Hansenula debayii 3cc20, a preparation method and application thereof

Abstract

This application provides a *Hansophila barley* 3CC20 strain, its preparation method, and its applications. The *Hansophila barley* 3CC20 strain has the accession number CGMCC28442. A bacterial suspension or starter culture is prepared using *Hansophila barley* 3CC20. The suspension or starter culture is then applied to meat pieces or ham at an inoculation rate of 3% or 5% of the meat weight, and fermented in a constant temperature and humidity incubator for a predetermined time. This application enables the extraction of *Hansophila barley* 3CC20 from the surface and shallow layers near the surface of fermented meat products. Furthermore, *Hansophila barley* 3CC20 exhibits high salt tolerance, nitrate tolerance, acid tolerance, strong acid production capacity, and strong antioxidant properties. *Hansophila barley* 3CC20 promotes meat product fermentation, shortens the meat product production cycle, ensures the hygiene and safety of meat products, and maintains the original unique flavor of fermented meat products.

Description

Technical Field

[0001] This application relates to the field of food microbiology technology, and more specifically, to a Hansenula d'Barry yeast 3CC20 and its preparation method and application. Background Technology

[0002] Cured meat products are outstanding representatives of traditional Chinese meat products, known for their rich aroma and long shelf life, making them popular among Chinese consumers. Traditional cured meat products are made from livestock and poultry meat, cured with salt and spices, and then processed through air-drying and maturation under suitable temperature conditions. Traditional cured meat products rely primarily on natural fermentation, resulting in a long production period and significant environmental influences.

[0003] In summary, there is a need to provide an improved technical solution that addresses the shortcomings of the existing technology. Summary of the Invention

[0004] The purpose of this application is to provide a Hansenula balizyme 3CC20 and its preparation method and application, which can extract Hansenula balizyme 3CC20 on the surface and shallow layer near the surface of fermented meat products, and can be applied to the preparation of fermented meat blocks, bacon or ham.

[0005] In a first aspect, a Hansenula d'Barry yeast strain 3CC20 is provided, the preservation number of which is CGMCC28442.

[0006] According to a second aspect of this application, a method for preparing Hansenula d'Barry yeast 3CC20 is also provided, comprising the following steps:

[0007] S1. Isolate the target strain;

[0008] S2. Purify the target strain and determine the species of the target strain.

[0009] In one feasible embodiment, step S1 includes at least the following:

[0010] S11, Enrichment culture of the target strain;

[0011] S12. Isolation of the target strain.

[0012] In one implementable manner, step S2 includes at least the following:

[0013] Physiological and biochemical identification and ITS rDNA identification of the strain were performed to determine the species of the target strain.

[0014] In one feasible manner, after step S2, the functional characteristics of Hansenula barley yeast 3CC20 are evaluated and various enzymatic properties of Hansenula barley yeast 3CC20 are determined.

[0015] In one feasible approach, evaluating the functional properties of *Hansophila barley* 3CC20 includes: determining the growth curve of *Hansophila barley* 3CC20; and determining the optimal growth temperature of *Hansophila barley* 3CC20 based on the growth curve, thus providing a basis for the application of *Hansophila barley* 3CC20.

[0016] According to a third aspect of this application, an application of Hansendberry yeast 3CC20 is also provided, including the following: using Hansendberry yeast 3CC20 to prepare fermented meat chunks, bacon, or ham.

[0017] In one feasible manner, the application of Hansenula d'Barry yeast 3CC20 in the preparation of fermented meat blocks specifically includes the following steps:

[0018] A bacterial suspension was prepared using Hansenula barley yeast 3CC20;

[0019] Prepare raw meat; marinate the raw meat; apply 3CC20 bacterial suspension to the marinated meat pieces at an inoculation rate of 5% of the meat weight; hang the inoculated meat pieces in a constant temperature and humidity incubator at 25℃ and 70% relative humidity for 60 days of fermentation.

[0020] In one feasible manner, the application of Hansenula de Barry yeast 3CC20 in the preparation of cured meat specifically includes the following steps:

[0021] A starter culture was prepared using Hansenula de Barry yeast 3CC20.

[0022] The smoked ingredients and the bacterial suspension of Hansenbali yeast 3CC20 were applied to the marinated meat pieces; the inoculated meat was then suspended in a constant temperature and humidity incubator at 25℃ and 60%-70% relative humidity for 30 days; the fermented meat was then dried in a 50℃ drying oven.

[0023] One feasible approach involves the application of Hansohde Barry yeast 3CC20 in the preparation of ham. Specifically, it includes the following:

[0024] A bacterial suspension was prepared using Hansenula barley yeast 3CC20;

[0025] Prepare the raw meat; marinate the raw meat; coat the marinated meat pieces with 3CC20 bacterial suspension at an inoculation rate of 3% of the meat weight; hang the inoculated meat pieces in a constant temperature and humidity incubator for fermentation for a predetermined time; the fermentation temperature is 25℃, the relative humidity is 60%-70%, and the fermentation time is 90 days.

[0026] Compared with the prior art, the beneficial effects of this application are as follows:

[0027] In the technical solution of this application, Hansenula d'Barry yeast 3CC20 can be extracted from the surface and shallow layer near the surface of fermented meat products. Furthermore, Hansenula d'Barry yeast 3CC20 possesses characteristics such as high salt tolerance, nitrate tolerance, acid tolerance, strong acid production capacity, and strong antioxidant properties. Hansenula d'Barry yeast 3CC20 promotes meat product fermentation, shortens the meat product production cycle, ensures the hygiene and safety of meat products, and maintains the original unique flavor of fermented meat products. Attached Figure Description

[0028] Figure 1 This is a colony morphology diagram of Hansenula d'Barry yeast 3CC20 according to an embodiment of the present invention.

[0029] Figure 2 This is a cell morphology diagram of Hansenula d'Barry yeast 3CC20 according to an embodiment of the present invention.

[0030] Figure 3 This is the phylogenetic tree of the Hansenula d'Barry yeast 3CC20 constructed according to an embodiment of the present invention.

[0031] Figure 4 This is the growth curve of Hansenula d'Barry yeast 3CC20 according to an embodiment of the present invention. Detailed Implementation

[0032] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0033] Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0034] According to the first aspect of this application, see Figure 1 Firstly, a strain of Debaryomyces hansenii, 3CC20, is provided. The preservation number of Debaryomyces hansenii is CGMCC NO.28442; the biological preservation unit is the China General Microbiological Culture Collection Center (CGMCC); the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; and the deposit date is September 12, 2023.

[0035] According to a second aspect of this application, a method for preparing Hansenula d'Barry yeast 3CC20 is also provided, comprising the following steps:

[0036] S1, Isolate the target strain.

[0037] Before step S1, obtain the bacterial strain.

[0038] Ham produced using traditional methods and sold in the market was used. Five processing groups were set up, and samples were taken from the four corners and the center of the ham in each group and mixed together, totaling 500g of sample. After being labeled, the samples were placed in a sampling box containing dry ice and stored at -20℃ in the laboratory until use.

[0039] Step S1 includes at least the following:

[0040] S11, Enrichment culture of the target strain.

[0041] Weigh 10g of the minced sample and place it in a sterile beaker. Add 90mL of 0.85% sterile saline, then homogenize at 5000rpm / min for 10min. Let it stand for 10min, collect the supernatant, and perform serial dilutions to a final concentration of 10. -3 10 -4 10 -5 Take 100 μL of the diluted solution and spread it onto yeast extract peptone glucose medium (PDA solid medium) using a spreader. Let it stand for 30 min, then invert it and incubate at 28°C for 48 h.

[0042] S12. Isolation and purification of the target strain.

[0043] Select single colonies for microscopic examination and choose strains with yeast colony morphology.

[0044] Single colonies were streaked onto PDA solid medium for isolation and incubated at 28°C for 48 hours. After streaking and isolation twice, the pure bacteria were inoculated into liquid medium and incubated at 28°C for 24 hours. The bacterial culture was then collected for preservation and stored at -80°C.

[0045] This strain exhibits raised, round colonies on PDA solid agar plates. Figure 1 and Figure 2 As shown, the colonies of the screened yeast strains are round, 2-3 mm in size, milky white, raised, viscous, smooth, opaque, moist and glossy. The cell morphology is mostly oval or spherical, and they reproduce by budding.

[0046] S2. Physiological and biochemical identification and ITS rDNA identification of the target strain to determine the species of the target strain.

[0047] Specifically, the Ezup column-based yeast genomic DNA extraction kit was used to extract DNA from the target strain. The ITS rDNA PCR gene was amplified using the yeast forward primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3' and the reverse primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3'. The gene sequencing results were assembled using DNAMAN 8.0 software. The assembled sequences were then subjected to BLAST alignment analysis using the NCBI database to determine the species. Phylogenetic trees were constructed using MEGA 7.0 software, selecting gene sequences from highly homologous type strains in the database and comparing them with the target strain sequences. Figure 3 As shown, the taxonomic position of the target strain was determined to be *Hansøe du Baléri*, and it was named *Hansøe du Baléri* 3CC20.

[0048] In one feasible approach, the functional characteristics of Hansson's barley yeast 3CC20 are evaluated and various enzymatic properties of Hansson's barley yeast 3CC20 are determined to confirm that Hansson's barley yeast 3CC20 can be used for ham fermentation, specifically including the following:

[0049] a. Determine the growth curve of Hansenula d'Barry yeast 3CC20, such as Figure 4 As shown in the figure. The optimal growth temperature of Hansenula baryonia 3CC20 was determined based on the growth curve, providing a basis for the application of Hansenula baryonia 3CC20.

[0050] The strain of *Hansophila barley* 3CC20 was inoculated into simulated broth liquid medium and cultured at 25°C for 48 hours. Using a 723 spectrophotometer at a wavelength of 600 nm, with a blank medium as a control, the OD value was measured every 4 hours. The results were recorded, and a growth curve was plotted. Based on the growth curve, the optimal growth temperature of *Hansophila barley* 3CC20 was determined to be 25°C, providing a basis for the application of *Hansophila barley* 3CC20.

[0051] b. Detect and obtain the salt tolerance, acid tolerance and acid production of Hansenula d'Barry yeast 3CC20.

[0052] Specifically, the salt tolerance of Hansenula balizyme 3CC20 was tested and obtained by inoculating Hansenula balizyme into YPD medium containing 10% sodium chloride at a 1% inoculum, culturing for 24 hours, and measuring the absorbance at 600 nm using a blank medium as a control.

[0053] The acid resistance of Hansenula d'Barry yeast 3CC20 was detected and obtained by inoculating it at a 1% inoculum into YPD liquid medium at pH 4.5 and culturing for 24 h. The absorbance was measured at 600 nm using a blank medium as a control.

[0054] The detection and acquisition of acid production of Hansenula d'Bary yeast 3CC20 included: inoculating 1% Hansenula d'Bary yeast 3CC20 into a pH 6.1 simulated broth medium, culturing for 48 h, and measuring the pH using a blank medium as a control.

[0055] c. Detect and obtain the DPPH removal rate of Hansenula d'Barry yeast 3CC20.

[0056] Hansenula d'Barry yeast 3CC20 was inoculated into YPD liquid medium at a 1% inoculum size. When the OD value reached approximately 0.8, the medium was centrifuged at 10,000 rpm for 10 minutes, washed twice with 0.1 mol / L phosphate buffer, and resuspended. Samples were added according to the settings in Table 1. After sample addition, the medium was incubated at 25°C for 30 minutes, centrifuged at 4,000 rpm for 5 minutes, and the OD value was measured at 517 nm using a 723 visible-ultraviolet spectrophotometer. The absorbance of anhydrous ethanol was used as the zero mark.

[0057] Table 1. DPPH scavenging rate of Hansenula d'Bary yeast 3CC20 (sample loading table)

[0058] Comparison experimental group blank Sample (ml) 0.5 0.5 0 Anhydrous ethanol (ml) 0.5 0 0.5 0.02 mol / L DPPH (ml) 0 0.5 0.5

[0059] d. The ability of Hansenula d'Bary yeast 3CC20 to inhibit hydroxyl radicals, total antioxidant capacity, and superoxide anion resistance were tested respectively. The results are shown in Table 2.

[0060] It should be noted that Table 2 also includes the test results of salt tolerance, acid tolerance, acid production, DPPH scavenging rate, and OD value of Hansenula d'Bary yeast 3CC20.

[0061] Table 2 Functional Evaluation of Hansenula d'Bary yeast 3CC20

[0062]

[0063] e. A study was conducted to determine the various enzyme activity characteristics of *Hansophila barley* 3CC20, specifically including: testing the protease, lipase, nitrite tolerance, nitrate reductase activity, and nitrite degradation capacity of *Hansophila barley* 3CC20 to determine its various enzyme activities, antioxidant capacity, and ability to act as a fermentation carbon source. The specific test results are shown in Table 3.

[0064] Table 3. Evaluation of the properties of Hansenula d'Bary yeast 3CC20

[0065]

[0066] It should be noted that the "+" reaction in Table 3 indicates a positive result, growth, or a very strong result; the "-" reaction in Table 3 indicates a negative result, no growth, or a very weak result.

[0067] In one feasible method, the agar perforation method is used for protease activity identification. The perforation diameter is 0.6 cm. 100 μL of activated Staphylococcus bacterial culture is added to the agar wells of the skim milk powder protease identification plate. The plate is allowed to diffuse statically at 4°C for 30 min, and then incubated at 30°C for 2 days. The presence of hydrolysis zones on the protease identification medium is then observed.

[0068] When identifying lipase, yeast cells cultured on plates for 24 hours were picked, and single colonies were inoculated onto tributyric acid lipase identification plates using the spot inoculation method. The plates were then incubated at 28°C for 2 days, and the presence of a clear zone around the colony was observed.

[0069] To identify nitrate reductase activity, the test strain is inoculated into nitrate-reducing medium and cultured at 30°C for 48 hours. Then, one drop each of Griess reagent A and B is added to the bacterial solution. If the solution turns pink, rose red, orange, or brown, it indicates the presence of nitrite and is a positive result for nitrate reduction.

[0070] To assess the nitrite degradation capacity, activated third-generation yeast was inoculated at a 1% inoculation rate into YPD medium containing 100 mg / L nitrite and cultured in a shaker at 28°C for 48 h.

[0071] For the determination of iron ion reducing capacity, the cultured bacterial solution was centrifuged at 10000 rpm for 8 min, resuspended in 0.2 mol phosphate buffered saline (PBS), and the bacterial concentration was adjusted to 10⁷ CFU / mL. 1 ml of sample, 1 ml of 0.1% potassium ferricyanide solution, and 1 ml of PBS were added sequentially to a 5 ml centrifuge tube, and the mixture was incubated in a 50°C water bath for 20 min. After rapid cooling, 1 ml of 10% trichloroacetic acid (TCA) solution was added, and the mixture was centrifuged at 3000 rpm for 5 min. 3 ml of the supernatant was added to a 5 ml centrifuge tube, and 0.4 ml of 0.1% ferric chloride was added. The reaction was allowed to proceed for 10 min, and the absorbance was measured at 600 nm using a UV-Vis spectrophotometer, expressed as L-cysteine ​​equivalents.

[0072] When determining the free radical scavenging ability of ABTS, follow the instructions in the kit.

[0073] In the nitrite tolerance assessment, Hansenula d'Barry yeast 3CC20 was inoculated into YPD liquid medium at an inoculation rate of 1% and cultured at 37°C for 48 h. The absorbance was measured at a wavelength of 600 nm using a UV-Vis spectrophotometer.

[0074] Based on the above tests, it was determined that the Hansenbali yeast 3CC20 provided in this application exists in large quantities on the surface and in the shallow layer near the surface of fermented meat products, and can produce the unique flavor of yeast.

[0075] In one feasible approach, *Hansophila barley* 3CC20 was also subjected to sugar fermentation and carbon source assimilation experiments to ensure its effectiveness in ham fermentation. The experimental results are shown in Tables 4 and 5, respectively.

[0076] Table 4 Results of sugar fermentation experiment

[0077]

[0078] It should be noted that "++" indicates a very strong positive reaction, meaning the sugar has a strong ability to ferment; "+" indicates a strong positive reaction, meaning the sugar has a strong ability to ferment; and "-" indicates a negative reaction, meaning the sugar cannot be fermented.

[0079] Table 5 Carbon source assimilation experiment

[0080]

[0081] It should be noted that "++" indicates a very strong positive result, meaning a strong ability to assimilate the carbon source; "+" indicates a strong positive result, meaning a strong ability to assimilate the carbon source; and "-" indicates a negative result, meaning the carbon source cannot be assimilated.

[0082] According to a third aspect of this application, an application of Hansenula d'Barry yeast 3CC20 is also provided, comprising the following:

[0083] Example 1:

[0084] The application of Hansenula d'Barry yeast 3CC20 in the preparation of fermented meat blocks includes the following steps:

[0085] Prepare the raw meat; marinate the raw meat; coat the marinated meat pieces with 3CC20 bacterial suspension at an inoculation rate of 5% of the meat weight; hang the inoculated meat pieces in a constant temperature and humidity incubator for fermentation for the predetermined time.

[0086] In this embodiment, 15 kg of pig hind leg was selected, washed with cold water to remove surface oil, dirt, connective tissue, tendons, and muscle membranes, drained, and cut into pieces of approximately 500 g each. The meat was then cured with standard salt at 8.0% of its weight, rubbed onto the surface, and cured at 4°C for one week. The cured meat pieces were rinsed with sterile water for 1 minute and blotted dry with filter paper. At an inoculation rate of 5% of the meat weight, the prepared 3CC20 bacterial suspension was thoroughly coated onto the meat pieces. The control group was not inoculated. The inoculated meat pieces were then suspended in a constant temperature and humidity incubator at 25°C and 70% relative humidity for 60 days of fermentation.

[0087] In one feasible manner, the results analysis of Hansenula de Barry yeast 3CC20 in ham fermentation specifically includes:

[0088] Meat chunks fermented for 60 days were used for color difference, volatile substances, sensory evaluation, and determination of malondialdehyde (MDA) content. The color difference comparison results are shown in Table 7, the sensory evaluation results are shown in Table 8, the determination results of malondialdehyde (MDA) content are shown in Table 9, and the volatile substances results are shown in Table 10.

[0089] Table 7 Comparison Results of Color Difference Values

[0090]

[0091] As shown in Table 7, the L value of the inoculated group was not significantly different from that of the control group, but the redness value was higher, indicating that Hansenula d'Barry yeast 3CC20 has a certain color-developing effect in meat. Furthermore, Hansenula d'Barry yeast 3CC20 can prevent excessive lipid oxidation.

[0092] Table 8 Sensory Evaluation Results

[0093]

[0094] As shown in Table 8, the inoculation group significantly improved the color, texture, and aroma of the fermented ham, provided a good flavor, and significantly improved the overall acceptability of the fermented ham.

[0095] The level of lipid oxidation can be detected by measuring the malondialdehyde (MDA) content in ham. The results of the MDA content determination are shown in Table 9.

[0096] Table 9 Comparison of malondialdehyde (MDA) content in fermented meat chunks

[0097] Measurement indicators control group Inoculation group Malondialdehyde content (mg / kg) <![CDATA[0.876±0.025 a ]]> <![CDATA[0.515±0.024 b ]]>

[0098] It should be noted that different lowercase letters in the same column of Tables 7, 8, and 9 indicate significant differences (p<0.05).

[0099] A higher malondialdehyde (MDA) content indicates a more severe oxidation process. As shown in Table 9, the MDA content in the inoculated group was significantly lower than that in the control group, indicating that the ability of Hansenula d'Barry yeast 3CC20 to resist superoxide anions, provide total antioxidant activity, inhibit hydroxyl radicals, and scavenge free radicals with DPPH plays an important role in the process of excessive lipid oxidation.

[0100] Volatile flavor compounds in fermented Xuanwei ham were detected. Headspace solid-phase microextraction (HSP) was used to extract the compounds from 60-day fermented ham blocks. 3.00 g of chopped ham was accurately weighed into a headspace vial, and 3 μL of 1,2-dichlorobenzene was added as an internal standard. The vial was then sealed with a polytetrafluoroethylene (PTFE) silicone septum. The vial was then placed in a shaker at 45°C for 10 min to equilibrate, followed by extraction at 45°C for 30 min. Finally, gas chromatography-mass spectrometry (GC-MS) was used for qualitative and quantitative analysis. A DB-Wax (0.25 mm × 0.25 μm × 30 m) capillary column was used. The injection port temperature was 250°C, and desorption at the SPME fiber-tipped GC port was performed for 5 min. The injection method was split injection with a split ratio of 1:5. The column oven temperature program was as follows: initial temperature 40℃, hold for 2 min, increase to 90℃ at 3℃ / min, hold for 5 min, then increase to 200℃ at 3℃ / min, and finally increase to 230℃ at 15℃ / min, hold for 10 min. Helium was used as the carrier gas, and the column flow rate was 1.5 mL / min. The interface temperature was 280℃, and the ion source temperature was 230℃.

[0101] Table 10 Comparison Table of Volatile Matter Measurements in Fermented Xuanwei Ham

[0102]

[0103]

[0104] As shown in Table 10, a total of 47 flavor compounds were detected in the inoculated group, including 11 esters, 5 aldehydes, 10 alcohols, 4 benzenes, 8 alkanes, 3 ketones, 3 pyrazines, 1 furan, 1 olefin, and 1 ether. A total of 46 flavor compounds were detected in the control group, including 8 esters, 5 aldehydes, 9 alcohols, 4 benzenes, 3 ketones, 3 pyrazines, 1 furan, 10 alkanes, 1 olefin, 1 ether, and 1 amine. The total amounts of aldehydes, alcohols, and esters in the inoculated group were significantly higher than those in the control group, indicating that 3CC20 yeast can significantly increase the content of esters, aldehydes, and alcohols in meat products. Compared with the flavor compounds of Xuanwei ham, the inoculated group had 18 identical flavor compounds, with a total similarity of 763.58 μg / kg, which was higher than the 590.01 μg / kg of the finished product. Although the control group had 15 identical flavor compounds to those of Xuanwei ham, the total similarity was less, at only 130.92 μg / kg.

[0105] Example 2:

[0106] The application of Hansenula d'Barry yeast 3CC20 in the preparation of cured meat includes the following steps:

[0107] Pork belly was used as the raw material and cut into multiple pieces, each 5cm wide and weighing 1kg. A starter culture was prepared using Hansenula polymorpha 3CC20. The pork was cured with standard salt at 3% of its weight, rubbed into the surface, and then cured at 4℃ for one week. Smoking materials and the prepared 3CC20 bacterial suspension were thoroughly coated onto the pork pieces. The inoculated pork slices were then hung in a constant temperature and humidity incubator at 25℃ and relative humidity controlled at 60%-70% for 30 days of fermentation. The fermented pork was then dried in a 50℃ drying oven.

[0108] It should be noted that a control group was set up during the preparation of cured meat, and the control group was not inoculated with bacteria.

[0109] In one feasible method, the preparation of the starter culture includes at least the following: activating and subculturing Hansenula baiji 3CC20 three times is inoculated into YPD liquid medium at a ratio of 1%, and cultured at 28°C and 200 rpm for 48 h to obtain the bacterial culture; the culture is then centrifuged at 4000 rpm for 10 min, resuspended in 0.85% sterile physiological saline, and the bacterial concentration is adjusted to 10⁷ CFU / ml to obtain the Hansenula baiji starter culture.

[0110] This embodiment also includes: sensory analysis of cured meat fermented with Hansendberry yeast 3CC20, and the analysis results are shown in Table 11.

[0111] Table 11 Sensory evaluation results

[0112] Organizational status color taste Flavor Total acceptability control group <![CDATA[6.44±0.57 b ]]> <![CDATA[7.72±0.63 a ]]> <![CDATA[6.75±0.55 b ]]> <![CDATA[6.98±0.75 b ]]> <![CDATA[6.23±0.28 b ]]> Inoculation group <![CDATA[7.81±0.29 a ]]> <![CDATA[8.05±0.72 a ]]> <![CDATA[7.78±0.73 a ]]> <![CDATA[7.55±0.86 a ]]> <![CDATA[7.29±0.78 a ]]>

[0113] It should be noted that the sensory evaluation method in Table 11 was as follows: a 20-person evaluation panel composed of food professionals scored the samples, with a scoring standard of 10 points. As shown in Table 11, the cured meat produced by fermentation with Hansenula d'Barry yeast 3CC20 in this embodiment showed a significantly better final flavor acceptability than the control group.

[0114] Specifically, a score of 1-4 indicates: loose cut surface; dull color; no aroma or rancid smell; a score of 4-6 indicates: slightly scattered cut surface; dark red cut surface; slight ham aroma; a score of 6-10 indicates: dense cut surface; elastic; red or rose red color; strong aroma.

[0115] Example 3:

[0116] The application of Hansenula bale yeast 3CC20 in the preparation of Xuanwei ham. Specifically, it includes the following:

[0117] Prepare the raw meat; marinate the raw meat; coat the marinated meat pieces with 3CC20 bacterial suspension at an inoculation rate of 3% of the meat weight; hang the inoculated meat pieces in a constant temperature and humidity incubator for fermentation for the predetermined time.

[0118] Specifically, in this embodiment, 10 kg of whole pig hind legs were selected, washed with cold water to remove surface oil, dirt, connective tissue, tendons, and muscle membranes, and drained. The meat was then cured with standard salt at 8.0% of its weight, rubbed onto the surface, and cured at 4°C for 3 weeks. The cured meat pieces were rinsed with sterile water for 1 minute and blotted dry with filter paper. The prepared 3CC20 bacterial suspension was thoroughly coated onto the meat pieces at an inoculation rate of 3% of the meat weight. The control group was not inoculated. The inoculated meat slices were suspended in a constant temperature and humidity incubator at 25°C and 60%-70% relative humidity for 90 days of fermentation.

[0119] Results analysis of Hansenula d'Barry yeast 3CC20 in ham fermentation was conducted, specifically including: determination of protein, color difference, sensory evaluation, and malondialdehyde (MDA) content in meat blocks fermented for 90 days. The comparison results of protein, color difference, and MDA content are shown in Table 12, and the sensory evaluation results are shown in Table 13.

[0120] Table 12 Physicochemical Indicators of Fermented Xuanwei Ham

[0121]

[0122]

[0123] Table 12 shows that the redness value of the inoculated group increased under the reducing effect of Hansenula baryoniae 3CC20. Under the antioxidant effect of Hansenula baryoniae 3CC20, the malondialdehyde content decreased significantly.

[0124] This embodiment also includes: sensory analysis of ham fermented with Hansendberry yeast 3CC20, and the analysis results are shown in Table 13.

[0125] Table 13 Sensory evaluation results

[0126]

[0127] Note: Different lowercase letters in the same row of the table indicate significant differences (p<0.05).

[0128] It should be noted that the sensory evaluation method in Table 13 is as follows: a 20-person evaluation panel composed of food professionals will score the samples, with a scoring standard of 10 points. 1-4 points indicate: loose cut surface; dull color; no aroma or off-odor; poor acceptability; 4-6 points indicate: slightly scattered cut surface; rose-red color; slight aroma; acceptable; 6-10 points indicate: dense cut surface; bright color; strong aroma; good acceptability.

[0129] As shown in Table 13, the ham produced by fermentation with Hansenula d'Barry yeast 3CC20 in this embodiment showed significantly better final flavor acceptability than the control group. It significantly improved the texture and structure of the fermented ham, provided a good flavor, and markedly increased the overall acceptability of fermentation in ham.

[0130] In summary, this application enables the extraction of *Hansenula d'Barry* 3CC20 from the surface and shallow near-surface layers of fermented meat products. Furthermore, *Hansenula d'Barry* 3CC20 exhibits characteristics such as high salt tolerance, nitrate tolerance, acid tolerance, strong acid production capacity, and strong antioxidant properties. *Hansenula d'Barry* 3CC20 promotes meat product fermentation, shortens the meat product processing cycle, ensures the hygiene and safety of meat products, and maintains the original unique flavor of fermented meat products.

[0131] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A type of Hansenula baiji ( Debaryomyces hansenii 3CC20, characterized in that, The Hansenula d'Barry yeast 3CC20 has the accession number CGMCC NO.28442.

2. An application of Hansenula d'Barry yeast 3CC20 as described in claim 1, characterized in that, include: Hansenula basil yeast 3CC20 can be used to prepare cured meat or ham.

3. The application of Hansenula d'Barry yeast 3CC20 according to claim 2, characterized in that, The application of Hansenula d'Barry yeast 3CC20 in the preparation of cured meat includes the following steps: A starter culture was prepared using Hansenula de Barry yeast 3CC20. The smoked ingredients and the bacterial suspension of Hansenbali yeast 3CC20 were applied to the marinated meat pieces; the inoculated meat was then suspended in a constant temperature and humidity incubator at 25℃ and 60%-70% relative humidity for 30 days; the fermented meat was then dried in a 50℃ drying oven.

4. The application of Hansenula d'Barry yeast 3CC20 according to claim 2, characterized in that, The application of Hansenula d'Barley yeast 3CC20 in the preparation of ham includes the following: A bacterial suspension was prepared using Hansenula barley yeast 3CC20; Prepare the raw meat; marinate the raw meat; coat the marinated meat pieces with 3CC20 bacterial suspension at an inoculation rate of 3% of the meat weight; hang the inoculated meat pieces in a constant temperature and humidity incubator for fermentation for a predetermined time; the fermentation temperature is 25℃, the relative humidity is 60%-70%, and the fermentation time is 90 days.

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