A strain of Meyer yeast and its application in sausage fermentation

Abstract

A strain of Meyerozyma guilliermondii Y27 and its use in sausage fermentation. The yeast is Pichia guilliermondii Y27, deposited with the China Center for Type Culture Collection on April 30, 2024, with the accession number CCTCC No. M2024843. When inoculated with the strain, the fermentation characteristics of the strain meet the requirements of a meat starter, making it suitable for meat fermentation. When used in sausage fermentation, the strain promotes the production of free amino acids, inhibits fat oxidation in the sausage, significantly improves the flavor quality of the fermented sausage, controls the accumulation of biogenic amines, and enhances the safety of the sausage.

Description

Technical Field

[0001] The invention relates to a strain of Meyerozyma guilliermondii and application of the same in sausage fermentation, belonging to the technical field of microbial fermentation. Background Art

[0002] Fermented sausage, a traditional meat product, has a long history. As early as the ancient Roman period, over 2,000 years ago, people knew how to make delicious sausages from ground meat, salt, sugar, and spices, and these products had a long shelf life. The processing of fermented sausages typically includes steps such as pre-processing the raw meat, mincing, seasoning, curing, filling, fermentation, and air-drying. Fermentation is a core step, determining the sausage's final flavor and quality. During the fermentation process, conditions such as temperature, humidity, and fermentation time must be carefully controlled to ensure a smooth fermentation process. Furthermore, to achieve better flavor and quality, appropriate amounts of salt, sugar, fermentation agents, spices, and other auxiliary ingredients are added to the mix.

[0003] For a long time, fermented sausage production relied primarily on natural fermentation. This traditional fermentation method presents numerous challenges, including long production cycles, significant seasonal variations, and unclear microbial origins. With the continued industrialization of fermented meat products, the introduction of meat-grade starter cultures has become necessary to improve color, shorten processing cycles, enhance flavor, and improve product safety. Lactic acid bacteria, coagulase-negative cocci, molds, and yeasts are the most commonly used starter cultures. These cultures, isolated from traditional foods, can promote the standardized production of fermented meat products and enhance product safety.

[0004] In fermented sausages, yeast can work synergistically with microorganisms such as lactic acid bacteria, utilizing carbohydrates in sausages to produce acid and alcohol, thus imparting a unique flavor. This mixed fermentation method can enhance the product's flavor and color compared to pure lactic acid fermentation. Yeast metabolic activity produces a variety of aromatic compounds, such as alcohols, acids, and heterocyclic compounds, which interact with other sausage components to create a complex and appealing flavor. Yeast also has the ability to reduce nitrates, which helps improve the sausage's color. Yeast can break down fats and proteins, producing antioxidants such as catalase, thereby delaying the rancidity process and extending its shelf life. Yeast also has a competitive inhibitory effect on harmful microorganisms such as Staphylococcus aureus, helping to maintain the sausage's microbial balance and safety. Corral et al. found that inoculating dry-fermented sausages with yeast significantly increased the free fatty acid content and ester levels in the product. Dura et al. found that yeast accelerated the degradation of myofibrillar proteins during the initial drying phase of dry-fermented sausages and promoted the production of free amino acids at the end of fermentation. Yeast's antioxidant activity also contributes to the formation of esters.

[0005] In recent years, there has been increasing interest in the role of microorganisms in regulating the quality of fermented meat products. Yeast is the dominant fungus in fermented meat products, significantly influencing the flavor and sensory properties of the products. However, the impact of yeast on sausage quality and the mechanisms involved are still in their infancy. Therefore, screening yeast strains with excellent fermentation and growth properties and applying them to fermented sausages is of great significance. This provides a theoretical basis and strain resources for improving sausage quality, shortening processing cycles, promoting standardized production, and enhancing product safety. Summary of the Invention

[0006] The present invention aims to provide a strain of Meyer yeast and its application in sausage fermentation.

[0007] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a strain of Meyer yeast, wherein the Meyer yeast is Pichia guilliermondii Y27 (Meyerozyma guilliermondii Y27), which has been deposited in the China Center for Type Culture Collection (CCTCC) with a deposit date of April 30, 2024, and a strain deposit number of CCTCCNO: M 2024843.

[0008] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: the application of the Pichia guilliermondii Y27 in sausage fermentation.

[0009] The preferred technical solution is: comprising the following steps:

[0010] Step 1: Preparation of Pichia guilliermondii Y27 culture: Inoculate a slant of Pichia guilliermondii Y27 into YPD liquid activation medium and culture with shaking at 25-30°C for 24-36 hours. Transfer the resulting culture to YPD liquid medium using an inoculating loop and culture at 25-30°C for 28-40 hours to obtain an inoculum.

[0011] Step 2: Preparation of bacterial suspension: The inoculum obtained in step 1 was centrifuged at 3000-8000 × g for 5-15 min at 2-5°C, the precipitate was collected, washed with physiological saline and resuspended in physiological saline to prepare a bacterial suspension. The bacterial count of the bacterial suspension cell pellet was adjusted to 10 6 -10 8 CFU / ml;

[0012] The preferred technical solution is: comprising the following steps:

[0013] a. Add seasonings and auxiliary materials to the raw meat and stir evenly to obtain marinated meat;

[0014] b: Inoculate the bacterial suspension into the cured meat, mix well, and make sausages;

[0015] c. Fermentation: Fermentation at 20-28°C and 80-90% relative humidity for 40-50 hours;

[0016] d: Air drying: Ferment at 10-20°C and relative humidity of 60-80% for 10-15 days.

[0017] The preferred technical solution is: in step a, the raw meat is fresh pork front leg lean meat and fat, which is divided into blocks, washed in 3-10°C water, blood clots and pig skin are removed, the meat is minced and mixed according to a mass ratio of pork front leg lean meat to fat of 4-10:3.

[0018] The preferred technical solution is: the auxiliary materials include salt, glucose, nitrite and thirteen spices; the added amounts are: 2-3% salt, 1.5-2.5% glucose, 0.005-0.008% nitrite, and 0.05-0.15% thirteen spices based on the mass of the raw meat.

[0019] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0020] 1. Compared with traditional fermented sausages, the present invention effectively improves the overall color of the sausages and inhibits fat oxidation in the sausages by inoculating the yeast Guillermo Mayer Y27 of the present invention.

[0021] 2. The sausage fermented with the yeast Guillermo Mayer Y27 of the present invention significantly improves the flavor quality of the fermented sausage and controls the accumulation of biogenic amines. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a picture of the cell morphology of Pichia guilliermondii Y27 under an optical microscope.

[0023] Figure 2A This is the growth of Pichia guilliermondii Y27 at different temperatures.

[0024] Figure 2B This is the growth of Pichia guilliermondii Y27 under different acidity.

[0025] Figure 2C The figure shows the production of neutral protease by Pichia guilliermondii Y27 at different times.

[0026] Figure 3 It is the change in pH during sausage processing.

[0027] Figure 4 The texture of sausages in the inoculated group and the control group after 14 days.

[0028] Figure 5 This is a flowchart of sausage production. DETAILED DESCRIPTION

[0029] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0030] See also Figure 1-5 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no substantive technical significance. Any modification of the structure, change in the proportional relationship or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the efficacy and purpose that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0031] Deposit of biological materials:

[0032] Pichia guilliermondii Y27 has been deposited in the China Center for Type Culture Collection (CCTCC) on April 30, 2024, with the strain accession number C CTCCNO: M 2024843. The deposit address is: China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0033] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0034] Example 1: A strain of Meyer yeast and its application in sausage fermentation

[0035] 1. The steps for screening yeast are as follows:

[0036] The 72 yeast strains currently in the laboratory were subjected to methylene blue staining microscopy, mucus production test, hemolysis test, indole test, glucose gas production test, H2S production test, arginine ammonia production test, amino acid decarboxylase test, Vogel test, salt tolerance test, and acid tolerance test. The results are shown in Table 1. One strain, Pichia guilliermondii Y27, was selected, and its colony morphology, size, and methylene blue staining results are shown in Table 1.

[0037] Table 1: Yeast physiological and biochemical screening results

[0038]

[0039]

[0040] 2. Strain Identification

[0041] The strain was sent to a biological sequencing company for sequencing identification. The sequencing results were compared and searched using the basic local alignment search tool (BLAST) in the GenBank data of the National Center of Biotechnology Information (NCBI). The ITS rDNA sequence of the model strain with higher homology was selected, and the strain was identified as Meyerozyma guilliermondii. It was deposited in the China Center for Type Culture Collection (CCTCC) on April 30, 2024, with the deposit number CCTCC NO.M2024843. The address of the China Center for Type Culture Collection is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0042] ITS rDNA sequence:

[0043]

[0044] 3. Production adaptability test of Pichia guilliermondii Y27

[0045] The growth of Pichia guilliermondii Y27 at different temperatures is shown in Figure 2. Figure 2A As shown in the results, the biomass of Pichia guilliermongensis Y27 was the highest at 0.705 at 25℃. At 20℃-40℃, the biomass of Pichia guilliermongensis Y27 first increased and then decreased with the increase of temperature. The growth of Pichia guilliermongensis Y27 under different acidity is shown in Figure 2B As shown in the figure, the biomass of Pichia guilliermonensis Y27 reached a maximum of 0.69 when pH was 6.0, and the growth inhibition degree of Pichia guilliermonensis Y27 increased with decreasing pH. The production of neutral protease by Pichia guilliermonensis Y27 at different times was shown in the figure. Figure 2C As shown, the results showed that the neutral protease activity of Pichia guilliermondii Y27 was the highest at 24 hours, and after 24 hours, the enzyme production ability weakened with the increase of time.

[0046] Example 2: A strain of Meyer yeast and its application in sausage fermentation

[0047] This example provides a strain of Pichia guilliermondii Y27, which has been deposited with the China Center for Type Culture Collection (CCTCC) on April 30, 2024, with a strain deposit number of CCTCC NO. M2024843. The deposit address is: China Center for Type Culture Collection (Wuhan, China).

[0048] Applied to the above-mentioned sausage fermentation, the steps are as follows:

[0049] 1. Preparation of fermentation agent

[0050] Step 1: Preparation of Pichia guilliermondii Y27 culture: Inoculate a slant of Pichia guilliermondii Y27 into YPD liquid activation medium and culture with shaking at 25°C for 24 hours. Transfer the resulting culture to YPD liquid medium using an inoculating loop and culture at 25°C for 28 hours to obtain an inoculum.

[0051] Step 2: Preparation of bacterial suspension: The inoculum was centrifuged at 6000 × g for 10 min at 4 °C, and the precipitate was collected. The precipitate was washed twice with saline and resuspended in saline to prepare a bacterial suspension. The bacterial count of the bacterial suspension cell pellet was adjusted to 10 7 CFU / ml, set aside.

[0052] 2. Preparation of fermented sausages

[0053] Fresh pig front leg lean and fat were cleaned with water, and blood clots and pig skin were removed. The meat was minced and mixed to a lean to fat ratio of 7:3. A mixture of 2.5% salt, 2% glucose, 0.008% nitrite, and 0.1% thirteen-spice seasoning and auxiliary ingredients, along with a bacterial suspension, was added and mixed evenly. Sausages were then hung in a constant temperature and humidity environment for fermentation. Fermentation was performed at 25°C and 90% relative humidity for 48 hours, followed by air drying at 15°C and 70% relative humidity for 12 days. The group inoculated with Meyer yeast Y27 was designated the MG group, while the group not inoculated with the yeast was designated the CK group.

[0054] 3. Physical and chemical properties of fermented sausages

[0055] (1) Color analysis of fermented sausage

[0056] The color of sausages from the CK and MG groups was measured after air-drying for 12 days. The sausage samples were cut into 1.5 cm thick slices and their lightness (L*), yellowness (b*), and redness (a*) values ​​were measured using a colorimeter, and the average values ​​were calculated.

[0057] The results are shown in Table 2. Compared with the CK group, the inoculation of Meyer yeast can significantly increase the redness of the fermented sausage, significantly reduce the brightness value of the fermented sausage, and effectively improve the color quality of the product.

[0058] Table 2: Colour of sausages at the end of drying

[0059] L* a* b* CK 41.38±0.72 8.92±1.02 10.23±0.5 MG 35.79±0.29 11.3±1.4 10.64±0.87

[0060] (2) Determination of pH value of fermented sausage:

[0061] The pH value of the sausages was measured on days 0, 2, 5, 8, 11, and 14. Accurately weigh 10.0 g of sample, mince it, add 100 mL of distilled water and mix thoroughly, homogenize at 8000 × g for 30 seconds, filter, and measure the supernatant with a pH meter. Repeat three times and take the average value.

[0062] The results are as follows Figure 3 As shown in the figure, the pH of the CK group and the MG group were similar on the 0th day. After 2 days of fermentation, the pH of the sausages dropped rapidly to about 5.0. After 5 days, the pH of the two groups of sausages began to gradually increase, and the pH of the MG group was always higher than that of the CK group, indicating that the inoculated Meyer yeast was likely to inhibit the growth of some miscellaneous bacteria with strong acid-producing ability.

[0063] (3) Determination of malondialdehyde and peroxide value in sausages

[0064] a. Determination of malondialdehyde content in sausages: Take sausages on the 12th day after the last day of air drying, and determine the malondialdehyde content in the sausages by spectrophotometry according to the second method in GB 5009.181-2016 "National Food Safety Standard - Determination of Malondialdehyde in Foods".

[0065] b. Determination of peroxide value in sausages: Take sausages on the 12th day after the last day of air drying, and determine the peroxide value in the sausages according to the first method titration method of GB 5009.227-2016 "National Food Safety Standard for Determination of Peroxide Value in Foods".

[0066] TBARS is widely used to indicate lipid oxidation, which can cause rancid odors and flavors in meat and meat products, as well as changes in texture, color, and nutritional status. Peroxide value (TBARS) is an indicator of the degree of oxidation of fats and fatty acids, indicating whether a sample has deteriorated due to oxidation. The results, as shown in Table 3, show that inoculation with the yeast Mycobacterium quaternii significantly reduced the malondialdehyde content and TBARS in sausages compared to the CK group.

[0067] Table 3: Malondialdehyde and peroxide values ​​of sausages at the end of air drying

[0068] Malondialdehyde content (mg / kg) Peroxide value (g / 100g) CK 2.87±0.06 0.31±0.01 MG 2.29±0.05 0.08±0.01

[0069] (4) Determination of sausage texture

[0070] Sausages from the last day of air-drying were cut into 1-cm-thick slices and analyzed using a two-stroke compression test using a P / 50A probe. The test conditions were: a pre-test rate of 5 mm / s, a test rate of 2 mm / s, a post-test rate of 2 mm / s, a compression degree of 50%, and a dwell time of 5 seconds. The results included seven indicators: hardness, stickiness, chewiness, elasticity, cohesion, adhesion, and recovery value.

[0071] The results of texture are as follows Figure 4 As shown in the figure: the hardness, viscosity, chewiness, elasticity, cohesion, adhesion and recovery value of the sausage in the inoculated group were higher than those in the control group, indicating that inoculation can promote the fermentation of sausage and improve the texture of sausage.

[0072] 4. Determination of biogenic amine content in fermented sausages

[0073] 1. Sample Preparation

[0074] Take 5.00 g of sausage from the last day of air-drying, mince it, and homogenize it in 10 mL of 0.6 M cold perchloric acid for 1 minute. Centrifuge the homogenate at 10,000 rpm for 15 minutes, and filter the supernatant into a 25 mL volumetric flask. Repeat the extraction with 10 mL of 0.6 M perchloric acid solution, and finally adjust the volume to 25 mL with 0.6 M perchloric acid. Add 0.2 mL of the sample to 40 μL of 2 M NaOH, 60 μL of saturated NaHCO₃, 400 μL of 10 mg / mL dansyl chloride, and then 20 μL of NH₄OH. Remove any residual dansyl chloride by adding 20 μL of 25% aqueous ammonia. Incubate in a 40°C water bath for 45 minutes. Incubate in the dark for 30 minutes, then add acetonitrile to 1 mL, filter, and analyze by HPLC.

[0075] 2. Chromatographic conditions

[0076] The chromatographic column was a Waters-symmetry C18 (4.6 mm × 250 mm, 5 μm) with a mobile phase consisting of ammonium acetate (0.1 M; solvent A) and acetonitrile (solvent B). Elution was performed using the following gradient: 0 min, 50% B; 25 min, 90% B; 35 min, 90% B; 45 min, 50% B. Samples were detected at 254 nm with a 10 μL injection volume, and identification was performed based on retention time.

[0077] Biogenic amines are a general term for a class of biologically active, nitrogen-containing, low-molecular-weight organic compounds. They are found in most foods and are primarily produced by the decarboxylation of amino acids by microbial amino acid decarboxylases. They are considered potentially harmful to humans. As shown in Table 4, sausages inoculated with the yeast Mycobacterium juncea significantly reduced histamine, tyramine, spermine, and putrescine compared to the CK group, with putrescine being the most significantly reduced, by 61.6%. The total biogenic amine content in the CK group was 200.16 mg / kg, while that in the MG group was 113.15 mg / kg, a significant decrease of 43.5%. Therefore, inoculation with the yeast Mycobacterium juncea effectively reduces the formation of biogenic amines in fermented sausages, improving product safety.

[0078] Table 4 Biogenic amine content of fermented sausages at the end of air drying

[0079]

[0080] 5. Determination of volatile compounds in fermented sausages

[0081] The volatile flavor compounds in fermented sausages were analyzed by headspace solid phase microextraction-gas chromatography-mass spectrometry. 2.0 g of sausages were taken from the last day of air drying, chopped, and placed in a 20 mL headspace sample vial. Subsequently, 10 μL of 2,4,6-trimethylpyridine at a concentration of 0.1 g / L was added as an internal standard. After equilibration at 60 ° C for 5 minutes, an SPME fiber coated with carbon / polydimethylsiloxane (CAR / PDMS) was inserted and adsorbed for 30 minutes. The CAR / PDMS fiber was then inserted into the air inlet and thermally desorbed at 230 ° C for 5 minutes.

[0082] The chromatographic column was DB-WAX (30 m × 0.25 mm × 0.25 μm); the injection port temperature was 250°C; injection was performed in splitless mode; helium was used as the carrier gas at a flow rate of 1 mL / min; the temperature program was as follows: initial column temperature 40°C, hold for 5 min; then increase the temperature at 2°C / min to 90°C without dwelling; then increase the temperature at 5°C / min to 180°C without dwelling; then increase the temperature at 10°C / min to 230°C, hold for 8 min. Mass spectrometry conditions included an ion source temperature of 230°C; and a mass scan range of 30–550 m / z.

[0083] The results, as shown in Table 5, showed significantly higher levels of volatile compounds in the MG group than in the CK group. While the CK group contained 20 volatile compounds with a total content of 4337.37 μg / kg, the MG group contained 33 compounds with a total content of 12119.52 μg / kg. Alcohols and acids, as precursors of esters, are likely converted into esters through yeast metabolism during fermentation. The alcohol content in the MG group was 1674.76 μg / kg, significantly higher than that in the CK group. 1-Octen-3-ol, which has a distinct mushroom aroma, is a unique flavor compound in fermented sausages. Esters are generally considered the most important flavor contributors to fermented meat products, imparting fruity, floral, and sweet aromas and helping to mask unpleasant odors. The uninoculated CK group had an ester content of 572.35 μg / kg, while the MG group had a high ester content of 2471.79 μg / kg. Ethyl acetate was the primary ester found in the MG group, at 1644.83 μg / kg. Guillermo Mayer yeast may significantly promote the production of ester compounds in sausages. Aldehydes, primarily derived from the oxidation of unsaturated fatty acids, were lower in the MG group than in the CK group, suggesting that their reduced content may be due to the ability of Guillermo Mayer yeast to inhibit fat oxidation. Therefore, inoculation with Guillermo Mayer yeast enhances the flavor of fermented sausages.

[0084] Table 5: Volatile compound content of fermented sausages at the end of air drying

[0085]

[0086]

[0087] 6. Determination of free amino acids in fermented sausages

[0088] Determination of Free Amino Acids in Sausages: The free amino acid content of fermented sausages was determined using a fully automated amino acid analyzer. An appropriate amount of sausage was minced and freeze-dried using a freeze dryer. A 0.1 g sample was accurately weighed and added to 4 mL of a 4% sulfosalicylic acid solution. The mixture was ultrasonically extracted for 30 minutes and allowed to stand for 10 minutes. After separation, 1.5 mL of the supernatant was collected and centrifuged at 12,000 × g and 4°C for 30 minutes. 1 mL of the supernatant was filtered through a 0.22 μm aqueous filter membrane and the free amino acid content was determined using a fully automated amino acid analyzer.

[0089] Because free amino acids are precursors to some flavor compounds, some branched-chain amino acids, such as valine, leucine, and isoleucine, can be further metabolized into methyl branched-chain aldehydes, alcohols, and acids, and phenylalanine can be converted into benzaldehyde. These contribute to the mature flavor of dry sausages. Table 6 shows that on the last day of fermentation, the free amino acid content of the inoculated sausages was higher than that of the uninoculated sausages, indicating that inoculation with Mycobacterium spp. promotes the formation of amino acids in the sausages, ultimately affecting their overall flavor.

[0090] Table 6: Free amino acid content of fermented sausage at the end of air drying (m g / 100 g )

[0091]

[0092]

[0093] Example 3: A strain of Meyer yeast and its application in sausage fermentation

[0094] This embodiment provides a strain of Pichia guilliermondii Y27, which is used in sausage fermentation, and the steps are as follows:

[0095] 1. Preparation of fermentation agent

[0096] Step 1: Preparation of Pichia guilliermondii Y27 culture: Inoculate a slant of Pichia guilliermondii Y27 into YPD liquid activation medium and culture with shaking at 25°C for 24 hours. Transfer the resulting culture to YPD liquid medium using an inoculating loop and culture at 25°C for 28 hours to obtain an inoculum.

[0097] Preparation of Lactobacillus sakei L48 culture liquid: Inoculate a slant of Lactobacillus sakei L48 into MRS liquid activated medium and culture with shaking at 37°C for 18 hours. Transfer the resulting culture to MRS liquid medium using an inoculating loop and culture at 37°C for 24 hours to obtain an inoculum.

[0098] Step 2: Preparation of bacterial suspension: The inoculum was centrifuged at 6000 × g for 10 min at 4 °C. The precipitate was collected, washed twice with saline and resuspended in saline to prepare a bacterial suspension. The final bacterial concentration was adjusted to 1 × 10 7 CFU / g, volume 1ml, set aside. Set aside.

[0099] 2. Preparation of fermented sausages

[0100] Fresh pig front leg lean and fat were cleaned with water, and blood clots and pig skin were removed. The meat was minced and mixed to a lean to fat ratio of 7:3. A seasoning of 2.5% salt, 2% glucose, 0.008% nitrite, and 0.1% thirteen spices was added, along with the bacterial suspension, and mixed evenly. Sausages were then hung in a constant temperature and humidity environment for fermentation. Fermentation was performed at 25°C and 90% relative humidity for 48 hours, followed by air drying at 15°C and 70% relative humidity for 12 days. The Lactobacillus sakei L48 group was designated the LS group, and the mixed inoculation group was designated the MGL group.

[0101] 3. Determination of biogenic amine content in fermented sausages

[0102] The experimental method is the same as that in Example 3.

[0103] As can be seen from Table 7, putrescine content in the sausage decreased significantly during the fermentation process. The putrescine content in the LS group was 20.02 mg / kg, and the putrescine content in the MGL group was 13.37 mg / kg, a decrease of 49.73%. The total biomass in the MGL group was lower than that in the LS group, indicating that the accumulation of biogenic amines was inhibited by the inoculated bacterial species, and the selected yeast was beneficial to inhibiting the production of biogenic amines.

[0104] Table 7: Biogenic amine content of fermented sausages at the end of air drying

[0105]

[0106] 4. Determination of volatile compounds in fermented sausages

[0107] The experimental method is the same as that in Example 3.

[0108] Esters are typically produced by esterification reactions between alcohols and organic acids, imparting floral and fruity aromas. Table 8 shows that ethyl acetate was the predominant ester found in the MGL group, at 1011.50 μg / kg. This is believed to be essential for achieving the desired aroma by adding fruity notes and masking sour and rancid flavors. The ester content in the MGL group was significantly higher than in the LS group. This result suggests that yeast contributes most to sausage esters. Among alcohols, the MGL group showed significantly higher levels of 1-octen-3-ol, 2,3-butanediol, and n-hexanol, respectively, compared to the LS group (442.77 μg / kg, 172.43 μg / kg, and 842.97 μg / kg). MGL can significantly increase the content of alcohols (1645.06 μg / kg), esters (1448.52 μg / kg), ketones (665.33 μg / kg), and hydrocarbons (224.67 μg / kg) in fermented sausages. Therefore, the addition of Meyer yeast Y27 can improve the flavor of sausages.

[0109] Table 8: Volatile compound content of fermented sausages at the end of air drying

[0110]

[0111]

[0112] 5. Determination of free amino acids in fermented sausages

[0113] The experimental method is the same as that in Example 3.

[0114] It can be seen from Table 9 that after the fermentation, the total amino acid content of the MGL group increased significantly compared with the control group, indicating that the inoculation of Y. meyerii yeast Y27 can promote the formation of amino acids in sausages and improve the flavor.

[0115] Table 9: Free amino acid content of fermented sausage at the end of air drying (mg / 100g)

[0116] Amino acid types LS group MGL group Asp (Aspartic acid) 9.26±1.06 15.45±0.99 Glu (glutamate) 13.73±3.29 18.04±2.41 Thr (Threonine) 24.54±3.93 38.35±2.08 Ser (serine) 88.95±3.22 130.26±28.35 Lys(lysine) 47.42±5.66 49.86±5.57 Gly(glycine) 31.12±0.88 53.18±7.00 Ala (alanine) 114.22±18.04 189.17±12.79 Cys (cysteine) 29.88±4.97 44.70±4.20 Val (valine) 13.91±3.26 22.42±2.98 Met (methionine) 24.29±4.28 32.39±2.57 Ile (Isoleucine) 42.46±2.94 57.06±1.63 Leu (leucine) 12.12±2.80 21.70±6.64 Tyr(Tyrosine) 27.97±3.32 32.80±2.04 Phe (phenylalanine) 200.60±27.37 274.55±14.88 His (histidine) 54.44±2.66 86.06±6.46 Arg (Arginine) 0.70±0.18 1.87±0.37 Total 735.6±73.20 1067.86±75.57

[0117] Example 4: A strain of Meyer yeast and its application in sausage fermentation

[0118] This embodiment provides a strain of Pichia guilliermondii Y27, which is used in sausage fermentation, and the steps are as follows:

[0119] 1. Preparation of fermentation agent

[0120] Step 1: Preparation of Pichia guilliermondii Y27 seed solution: Inoculate a slant of Pichia guilliermondii Y27 into YPD liquid activation medium and culture with shaking at 25°C for 24 hours. Transfer the resulting culture to YPD liquid medium using an inoculating loop and culture at 25°C for 28 hours to obtain an inoculum.

[0121] Step 2: Preparation of bacterial suspension: The inoculum was centrifuged at 6000 × g for 10 min at 4°C, and the precipitate was collected. The precipitate was washed twice with saline and resuspended in saline to prepare a bacterial suspension. The bacterial count of the bacterial suspension cell pellet was adjusted to 10 8 CFU / ml, set aside.

[0122] 2. Preparation of fermented sausages

[0123] Fresh pork front leg lean and fat are cleaned with water, and blood clots and pig skin are removed. The meat is minced and the lean to fat ratio is adjusted to 7:3. A seasoning of 2.5% salt, 2% glucose, 0.008% nitrite, and 0.1% thirteen spices is added, along with the supplementary ingredients and bacterial suspension, and mixed evenly. The sausages are then hung in a constant temperature and humidity environment for fermentation. Fermentation is performed at 20°C and 90% relative humidity for 48 hours, followed by air drying at 15°C and 70% relative humidity for 12 days.

[0124] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A strain of Meyer yeast ( Meyerozyma guilliermondii ), characterized by: The Meyer yeast of Guillermo guillermoi is Pichia guillermoi Y27, which has been deposited in the China Center for Type Culture Collection (CCTCC) with a deposit date of April 30, 2024, and a strain deposit number of C CTCCNO: M 2024843.

2. Use of Pichia guillermongi Y27 according to claim 1 in sausage fermentation.

3. The use of Pichia guilliermondii Y27 in sausage fermentation according to claim 2, characterized in that: The following steps are involved: Step 1: Preparation of Pichia guilliermondii Y27 culture: Inoculate a slant of Pichia guilliermondii Y27 into YPD liquid activation medium and culture with shaking at 25-30°C for 24-36 hours. Transfer the resulting culture to YPD liquid medium using an inoculating loop and culture at 25-30°C for 28-40 hours to obtain an inoculum. Step 2: Preparation of bacterial suspension: The inoculum obtained in step 1 was centrifuged at 3000-8000 × g for 5-15 min at 2-5°C, the precipitate was collected, washed with physiological saline and resuspended in physiological saline to prepare a bacterial suspension. The bacterial count of the bacterial suspension cell pellet was adjusted to 10 6 -10 8 CFU / ml.

4. The use of Pichia guilliermondii Y27 in sausage fermentation according to claim 3, characterized in that: The following steps are involved: a. Add seasonings and auxiliary materials to the raw meat and stir evenly to obtain marinated meat; b: Inoculate the bacterial suspension into the cured meat, mix well, and make sausages; c. Fermentation: Fermentation at 20-28°C and 80-90% relative humidity for 40-50 hours; d: Air drying: Ferment for 10-15 days at 10-20℃ and relative humidity 60-80%.

5. The use of Pichia guillermongis Y27 in sausage fermentation according to claim 4, characterized in that: In step a, the raw meat is fresh pork front leg lean meat and fat meat, which is cut into pieces, washed in 3-10°C water, blood clots and pig skin are removed, the meat is minced and mixed according to a mass ratio of pork front leg lean meat to fat meat of 4-10:

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6. The use of Pichia guillermongis Y27 in sausage fermentation according to claim 4, characterized in that: The auxiliary materials include salt, glucose, nitrite and thirteen spices; the added amounts are: 2-3% of the mass of the raw meat of salt, 1.5-2.5% of glucose, 0.005-0.008% of nitrite, and 0.05-0.15% of thirteen spices.

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