A complex microbial agent of staphylococcus epidermidis M18-2 and hansenula debaryi P13-1, a preparation method and application thereof

Abstract

The application discloses a Hansenula polymorpha P13-1 and Staphylococcus epidermidis M18-2, the Hansenula polymorpha P13-1 has a preservation number of GDMCC No: 66622, and the Staphylococcus epidermidis M18-2 has a preservation number of GDMCC No: 66709.The Hansenula polymorpha P13-1 and the Staphylococcus epidermidis M18-2 complex microbial agent provided by the application can significantly improve the flavor quality of Panxian ham, and the total amount, type and characteristic aroma components of flavor substances are significantly better than natural fermentation, effectively solving the problems of unstable quality and single flavor of traditional natural fermentation.

Description

Technical Field

[0001] This invention relates to the field of food microbiology, specifically to a compound microbial agent of Staphylococcus epidermidis M18-2 and Saccharomyces hansenulatus P13-1, its preparation method, and its application. Background Technology

[0002] Dry-cured ham is an outstanding representative of traditional fermented meat products in my country, renowned for its unique flavor, firm texture, and long shelf life. Panxian ham, relying on the unique high-altitude mountain climate and abundant natural microbial resources of the region, has developed the quality characteristics of firm meat, reddish-brown color, white fat, and a fresh, sweet aftertaste. Traditional Panxian ham production relies entirely on spontaneous, multi-strain fermentation by the microbial community in the natural environment. However, natural fermentation suffers from problems such as poor quality stability, long fermentation cycles, low production efficiency, and the potential for contamination by opportunistic pathogens that produce hemolysins and bioamines.

[0003] Studies have shown that coagulase-negative staphylococci possess strong protease and lipase activities, effectively breaking down proteins and fats and promoting flavor formation. However, their flavor contribution is primarily biased towards producing richer, more robust flavors such as fatty and cheesy aromas, with relatively limited ability to develop delicate flavor layers such as freshness, fruitiness, and floral notes. Yeasts such as *Hansophila d'Barry* have also been reported to tolerate high-salt, low-temperature environments, secreting extracellular enzymes and producing aroma compounds such as alcohols and esters, showing potential in improving the flavor of dry-cured hams. However, their primary ability to break down proteins and fats is limited, often requiring longer fermentation times to produce the desired flavor.

[0004] Current technologies still largely focus on the function of single bacterial strains, neglecting the synergistic metabolic relationships between bacteria and yeasts in natural fermentation systems. However, in actual natural fermentation, Staphylococcus and yeast coexist and interact, exhibiting significant complementarity in flavor generation pathways. Furthermore, existing studies often directly use reported strain combinations when constructing compound microbial agents, lacking the ability to isolate and screen new strains with unique metabolic characteristics from natural fermentation systems in specific production areas. This makes it difficult for the performance of compound microbial agents to overcome the limitations of current technologies. Summary of the Invention

[0005] The present invention aims to provide a compound microbial agent of Staphylococcus epidermidis M18-2 and Saccharomyces hanseniifolius P13-1, as well as its preparation method and application. The two have complementary metabolisms, which can improve the safety, flavor and nutritional quality of cured meat products.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a compound microbial agent containing Hansenula barley yeast and Staphylococcus epidermidis, the compound microbial agent comprising: Hansenula barley yeast P13-1, whose preservation number is GDMCC No: 66622; and Staphylococcus epidermidis M18-2, whose preservation number is GDMCC No: 66709.

[0007] Preferably, the ratio of viable cells of Hansenula d'Barry yeast P13-1 to Staphylococcus epidermidis M18-2 is 1:10 to 10:1, more preferably 1:5 to 5:1, and most preferably 1:1 to 1:2.

[0008] Preferably, the compound bacterial agent is in the form of a liquid bacterial suspension, freeze-dried bacterial powder, or frozen bacterial liquid.

[0009] In a second aspect, the present invention provides a method for preparing the compound microbial agent containing Staphylococcus epidermidis and Hansenula baileyi, comprising the following steps: (1) inoculating Hansenula baileyi P13-1 into a culture medium for activation culture, collecting the cells, and preparing a yeast culture solution; (2) inoculating Staphylococcus epidermidis M18-2 into a culture medium for activation culture, collecting the cells, and preparing a Staphylococcus culture solution; (3) mixing the yeast culture solution and the Staphylococcus culture solution according to the above-mentioned live cell ratio to obtain the compound microbial agent.

[0010] Thirdly, the present invention provides the application of the compound microbial agent in the production of fermented meat products.

[0011] Preferably, the fermented meat product is dry-cured ham, and more preferably, it is Panxian ham.

[0012] Preferably, during the curing or air-drying stage of the dry-cured ham, the compound microbial agent is inoculated onto the surface of the ham by spraying, soaking, or coating, so that the bacterial concentration on the ham surface reaches 10 after inoculation. 5 ~10 7 CFU / g.

[0013] Fourthly, the present invention provides a Panxian ham produced using the aforementioned compound microbial agent.

[0014] Working principle and beneficial effects of the present invention: The compound inoculant of Hansenula d'Barry yeast P13-1 and Staphylococcus epidermidis M18-2 provided in this application can significantly improve the flavor quality of Panxian ham. It is significantly superior to natural fermentation in terms of total amount, variety and characteristic aroma components of flavor substances, and effectively solves the problems of unstable quality and single flavor of traditional natural fermentation. Attached Figure Description

[0015] Figure 1 The chromatogram for simulated fermentation in blank group 1; Figure 2Chromatogram of simulated fermentation of Hansenula barley yeast P13-1; Figure 3 The chromatogram for simulated fermentation in blank group 2; Figure 4 Chromatogram of simulated fermentation of Staphylococcus epidermidis M18-2; Figure 5 This is the actual fermentation chromatogram of the blank group; Figure 6 The actual fermentation chromatogram of the compound fermentation agent group (M18-2+P13-1) is shown. Detailed Implementation

[0016] The following detailed description illustrates the specific implementation method: Example 1: Panxian ham from Panzhou City, Guizhou Province, which has been naturally fermented for more than 7 months, was used. Under aseptic conditions, the surface and deep muscle and fat tissues were cut off, 5 g of the ham was chopped and weighed, and then added to 45 mL of sterile physiological saline. After homogenization, 10 g of the ham was prepared. -1 Diluent, continue serial dilution to 10. -3 .

[0017] 0.1 mL of each serially diluted solution was plated onto potato dextrose agar (PDA) plates (containing chloramphenicol) and incubated upside down at 28°C for 48-72 h. Single, round, milky-white colonies were picked for purification. Identification was performed by methylene blue staining under a microscope (elliptical shape, budding) and ITS rDNA sequencing (primers ITS1 / ITS4). A total of 51 suspected strains were obtained, of which 42 were identified as yeasts. After safety and functional screening, one strain of *Hansophila haemolyticus* was obtained. Debaryomyces hansenii ), named P13-1.

[0018] 0.1 mL of each serially diluted solution was plated onto mannitol high-salt agar (MSA) plates and incubated upside down at 37°C for 24-48 h. Strains were picked for four-zone streak purification, and the morphology and color of single colonies were observed after incubation. Identification was performed by Gram staining microscopy (purple, spherical, grape-like arrangement) and 16S rDNA sequencing (forward primer 27F, reverse primer 1492R). A total of 42 suspected strains were obtained, of which 38 were identified as Staphylococcus. After safety and functional screening, one strain of Staphylococcus epidermidis (…) was obtained. Staphylococcus epidermidis ), named M18-2; The two strains mentioned above have been deposited with the Guangdong Provincial Microbial Culture Collection Center (GDMCC): Hansenula d'Barry yeast P13-1, with accession number GDMCC No: 66622, deposit date: July 2, 2025; and Staphylococcus epidermidis M18-2, with accession number GDMCC No: 66709, deposit date: July 18, 2025.

[0019] Example 2: Safety evaluation of the strain 1. Hemolytic activity test: Hansenula d'Barry yeast P13-1 and Staphylococcus epidermidis M18-2 were inoculated onto blood agar plates and incubated at 37℃ / 28℃ for 24 h. The results showed that no transparent or grass-green hemolytic zone (γ-hemolysis) appeared around the colonies of either strain, indicating that no hemolysin was produced; 2. Plasma coagulase test: Fresh rabbit plasma was diluted 4-fold, and bacterial suspensions of the two strains were added separately. The mixtures were incubated in water at 37℃ / 28℃ for 4 hours, and observed continuously for 24 hours. The results showed that the liquids in the test tubes remained fluid and no coagulation occurred. This indicates that neither strain of bacteria produces plasma coagulase.

[0020] 3. Amino acid decarboxylase test: The two bacterial strains were inoculated into a liquid culture medium containing decarboxylase such as L-lysine and cultured at 30°C for 24 h. The culture medium remained yellow (negative) and did not turn purple (positive). This indicates that the two bacterial strains do not possess amino acid decarboxylase activity and do not produce biogenic amines.

[0021] In conclusion, both P13-1 and M18-2 are safe food-grade strains.

[0022] Example 3: Determination of Enzymatic Activity of Strains 1. Catalase activity: Add 3% hydrogen peroxide solution to a glass slide and smear fresh bacterial growth on it. Both strains immediately produced a large number of bubbles, indicating strong catalase activity.

[0023] 2. Protease activity: The strains were inoculated onto MSA / PDA plates containing 15% skim milk powder. After cultivation, the ratio of the diameter of the clear zone (H) to the colony diameter (C) (HC value) was measured. The results showed that both strains exhibited strong protease activity.

[0024] 3. Lipase activity: The strains were inoculated into a culture medium containing glycerol tribose emulsion, and the HC value was measured after incubation. The results showed that both strains had strong lipase activity.

[0025] Example 4: Aroma Production Performance of Simulated Fermentation by a Single Strain P13-1 was inoculated into meat broth medium and cultured at 28℃ for 5 days; M18-2 was inoculated into meat broth medium and cultured at 37℃ for 5 days. Simulated fermentation and flavor analysis were performed, and the data are shown in Tables 1 and 2 below.

[0026] Table 1 - Flavor data from simulated fermentation of Hansenula d'Bary yeast P13-1

[0027] Table 2 - Flavor data from simulated fermentation of Staphylococcus epidermidis M18-2

[0028] Analysis of the simulated fermentation data in Tables 1 and 2 shows that *Hansenula d'Barry* P13-1 and *Staphylococcus epidermidis* M18-2 exhibit significant complementarity in flavor compound production, as specifically demonstrated below: (1) Hansenula d'Bary yeast P13-1 mainly contributes to the upper aromas such as floral, fruity, smoky, and malty aromas.

[0029] This strain uniquely produces phenethyl alcohol (average 1203.89 μg / L), imparting a rich rose and honey aroma to the product; uniquely produces isoamyl alcohol (average 1318.38 μg / L), contributing malty and whiskey aromas; uniquely produces isoamyl acetate (average 108.74 μg / L), exhibiting typical banana fruit and sweet aromas; uniquely produces 2-methoxy-4-vinylphenol (average 74.34 μg / L), contributing smoky and clove spice; uniquely produces phenethyl acetate (average 58.03 μg / L), contributing rose and honey aromas; uniquely produces 3-furanethanol (average 59.40 μg / L), presenting spicy and smoky aromas; uniquely produces 2-acetylfuran (average 26.47 μg / L), contributing nutty and roasted aromas; uniquely produces 1-nonanol (average 14.63 μg / L), contributing rose and citrus aromas; and uniquely produces furfural (average 16.60 μg / L). It has a sweet and caramel aroma, and also uniquely produces octanoic acid (average 198.28 μg / L), hexanoic acid (average 70.62 μg / L), decanoic acid (average 21.95 μg / L), and nonanoic acid (average 18.85 μg / L), which impart cheese and fatty aromas to the product and provide important precursors for ester synthesis.

[0030] (2) Staphylococcus epidermidis M18-2 mainly contributes to the base aromas such as cream, fat, fruit and mushroom.

[0031] This strain uniquely produces methyl palmitate (average 471.24 μg / L), contributing a rich fatty and fruity aroma; uniquely produces acetoin (average 197.72 μg / L), presenting a creamy and fatty aroma; uniquely produces methyl myristate (average 73.09 μg / L), contributing a waxy and fruity aroma; uniquely produces methyl decanoate (average 50.87 μg / L), presenting a fruity and ester aroma; uniquely produces methyl laurate (average 43.10 μg / L), contributing a fruity and sweet aroma; uniquely produces methyl transoleate (average 35.89 μg / L) and methyl stearate (average 19.40 μg / L), presenting a fatty aroma; uniquely produces methyl linoleate (average 9.78 μg / L), contributing a fruity and sweet aroma; and uniquely produces 1-octen-3-ol (average 19.36 μg / L). M18-2 uniquely produces 2-ethyl-1-hexanol (average 21.12 μg / L), contributing unique mushroom and earthy aromas; it uniquely produces decanal (average 21.12 μg / L), presenting citrus and grassy aromas; it uniquely produces methyl palmitate (average 15.75 μg / L), contributing fatty and fruity aromas; it uniquely produces p-cresol (average 8.43 μg / L), presenting smoky and medicinal aromas; and it uniquely produces 3,4-dimethylbenzyl alcohol (average 4.04 μg / L), contributing floral and sweet aromas. In addition, M18-2 also produces 2-ethyl-1-hexanol (average 2789.70 μg / L, compared to only about 18.82 μg / L for P13-1, contributing strong floral and fruity aromas).

[0032] (3) In terms of the differences in the content of common flavor substances, the content of 1-hexanol in P13-1 is about 42.44 μg / L, which is about 3.8 times that of M18-2, further supplementing the grassy and fruity aromas; while the content of 2-octanone in M18-2 is about 1764.60 μg / L, which is significantly higher than that of P13-1 (527.36 μg / L). The former contributes to the floral and fruity aromas and fatty aromas; the content of isophorone in M18-2 is about 2744.41 μg / L, which is slightly higher than that of P13-1 (2045.62 μg / L). Both contribute to the camphor and minty aromas.

[0033] (4) P13-1 excels at imparting aroma characteristics, primarily floral, fruity, smoky, and malty, to products through the generation of alcohols, phenols, and acids, belonging to the upper-layer aroma; while M18-2 excels at imparting aroma characteristics, primarily creamy, fatty, fruity, and mushroom, to products through the generation of esters, ketones, and long-chain alcohols, belonging to the lower-layer aroma. When the two are combined, a complete flavor layer coverage is achieved, from floral, fruity, and smoky aromas to creamy, fatty, and mushroom aromas. The aroma characteristics complement each other, and the flavor types are highly superimposed, making it an ideal combination of compound fermentation agents.

[0034] Example 5: Aroma Production Performance of Compound Microbial Agent Fermentation A compound inoculum was prepared by mixing *Hansenula d'Barry* P13-1 and *Staphylococcus epidermidis* M18-2 at a viable count ratio of 1:1. The viable count was then increased to 10⁻⁶. 7 CFU / g was inoculated onto Panxian ham, and the fermentation time was 8 months. The blank group was naturally fermented. The blank group and the compound starter culture group were fermented under the same fermentation conditions. After fermentation, samples of biceps femoris muscle were taken for testing, and the data are shown in Table 3 below.

[0035] Table 3 - Actual Fermentation Flavor Data of Compound Fermentation Agent

[0036] The following conclusions can be drawn from analyzing Table 3: (1) The content of total volatile flavor compounds was significantly increased. The total content in the three replicates of the control group was 77.19, 80.89, and 76.52 mg / kg, with an average of 78.20 mg / kg. The total content in the three replicates of the starter culture group was 154.94, 158.3, and 139.95 mg / kg, with an average of 151.06 mg / kg. The starter culture group showed a 1.93-fold increase compared to the control group, indicating that the compound microbial agent significantly promoted the formation of flavor compounds.

[0037] (2) The variety of flavor compounds is significantly rich. The control group showed 33 volatile flavor compounds, while the starter culture group showed 40, a net increase of 7. A total of 24 substances were found that were unique to the starter culture group but completely absent in the control group, including: hexanal (average 18.44 mg / kg), (E)-2-heptenal (average 7.39 mg / kg), methyl hexanoate (average 6.77 mg / kg), trimethylpyrazine (average 6.48 mg / kg), tetramethylpyrazine (average 4.06 mg / kg), 2-propylfuran (average 3.02 mg / kg), and 3,5-octadien-2-one (average 3.30 mg / kg). These newly added substances contribute to a richer and more complex aroma profile in the ham.

[0038] (3) Significantly enhanced characteristic aroma components Floral aroma (phenylethanol): The average concentration in the blank group was 0.46 mg / kg, while the average concentration in the fermentation group was 12.16 mg / kg, representing an increase of approximately 2543% (approximately 26 times).

[0039] Mushroom flavor (1-octen-3-ol): The average concentration in the blank group was 3.10 mg / kg, and the average concentration in the starter culture group was 15.93 mg / kg, representing an increase of approximately 414% (approximately 5 times).

[0040] Fruity / sweet aroma (esters): New additions include methyl hexanoate (average 6.77 mg / kg), ethyl 4-ethoxy-2-oxo-3-butenoate (average 1.62 mg / kg), and methyl palmitate (average 0.38 mg / kg).

[0041] Roasted / Nutritious Aroma (Pyrazines): Trimethylpyrazine (average 6.48 mg / kg) and tetramethylpyrazine (average 4.06 mg / kg) were detected only in the starter culture group, with 0 in the blank group, demonstrating that the compound microbial agent produced a unique aroma dimension that cannot be achieved by natural fermentation.

[0042] 4. Significant accumulation of flavor precursors (acids) Acetic acid: 2.60 mg / kg for blank control group, 6.69 mg / kg for starter culture group; Meanwhile, the addition of 3-methylbutyric acid (average 5.52 mg / kg), octanoic acid (average 3.63 mg / kg), and decanoic acid (average 1.25 mg / kg) provides sufficient precursors for ester synthesis.

[0043] In summary, the Staphylococcus epidermidis M18-2 and Saccharomyces hansenulatus P13-1 compound inoculum provided by this invention (live cell ratio 1:1, inoculation amount 10) 7 The CFU / g of this compound microbial agent can increase the total volatile flavor compounds content of Panxian ham. The above data fully demonstrates that this compound microbial agent has a significant synergistic effect, effectively solving the problems of unstable quality and monotonous flavor in traditional natural fermentation, and has good prospects for industrial application.

[0044] The above description is merely a preferred embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the inventive concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A Hansenula d'Barry yeast P13-1 and Staphylococcus epidermidis M18-2, characterized in that, Hansenula d'Barry yeast P13-1, with accession number GDMCC No: 66622; Staphylococcus epidermidis M18-2, with accession number GDMCC No: 66709.

2. A compound microbial agent comprising Hansenula d'Barry yeast P13-1 and Staphylococcus epidermidis M18-2 as described in claim 1.

3. The compound microbial agent according to claim 2, characterized in that, The ratio of viable cells of *Hansophila barley* P13-1 to *Staphylococcus epidermidis* M18-2 is 1:10 to 10:

1.

4. The compound microbial agent according to claim 3, characterized in that, The compound microbial agent is available in the form of a liquid bacterial suspension, freeze-dried bacterial powder, or frozen bacterial liquid.

5. The method for preparing the compound microbial agent according to any one of claims 1 to 4, characterized in that, The steps include: (1) inoculating Hansenula basilica P13-1 into a culture medium for activation culture, collecting the cells, and preparing yeast culture solution; (2) inoculating Staphylococcus epidermidis M18-2 into a culture medium for activation culture, collecting the cells, and preparing Staphylococcus culture solution; (3) mixing Staphylococcus culture solution and yeast culture solution according to the above live cell ratio to obtain the compound microbial agent.

6. Application of a compound microbial agent in the production of fermented meat products.

7. The application according to claim 6, characterized in that, The fermented meat product is dry-cured ham.

8. The application according to claim 7, characterized in that, During the curing or air-drying stage of dry-cured ham, the compound microbial agent is inoculated onto the surface of the ham by spraying, soaking, or coating, so that the bacterial concentration on the ham surface reaches 10 after inoculation. 5 ~10 7 CFU / g.

9. A type of Panxian ham produced using the compound microbial agent described in any one of claims 2-4.

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