD, PDA and MRS agar plates respectively, and the total number of yeast, mold and bacteria colonies in the samples were counted.

[0051] Changes in total yeast colony count as follows Picture 5 As shown in Figure A, the yeast cells in each group rapidly multiplied and their numbers gradually increased as fermentation progressed. Groups FK and FO, due to the initial inoculation with a large number of yeast cells, showed the highest growth rates. D. hansenii The yeast colony count in group 9F was significantly higher than that in groups CO and CK from day 0 to day 3 (P < 0.05). From day 3 to day 15, the total yeast count in groups FK and FO remained relatively stable, while the yeast count in groups CK and CO continued to show a rapid growth trend. At the fermentation endpoint (day 21), there were significant differences in the total yeast count among the groups (P < 0.05), with the order being FK > FO > CO > CK. This indicates that inoculation with exogenous yeast can significantly increase and maintain the yeast count in sausages. Meanwhile, the yeast count in group CO was significantly higher than that in group CK (P < 0.05), suggesting that the presence of OTA may have stimulated the growth and metabolism of other endogenous yeasts to some extent.

[0052] Changes in total mold colony count, as shown Picture 5 As shown in Figure B, during the initial fermentation stage, due to suitable temperature and abundant nutrients, the total number of mold colonies in all treatment groups continued to increase. Towards the mid-to-late fermentation stage (days 15-21), as the water activity (Aw) of the fermented sausage continued to decrease, the germination and colonization of mold spores were inhibited, leading to a gradual stabilization of the mold count. Notably, throughout the entire fermentation process, the total number of mold colonies in the FK and FO groups was significantly lower than that in the CK and CO groups (P < 0.05). This is because the inoculated... D. hansenii 9F rapidly occupied its nutrient and spatial niches, effectively inhibiting the growth of other molds. This indicates that strain 9F... D. hansenii 9F can significantly reduce the risk of sausages being contaminated with mold during fermentation.

[0053] Changes in total bacterial count, as shown Picture 5 As shown in Figure C, during the initial fermentation stage (day 3), the total bacterial count in all groups increased rapidly, but the growth rates of the FK and FO groups were significantly lower than those of the CK and CO groups, indicating that... D. hansenii9F strain exhibited a certain inhibitory effect on bacterial growth in the early stage of fermentation. However, due to the vigorous growth of yeast consuming a large amount of oxygen, favorable conditions may have been created for the growth of some facultative anaerobes. Therefore, the total bacterial count in the FK and FO groups continued to increase during the middle stage of fermentation (days 3 to 15), reaching approximately 8.40 Log CFU / g. As nutrients were consumed and water activity decreased in the fermented sausage, the total bacterial count in each group remained relatively stable from day 15 to 21, with no significant differences among the treatment groups (P > 0.05). The experimental results indicate that the strain... D. hansenii 9F temporarily affected bacterial growth in the early stages of fermentation, but in the middle and later stages of fermentation, due to the complex interaction between fungi and bacteria, the total number of bacterial colonies eventually became consistent with the control group.

[0054] 4. Changes in protease and lipase activity during fermentation. Changes in protease activity, such as Picture 6 As shown in Figure A, the protease activity in all treatment groups gradually increased with prolonged fermentation time. This is partly due to the activation of endogenous proteases in the meat by temperature, humidity, and pH conditions during fermentation; and partly due to the replenishment of exogenous proteases by microbial metabolic activities, which accelerated protein degradation. Notably, in this example, the protease activities of the FK and FO groups were significantly higher than those of the CK and CO groups on day 3 (P < 0.05), indicating that inoculation... D. hansenii 9F helps to enhance the activity of proteases in sausages during the early stages of fermentation, thereby promoting the formation of flavor precursors in the early stages of fermentation.

[0055] Changes in lipase activity, such as Picture 6 As shown in Figure B, the lipase activity in all treatment groups gradually increased throughout the fermentation process. This may be attributed to the combined effects of environmental conditions and microbial metabolism, which produced more exogenous lipase to degrade lipids and meet the microorganisms' carbon source requirements. Notably, the lipase activities of the FK and FO groups were significantly higher than those of the CK and CO groups throughout the fermentation cycle (P < 0.05), indicating that the strain... D. hansenii 9F helps to continuously increase lipase activity in fermented sausages, promoting fat hydrolysis and flavor formation. Furthermore, the addition of OTA may, to some extent, promote the production of more lipases by microorganisms in the CO group, but this has limited effect on... D. hansenii The lipase-promoting activity of 9F was not significantly affected.

[0056] 5. Changes in the physicochemical properties of sausages during fermentation. Changes in the water activity (Aw) of the sausage during fermentation are as follows: Picture 7As shown in Figure A, the water activity of sausages in all treatment groups continuously decreased throughout the fermentation process, especially after 3 days, where the downward trend was more pronounced. This was due to the decrease in relative humidity in the fermentation environment, which drove the internal water of the sausage to continuously diffuse and evaporate from the center to the surface under the combined action of concentration gradient and capillary force. By day 21 of fermentation, the water activity of the yeast-inoculated treatment groups (FK and FO groups) was significantly higher than that of the uninoculated control groups (CK and CO groups) (P < 0.05). At the end of fermentation, the water activity of all groups dropped below 0.8. This Aw level can effectively inhibit the growth activity of most microorganisms, which is beneficial to extending the shelf life of the product.

[0057] Water holding capacity (WHC) changes as follows Picture 7 As shown in Figure B, the water-holding capacity of the fermented sausages in all groups showed an overall decreasing trend. Although the high-salt environment promotes the dissolution of salt-soluble proteins and the formation of a gel network structure, the high osmotic pressure environment also causes water to permeate from the muscle cells to the outside, leading to a decrease in water-holding capacity. Notably, from day 15 to 21, the water-holding capacity of the CO and FO groups was significantly higher than that in the earlier and middle stages (P < 0.05). This may be because the toxic effect of OTA inhibited the growth of competing microorganisms to some extent, altered microbial metabolism, and avoided excessive breakage of the gel network; at the same time, D. hansenii The secreted proteases and lipases degrade myofibril proteins and fats, which also helps form a three-dimensional network gel structure, thereby improving the water-holding capacity of the sausage.

[0058] Changes in moisture content, such as Picture 7 As shown in Figure C, due to continuous moisture evaporation, the moisture content of the fermented sausage decreases steadily from day 0 to day 7, stabilizing at around 25% after day 15. This final moisture content range meets the quality requirements of typical dry fermented sausages, which helps inhibit the growth of harmful microorganisms, ensures food safety, and concentrates flavor substances, giving the product a rich, complex, and unique flavor.

[0059] Changes in salt (NaCl) content are as follows: Picture 7 As shown in Figure D, the salt content of the sausages in all groups showed a continuous decreasing trend during fermentation, gradually decreasing from approximately 3% initially to around 5%. This is because internal moisture migrates to the surface and evaporates during fermentation, while the non-volatile nature of salt causes it to remain inside the sausage. There was no significant difference in salt content among the treatment groups (P > 0.05), indicating that... D. hansenii Neither 9F nor OTA significantly affected the final salt content of the fermented sausage.

[0060] pH changes as follows Picture 7As shown in Figure E, the pH value of sausages in all treatment groups showed a decreasing trend in the early stage of fermentation (days 0 to 3), gradually stabilizing from day 15 to 21. The pH decrease in the early stage of fermentation was due to the decomposition of carbon sources such as glucose and glycogen in the meat into small molecule organic acids by microorganisms, as well as the secretion of organic acids such as acetic acid, lactic acid, and citric acid during microbial metabolism. As the carbon sources were depleted and the metabolic activity of microorganisms weakened, the pH gradually stabilized between 5.3 and 5.4. There was no significant difference in pH among the different treatment groups (P > 0.05), indicating that OTA and D. hansenii 9F does not significantly alter the acid-producing metabolic activities of other microorganisms, while D. hansenii 9F itself does not have a significant acid-producing ability.

[0061] 6. Texture analysis of fermented sausages Table 1. Texture properties of fermented sausages Note: Different letters a to b indicate significant differences between groups.

[0062] The textural properties of fermented sausages in each treatment group at the fermentation endpoint (day 21) are shown in Table 1. Regarding firmness, the inoculation... D. hansenii The sausage firmness of the FK and FO groups (9F) was significantly lower than that of the uninoculated CK and CO groups (P < 0.05). This may be due to... D. hansenii During fermentation, to resist high salt osmotic pressure, the microorganisms metabolize sugars to produce glycerol, inhibiting water loss. Simultaneously, the secreted proteases hydrolyze some muscle proteins, weakening the protein gel network structure and leading to softening of the texture. Regarding chewiness, the CO group sausages were significantly chewier than other treatment groups (P < 0.05), possibly because the presence of OTA altered microbial metabolism and increased sausage firmness by inhibiting protease activity. In contrast, the FO group... D. hansenii The protease secreted by 9F weakened the hardening effect induced by OTA, effectively softening the sausage texture. There were no significant differences in viscosity, cohesion, and elasticity among the groups (P > 0.05), indicating... D. hansenii 9F and OTA had no significant effect on the viscosity, cohesiveness and elasticity of fermented sausages.

[0063] 7. Electronic sensory evaluation Electronic nose analysis: An electronic nose was used to macroscopically detect and compare the volatile components of fermented sausages from each treatment group on day 21. Three parallel samples were randomly selected from each treatment group for measurement. Principal component analysis (PCA) was used to establish a response model, and the results are as follows: Picture 8As shown in Figure A, PC1 and PC2 contributed 56.3% and 17.4% respectively, accounting for a cumulative 73.4% of the total variance, indicating that these two principal components could capture most of the information in the sample. The PCA score plot showed that the yeast-inoculated treatment groups (FK and FO groups) and the uninoculated control groups (CK and CO groups) were clearly separated along the PC1 and PC2 directions, with virtually no overlap, indicating a significant difference in their volatile flavor profiles. However, there was no significant separation between the OTA group and the group without OTA, suggesting that OTA itself does not significantly alter the overall flavor profile of the fermented sausage.

[0064] Table 2 Electronic sensor response values Note: Letters a through c indicate significant differences between groups; CK: control group (PBS + saline); CO: OTA group (OTA + saline); FK: yeast group (PBS + saline) D. hansenii 9F); FO: OTA+ Yeast Group (OTA+ D. hansenii 9F).

[0065] The specific differences in the response values ​​of each sensor are shown in Table 2 and Picture 8 As shown in Figure B, there were significant differences (P < 0.05) between the yeast groups (FK and FO groups) and the non-yeast groups (CK and CO groups) in sensor response values ​​for benzene aromatic compounds, nitrogen oxides, ammonia aromatic compounds, methyl compounds, inorganic sulfides, alcohols and aldehydes / ketones, and organic sulfides. In the non-yeast group, methyl compounds, inorganic sulfides, and organic sulfides all had high sensor response values, indicating they are the main and stable volatile components. However, excessive methyl compound content produces a pungent "alcoholic" or "ether" odor, masking the natural aroma of meat products; sulfur compounds can exhibit a "rotten egg" odor even at extremely low thresholds, especially when combined with fat oxidation products, significantly amplifying the "rancid" sensation. In contrast, the yeast group significantly reduced the response values ​​of the aforementioned undesirable flavor compounds in fermented sausages, with reductions of 54.87%, 59.34%, and 51.16% for methyl compounds, inorganic sulfides, and organic sulfides, respectively. Furthermore, inoculation... D. hansenii 9F also significantly reduced the content of nitrogen oxides, alcohols, aldehydes, and ketones, which easily lead to lipid rancidity and unpleasant rancid flavors, while significantly increasing the response values ​​of benzene and ammonia aromatic compounds. Benzene aromatic compounds mainly contribute positive aromas such as nuts, caramel, and floral / fruity notes, while ammonia aromatic compounds can enhance the aroma complexity and enrich the flavor. In summary, strain 9F... D. hansenii 9F can significantly improve the flavor characteristics of fermented sausages, specifically by reducing a variety of unpleasant flavor substances and increasing the level of beneficial aromatic compounds.

[0066] Electronic tongue analysis: The flavor profiles of fermented sausages in each treatment group on day 21 were analyzed using an electronic tongue. The PCA model analysis results are as follows: Picture 8 As shown in Figure C, PC1 and PC2 contributed 59.5% and 25.4% respectively, accounting for a cumulative 84.9% of the total variance. The PCA score plot shows that the FO group is clearly separated from the other three groups (CK, CO, FK) with no overlap, indicating that the overall taste characteristics of the FO group are fundamentally different from the other groups. This may be due to... D. hansenii During the degradation of high-concentration OTA, the metabolic pathways or products of 9F changed, producing key substances that affect the final taste. No significant separation was observed between the FK group and the CK and CO groups, indicating that individual inoculation... D. hansenii 9F does not fundamentally change the flavor profile of fermented sausage.

[0067] The response values ​​of each flavor index are compared in Table 2 and... Picture 8 As shown in D, the FO group differed significantly from the other three groups in terms of sourness, bitterness, astringency, bitter aftertaste, sour aftertaste, umami, and flavor richness (P < 0.05). Specifically, all treatment groups had lower sourness response values, while the FO group had an even lower sourness value; simultaneously, the FO group also had lower bitterness, astringency, bitter aftertaste, and sour aftertaste values, as well as higher flavor richness. This indicates that when... D. hansenii When 9F coexists with high concentrations of OTA, it can significantly improve the flavor quality of fermented sausages, reduce the intensity of various unpleasant flavors, and enhance the overall richness and complexity of the flavor. This may be because the presence of OTA stimulates... D. hansenii 9F produced more hydrolytic enzymes, promoting protein and fat metabolism and generating a large amount of esters that contribute fruity and sweet aromas while masking bitterness. Furthermore, the saltiness value of the FK group was lower than that of the CK group (P < 0.05), which may be related to... D. hansenii 9F increases the water activity of the sausage, which is related to the dilution of saltiness perception.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Hansenbali yeast ( Debaryomyces hansenii )9F, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 2026936.

2. A microbial preparation, characterized in that, It contains the Hansenula balizyme 9F as described in claim 1.

3. The use of the Hansenula d'Barry yeast 9F as described in claim 1 or the microbial preparation as described in claim 2 in the degradation of ochratoxin A.

4. The application according to claim 3, characterized in that, The active substance that degrades ochratoxin A is a metalloproteinase within the cells of *Hansøe du Balémys 9F*.

5. The application according to claim 3, characterized in that, The pH conditions for the degradation are 6 to 8.

6. The use of the Hansenula d'Barry yeast 9F as described in claim 1 or the microbial preparation as described in claim 2 in the preparation of fermented meat products.

7. The application according to claim 6, characterized in that, The fermented meat product is a fermented sausage.

8. A method for preparing fermented sausage, characterized in that, This includes inoculating minced meat with the Hansenula d'Barry yeast 9F as described in claim 1 or the microbial preparation as described in claim 2, and then fermenting it.

9. The preparation method according to claim 8, characterized in that, The inoculum amount of *Hansenula d'Barry* 9F was 3% of the weight of the minced meat, and the concentration of the inoculum suspension was 10. 8 CFU / mL.

10. The preparation method according to claim 8, characterized in that, The fermentation conditions are as follows: ferment at 28℃ and 85%~90% relative humidity for 3 days, and then ferment at 15℃ and 75%~85% relative humidity for 18 days.

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