Debaryomyces hansenii with high yield of umami peptide and application of debaryomyces hansenii in improving umami of low-salt fermented sausage

Abstract

The invention discloses debaryomyces hansenii with high yield of umami peptide and application of the debaryomyces hansenii in improving the umami of low-salt fermented sausages, the debaryomyces hansenii is separated from the anglerkang sausages, is preserved in the China General Microbiological Culture Collection Center on September 30, 2025, and has the preservation number of CGMCC (China General Microbiological Culture Collection Center), and the preservation number of the debaryomyces hansenii HYYAKS044 is CGMCC NO. The biological preservation number is CGMCC (China General Microbiological Culture Collection Center) The content of peptides below 3kDa of the sausage inoculated with the debaryomyces hansenii disclosed by the invention is obviously more than that of a non-inoculated group, and six umami peptides, namely DDHFLFDKP, DPTPSDGKL, FPPDVGGN, ISEQFTAMFR, PETPPP and VDNPGHPFI, can be screened from the peptides below 3kDa, and the six umami peptides are respectively DDHFLFDKP, DPTPSDGKL, FPPDVGGN, ISEQFTAMFR, PETPPP and VDNPGHPFI.

Description

Technical Field

[0001] This invention relates to the field of food microbiology, and in particular to a strain of Hansenula de Barry yeast that produces high levels of umami peptides and its application in enhancing the umami flavor of low-salt fermented sausages. Background Technology

[0002] Fermented sausages, as a typical traditional meat product, are highly favored by consumers for their unique flavor, texture, and color. Their production typically uses minced pork and fat as the main raw materials, supplemented with salt, nitrites, sugars, and various seasonings, which are then fermented until mature. Salt not only provides the product with its necessary saltiness but also plays a crucial role in inhibiting the growth of spoilage and pathogenic microorganisms, maintaining product safety and quality stability. However, research shows that a high-salt diet is closely related to chronic diseases such as hypertension and cardiovascular disease. Therefore, reducing the salt content of fermented sausages to meet the demands of a healthy diet has become a pressing technical bottleneck and development direction for the meat products industry.

[0003] Currently, the main techniques for reducing salt in fermented meat products include direct salt reduction, partial salt replacement with alternative salts, and the addition of flavor enhancers. Direct salt reduction has advantages due to its ability to meet "clean label" requirements, but it often results in a decline in the sensory quality of the product, including weakened flavor, lighter color, and reduced texture. Existing research shows that when the NaCl content in fermented sausages is below 1.75%, their chewiness, color intensity, and flavor scores all decrease significantly. Therefore, developing novel technical solutions that can compensate for flavor loss under low-salt conditions is of great significance for improving the sensory quality of low-salt fermented sausages.

[0004] Existing research indicates that the application of starter cultures is one of the effective ways to improve the quality of low-sodium fermented meat products. Yeast, as a key component of starter cultures, is widely present in foods such as dry-aged beef, fermented sausages, and dried ham, and promotes the generation of flavor compounds and improves texture during fermentation through sugar metabolism, protein hydrolysis, and lipolysis. Existing technologies include studies on improving the quality of low-sodium meat products through inoculation with microbial starter cultures. For example, CN114634881A discloses a strain of *Hansophila haemolyticus* SH4, which can enhance the volatile flavor compounds and improve the texture of low-sodium dried sausages. However, this existing technology mainly focuses on increasing the total amount of volatile compounds such as aldehydes, ketones, acids, and esters and improving physical texture, without addressing the targeted enhancement of the core flavor of fermented sausages—umami—nor revealing the effect and mechanism of microbial inoculation on the production of specific umami peptides.

[0005] Furthermore, developing and screening umami peptides is also an effective way to improve the quality of low-salt fermented meat products. In recent years, various machine learning models, such as BIOPEP-UWM, Umami-YYDS, Umami-MRNN, and the TastePeptides database, have been used for the prediction and screening of potential umami peptides. Although machine learning models have been developed to predict the flavor characteristics of peptides, research on how to directionally generate and enrich umami-active peptides in low-salt meat product systems through specific microbial fermentation remains lacking. Effective microbial resources and technical solutions are needed to systematically address the specific problem of insufficient umami flavor in low-salt fermented sausages.

[0006] Therefore, the purpose of this invention is to provide a novel strain of Hansenula d'Barry yeast that can not only compensate for the loss of general flavor caused by low salt, but also specifically promote the production of umami peptides and work synergistically with umami nucleotides and amino acids to achieve targeted enhancement of the umami intensity of products, thereby making up for the shortcomings of the prior art. Summary of the Invention

[0007] This invention addresses the limitations of existing salt reduction technologies in compensating for the loss of umami flavor in fermented sausages by providing a strain of *Hansøe du Barry* yeast that produces high levels of umami peptides and its application in enhancing the umami flavor of low-salt fermented sausages. The core innovation of this strain lies in its powerful proteolytic ability, which allows for the targeted release of large amounts of small molecule peptides, generating multiple specific umami peptides not previously reported in existing technologies, thereby directly enhancing the perceived umami flavor of the product at the molecular level.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A strain of *Hansordebali* that produces high levels of umami peptides was isolated from Ankang sausage. Debaryomyces hansenii HYYAKS044 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 30, 2025, with the accession number CGMCC NO.35995.

[0009] The yeast of the present invention can be used to enhance the umami flavor of low-salt fermented sausages, and the number of peptides <3 kDa increases in fermented sausages inoculated with the yeast.

[0010] Furthermore, the <3 kDa peptides include at least one of the umami peptides shown in SEQ ID NO:1-6.

[0011] Furthermore, in the fermented sausage inoculated with the yeast, the content of umami nucleotides and / or umami free amino acids was significantly increased; the total content of volatile ester flavor compounds was also significantly increased.

[0012] The present invention also provides a method for enhancing the umami flavor of low-salt fermented sausages, specifically by inoculating the yeast described in the present invention during the sausage fermentation process.

[0013] The present invention also provides a umami peptide composition comprising at least one umami peptide selected from the amino acid sequences shown in SEQ ID NO:1-6; the umami peptide composition is derived from the fermentation process of low-salt meat products by Hansenula de Barry yeast HYYAKS044.

[0014] The umami peptide composition described in this invention can be used to enhance the umami flavor of low-salt fermented sausages.

[0015] Compared with the prior art, the outstanding effect of the present invention is as follows: (1) Good fermentation characteristics: The present invention isolated a strain of Hansenbali yeast HYYAKS044 from regional characteristic fermented meat products such as Ankang sausage in Shaanxi Province. It has excellent tolerance and good fermentation characteristics. When the yeast is inoculated into fermented sausage, it can enhance the umami and flavor.

[0016] (2) Targeted production of umami peptides: The content of peptides below 3kDa in sausages inoculated with the Hansenula de Barry yeast of the present invention was significantly higher than that in the uninoculated group. Using various machine learning models (BIOPEP-UWM, Umami-YYDS, etc.), six characteristic peptides with a umami prediction probability greater than 95% (SEQ ID NO:1-6) were screened out, namely DDHFLFDKP, DPTPSDGKL, FPPDVGGN, ISEQFTAMFR, PETPPP and VDNPGHPFI, which are completely undisclosed in the prior art.

[0017] (3) Synergistic enhancement of umami flavor: Inoculation with the Hansenula d'Barry yeast of this invention can increase the level of umami nucleotides, thereby improving the umami flavor of fermented sausages, promoting lactic acid accumulation, enhancing the sourness and flavor complexity of fermented sausages, promoting protein degradation in fermented sausages, increasing the content of TCA-soluble peptides and amino acids, increasing the umami and sweet amino acid content of low-salt fermented sausages, and compensating for the flavor profile of low-salt sausages. This strain can significantly increase the content of umami nucleotides (such as IMP) and key umami free amino acids (such as glutamic acid Glu) in fermented sausages, and significantly improve their taste activity value (TAV), thus enhancing umami flavor from the basis of flavor substances.

[0018] (4) Enriching the flavor profile: While enhancing umami, this strain can also promote the synthesis of pleasant volatile flavor compounds such as esters (especially ethyl hexanoate and methyl hexanoate), and convert some aldehydes and acids that may produce off-flavors, making the overall flavor more harmonious and complex. Hansenula d'Barry yeast HYYAKS44 can significantly increase the types and content of ester flavor compounds in low-salt fermented sausages, while promoting the conversion of aldehydes, ketones, and acids into esters. Compared with the uninoculated group, Hansenula d'Barry yeast HYYAKS44 significantly increased the synthesis of esters in fermented sausages, especially key flavor esters such as ethyl hexanoate, methyl hexanoate, ethyl butyrate, methyl butyrate, and ethyl acetate, optimizing the flavor profile of low-salt fermented sausages, thereby enhancing the complexity and stability of sausage aroma, and reducing the risk of oxidative off-flavors and rancidity. Furthermore, the formation of characteristic ketones and alcohols in the inoculated group further enriched its aroma layers, indicating that this strain has significant technical advantages in flavor regulation of low-salt fermented sausages.

[0019] (5) Optimized protein hydrolysis: The content of trichloroacetic acid (TCA) soluble peptides in the inoculated group was significantly higher than that in the control group, indicating that the protease secreted by this strain effectively promoted the degradation of meat protein and provided rich precursors for the formation of umami peptides.

[0020] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the Hansendberry yeast strain, which produces high levels of umami peptides, and its application in enhancing the umami flavor of low-salt fermented sausages.

[0021] Biological Preservation

[0022] Hansenula d'Barley yeast HYYAKS044, classified and named Debaryomyces hansenii It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is September 30, 2025, and the accession number is CGMCC NO. 35995. Attached Figure Description

[0023] Figure 1 The content of TCA-soluble peptides in different fermented sausages.

[0024] Figure 2 These are the flavor-enhancing free amino acids with TAV > 1 in four different fermented sausages.

[0025] Figure 3 The distribution of 3kDa peptides in three groups of fermented sausages.

[0026] Figure 4 The distribution of peptides below 3kDa in sausages containing 2.5% NaCl.

[0027] Figure 5 The distribution of peptides below 3kDa in sausages containing 1.5% NaCl.

[0028] Figure 6 The distribution of peptides below 3kDa in sausages from the 1.5% NaCl-DH group. Detailed Implementation

[0029] I. Experimental Materials Sample: Ankang sausage from Shaanxi Province. YPD liquid culture medium: 3 g beef extract, 10 g peptone, 10 g NaCl, 10 g D-mannitol, 1000 mL distilled water, adjusted to pH 7.2, sterilized at 121℃ for 15 min. YPD solid culture medium: 3 g beef extract, 10 g peptone, 10 g NaCl, 10 g D-mannitol, 1000 mL distilled water, 18 g agar powder, adjusted to pH 7.2, sterilized at 121℃ for 15 min.

[0030] II. Experimental Methods 1. Sample processing Cured meat samples were collected from sausages made by local residents in Ankang City, Shaanxi Province, as experimental materials. 5 g of the sample was taken using sterile scissors, chopped, and placed in a sterile container containing 45 mL of physiological saline (0.9% NaCl). The mixture was then homogenized for 2 min in a homogenizer (model: SCIENTZ-09, manufacturer: SCIENTZ, Ningbo, China). 1 mL of the homogenized solution was taken as the test sample and serially diluted 10-fold with physiological saline. 100 μL of the appropriate dilution was evenly spread on the surface of YPD solid medium and incubated at 30 ℃ for 72 h for bacterial isolation. After incubation, colonies with distinct morphological characteristics were randomly picked from the medium surface and subjected to three consecutive purification cultures using streak plating to obtain morphologically stable, single-source purified strains. Identification revealed that the yeast in Ankang sausages from Shaanxi Province contains… Debaryomyces hansenii (Hansenide barley yeast). Candida zeylanoides (Candida salivaria) Kodamaea ohmeri (Omkoda yeast).

[0031] 2. Sequencing of the strain's 18S rDNA Primers NL1-F (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4-R (5′-GGTCCGTGTTTCAAGACGG-3′), sequences shown in SEQ ID NO:7-8, were used to amplify the 18S rDNA of the target strain by PCR. The PCR reaction conditions were as follows: initial denaturation at 95 ℃ for 5 min; followed by 34 cycles, including denaturation at 95 ℃ for 15 s, annealing at 55 ℃ for 15 s, and extension at 72 ℃ for 1 min; after the cycles, an extension at 72 ℃ for 5 min was performed, followed by storage at 4 ℃ for analysis. The amplified products were detected by 1% agarose gel electrophoresis to confirm fragment length and amplification specificity. The confirmed amplified products were submitted for sequencing to obtain the 18S rDNA sequence information of *Hansophila haemolyticus* strain HYYAKS044. The identified nucleotide sequence was compared with that in the GenBank database using the BLAST procedure (…). https: / / blast.ncbi.nlm.nih.gov / Blast.cgi The isolates were compared and classified based on the highest sequence homology (99–100%).

[0032] 3. Preparation of Fermented Sausage Pork hind leg meat was selected as the main raw material and processed into uniform granules using a meat grinder. Lean meat and pork back fat were mixed at a mass ratio of 9:1, and the following ingredients (g / kg) were added per kilogram of minced meat: monosodium glutamate 3.0 g, glucose 50 g, sodium nitrite 0.09 g, cooking wine 10.0 g, sodium ascorbate 0.10 g, water 50 g, and sodium chloride (NaCl). Three treatment groups were set up in the experiment: Traditional formula group: contains 2.50% sodium chloride (2.50% NaCl); Salt-reduced control group: Contains 1.50% sodium chloride and no added starter culture (1.50% NaCl); Salt-reduced fermentation group: containing 1.50% sodium chloride and inoculated with Hansenula d'Barry yeast. Debaryomyces hansenii HYYAKS44 (1.50% NaCl-DH). All sausages in the above treatment groups were fermented under specified conditions for 12 days, and then frozen at −80 °C after fermentation.

[0033] 4. Determination of nucleotide and organic acid content Take 5.0 g of chopped fermented sausage sample, add 10 mL of pre-cooled 5% HClO4 solution, and homogenize thoroughly for 1 min. Centrifuge the mixture at 11,000 × g for 10 min at 4℃, and collect the supernatant. Repeat the above extraction step twice, and combine the obtained supernatants. Adjust the pH of the combined supernatant to 6.5 with 1 mol / L KOH, and make up to 50 mL with ultrapure water. Centrifuge again under the same conditions to remove the precipitate. Take 1 mL of the final supernatant, filter it through a 0.22 μm microporous membrane, and inject 10 μL of the filtrate into a high-performance liquid chromatography (HPLC) system (LC-2020C-HT, Shimadzu, Japan). The chromatographic column used is a Shim-pack GIST C18-AQ column (4.6 × 150 mm, 3 μm). The mobile phase is 25 mM PBS (pH 6.7), the column temperature is set to 40℃, and the detection wavelength is 260 nm. Nucleotide content is expressed in mg / g.

[0034] Take 5.0 g of chopped fermented sausage sample, add 20 mL of pure water, and homogenize using a high-speed homogenizer at 15,000 rpm for 2 min. Centrifuge the homogenate at 1,800 × g for 5 min at 4℃ and collect the supernatant. Repeat the above extraction step twice, combine the obtained supernatants, and make up to 50 mL with pure water. Filter the combined supernatant through a 0.45 μm microporous membrane, and inject 10 μL of the filtrate into a high-performance liquid chromatography (HPLC) system (LC-2020C-HT, Shimadzu, Japan). The chromatographic column used is a C18 column (4.6 × 250 mm, 5 μm). The mobile phase is methanol / 0.1% phosphoric acid solution (v / v 97.5:2.5). v / v The solution was prepared using methanol and a flow rate of 0.5 mL / min, a column temperature of 30 °C, and a detection wavelength of 210 nm. The organic acid content is expressed in mg / g.

[0035] 5. Determination of TCA soluble peptide content Take 10 g of chopped fermented sausage, add 50 mL of 50 mM PBS buffer (pH 7.0), homogenize at 12,000 rpm for 30 s, and incubate at 4℃ for 2 h. Centrifuge the homogenate at 10,000 × g for 20 min (4℃), collect the supernatant and filter, then mix with an equal volume of 15% ( w / v The mixture was mixed with TCA. After reacting at 37°C for 30 min, the mixture was centrifuged at 5,000 × g for 10 min, and the supernatant was collected as the peptide fraction. The peptide concentration was determined at 540 nm and expressed in mg / mL.

[0036] 6. Determination of free amino acid (FAAs) content and calculation of taste activity value (TAV) 0.2 g of chopped fermented sausage was mixed with 10 mL of 6 mol / L HCl (containing 0.1% phenol) and hydrolyzed at 110 °C for 24 h under nitrogen protection. After cooling, 2 mL of α-aminobutyric acid (2 mg / mL) was added as an internal standard, and the solution was brought to a final volume of 25 mL with 0.1 mol / L HCl. The supernatant was evaporated to dryness and then redissolved in 0.1 mol / L HCl. 0.2 mL of the redissolved solution was derivatized with 100 mmol / L carbonate buffer (pH 9.0) and 3% (v / v) 2,4-dinitrochlorophenylacetonitrile (soluble in acetonitrile), and reacted at 95 °C in the dark for 150 min. The reaction was then terminated by adding 10% (w / v) acetic acid. The reaction solution was filtered through a 0.22 μm filter, and 5 μL was analyzed by high-performance liquid chromatography (HPLC). Results are expressed as mg / g sample.

[0037] Taste Activity Value (TAV) is calculated using the following formula: ; Wherein, C1 is the concentration of the compound in the sample (mg / 100 g), and C2 is its taste threshold (mg / 100 mL). Compounds with TAV < 1 are considered to contribute little to the taste; compounds with TAV ≥ 1 are considered to be the main taste contributors.

[0038] 7. Peptide Identification 10 g of chopped fermented sausage was extracted sequentially with methanol (30 mL), chloroform (10 mL), and water (40 mL). The extract was dried, reconstituted with distilled water, and filtered through a 10 kDa ultrafiltration membrane. The filtrate was adjusted to pH < 3 with 20% trifluoroacetic acid, desalted using a SOLAu HRP desalting column, and then lyophilized. The lyophilized product was reconstituted with 50 μL of 0.1% formic acid before LC-MS / MS analysis. Peptide separation and detection were performed using a Vanquish Neo liquid chromatography system coupled with an Astral mass spectrometry system, C18 column (100 μm × 15 cm, 1.7 μm). The mobile phases were: A: 2% acetonitrile containing 0.1% formic acid; B: 80% acetonitrile containing 0.1% formic acid. Gradient elution: 0 min 8% B (1 μL / min); 1 min 17% B (1 μL / min); 5.5 min 55% B (0.4 μL / min); 7 min 99% B (0.7 μL / min); 8 min 99% B (1 μL / min). In data-dependent acquisition (Top 100) mode, the top 20 precursor ions by intensity were scanned by MS / MS. MS1 scan range 380–980 m / z, MS2 scan range 150–2000 m / z, with dynamic exclusion for 12 s. Peptide and protein identification were performed using PEAKS Studio 12.0 (Bioinformatics Solutions Inc., Waterloo, Canada), and S... pig's ear The UniProt database (http: / / www.uniprot.org / ) considers protein identification reliable when the false positive rate (FDR) is less than 1% and each protein matches at least one unique peptide.

[0039] 8. Virtual screening of umami peptides This invention uses fermented sausage hydrolysates to screen for potential umami peptides. Candidate peptides were predicted for their umami properties using various computational tools, including the BIOPEP-UWM, Umami-MRNN, Umami-YYDS, and TastePeptides databases. Only peptides predicted to be umami with a probability exceeding 95% were considered. Furthermore, peptides needed a PeptideRanker score greater than 0.5 to characterize potential biological activity. Given the higher biological activity of short peptides, peptides longer than 10 amino acids were excluded.

[0040] 9. Determination of volatile flavor compounds Volatile flavor compounds in fermented sausages were analyzed using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). 2.0 g of chopped sausage sample was placed in a 20 mL headspace vial, and 4 μL of 2,4,6-trimethylpyridine (0.01 g / L) was added as an internal standard. After equilibration at 60 °C for 5 min, a pre-aged 75 μm CAR / PDMS composite extraction needle was inserted for adsorption for 30 min. Immediately after extraction, the extraction tip was inserted into the GC-MS inlet for 5 min of analysis.

[0041] GC-MS analysis was performed using a 5977B system (Agilent) with a DB-WAX column (30 m × 0.25 mm × 0.25 μm). The injection port temperature was set to 250 °C, splitless injection was used, helium was used as the carrier gas, and the flow rate was 1 mL / min. The temperature program was as follows: initial temperature 40 °C, hold for 5 min; increase to 90 °C, rate 2 °C / min, no pause; increase to 180 °C, rate 5 °C / min, no pause; increase to 230 °C, rate 10 °C / min, hold for 8 min. Mass spectrometry conditions: ion source temperature 230 °C, scan range 30–550 m / z. Volatile compounds were identified by comparison with the NIST 20 mass spectrometry library; a similarity ≥80% was used to determine the component. Quantification was performed using the internal standard method, with 2,4,6-trimethylpyridine dissolved in n-hexane as the internal standard. Results are expressed in μg / kg.

[0042] III. Experimental Results 1. Nucleotide and organic acid content analysis Nucleotides are key flavor-active substances in the formation of umami flavor in fermented sausages. This invention determined the contents of inosine monophosphate (IMP), guanylic acid (GMP), adenosine monophosphate (AMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), adenosine triphosphate (ATP), adenosine diphosphate (ADP), inosine monophosphate (HxR), and inosine (Hx). As shown in Table 1, the total nucleotide content of the 1.50% NaCl and 1.50% NaCl-DH treatment groups was significantly higher than that of the 2.50% NaCl treatment group (…). P < 0.05), indicating that salt reduction is beneficial for nucleotide accumulation regardless of yeast inoculation. GMP, AMP, and IMP are umami nucleotides that play a key role in overall umami perception and can synergistically enhance the effects with free amino acids. The AMP content in the 1.50% NaCl-DH group was higher than that in the 2.50% NaCl group, but lower than that in the 1.50% NaCl group ( P < 0.05). The IMP content in the 1.50% NaCl-DH group was higher than that in the 2.50% NaCl group. P < 0.05), but there was no significant difference compared to the 1.50% NaCl group (P > 0.05). The results show that under reduced salt conditions... D. hansenii HYYAKS44 inoculation can effectively increase the accumulation of umami nucleotides, thereby enhancing the umami characteristics of fermented sausages.

[0043] Organic acids play a crucial role in both the flavor and microbial stability of fermented meat products. As shown in Table 1, lactic acid was detected in all sausage samples. The lactic acid contents in the 2.50% NaCl, 1.50% NaCl, and 1.50% NaCl-DH groups were 24.29, 24.49, and 26.93 mg / g, respectively. The lactic acid level in the 1.50% NaCl-DH group was significantly higher than that in the 2.50% NaCl and 1.50% NaCl groups. P < 0.05). These results indicate that reduced-salt inoculation with Saccharomyces hyyakS44 promotes lactic acid accumulation and enhances the sourness of fermented sausages.

[0044] Table 1. Nucleotide and organic acid content (mg / g) in different fermented sausages at the end of fermentation. ; Note: nd: Not detected. Results are expressed as mean ± standard error (SE). Different letters (a–c) indicate significant differences between groups. P < 0.05).

[0045] 2. TCA-soluble peptide content analysis Protein hydrolysis in fermented meat products is mainly catalyzed by endogenous proteolytic enzymes, including endopeptidases (such as calpain and cathepsin) and exopeptidases (such as peptidases and aminopeptidases). The content of soluble peptides in total amino acids (TCAs) reflects the abundance of small peptide molecules and is an important indicator of the degree of protein hydrolysis.

[0046] like Figure 1 As shown, the soluble peptide contents of TCA in the three treatment groups (2.50% NaCl, 1.50% NaCl, and 1.50% NaCl-DH) were 3.99, 5.25, and 7.27 mg / mL, respectively. The soluble peptide level in the 1.50% NaCl-DH treatment group was significantly higher than that in the other two groups. P The result (< 0.05) indicates that the microbial protease secreted by *Hansophila haematomarginatus* HYYAKS44 effectively promotes protein degradation and the generation of soluble peptides. These results demonstrate that inoculation with *Hansophila haematomarginatus* HYYAKS44 can significantly accelerate the protein hydrolysis process, providing richer small-molecule peptide precursors for the flavor formation of fermented meat products.

[0047] The Angel high-activity dry yeast (produced by Hubei Angel Yeast Co., Ltd.) used in Zhang Chaomin's research institute had a soluble peptide content of 258.50 μg / mL in its yeast fermentation broth (Zhang Chaomin, Duan Yajie, Ren Meiyan, et al. Effects of yeast and lactic acid bacteria fermentation on flavor characteristics of anchovy enzymatic hydrolysate [J]. China Brewing, 2025, 44(11): 48-54.). In contrast, the soluble peptide content of the 1.50% NaCl-DH group in this invention reached 7.27 mg / mL. Compared with commercially available yeast preparations, the Hansenula d'Barry yeast HYYAKS44 used in this invention exhibits an order-of-magnitude higher hydrolysis efficiency in soluble peptide generation, demonstrating significant technical advantages and application potential.

[0048] 3. Analysis of free amino acid content In fermented meat products, meat protein is first degraded into peptides by endogenous proteases, and then further hydrolyzed by microbial enzymes to generate free amino acids. Some amino acids themselves have characteristic flavors, while others act as flavor precursors and can be further converted into methyl branched aldehydes, alcohols, and acids, thereby enhancing the overall flavor. Table 2 shows the amino acid composition and content of different treatment groups. The amino acids with the highest content in the three sausage groups were Glu (5.23–5.77 mg / g), Arg (8.64–9.08 mg / g), Gly (1.35–1.66 mg / g), Ala (1.16–1.31 mg / g), and Tyr (0.83–0.94 mg / g). The total amino acid content of the 1.50% NaCl-DH group (22.11 mg / g) was significantly higher than that of the 2.50% NaCl group (21.03 mg / g) and the 1.50% NaCl group (21.57 mg / g). P < 0.05), indicating that reduced-salt inoculation with Hansenula d'Barry yeast HYYAKS44 can promote the release of amino acids in fermented sausages.

[0049] Compared with literature reports, Shen Hongyun inoculated low-salt air-dried chickens D. hanseniiThe total amino acid content of ACCC20010 was 8.56 mg / g (Shen Hongyun. Study on the effect of lactic acid bacteria and yeast inoculation and fermentation on the quality improvement of low-salt air-dried chicken [D]. Central South University of Forestry and Technology, 2025.), which was significantly lower than the value measured by the 1.50% NaCl-DH group in this invention, indicating that the HYYAKS44 strain used in this invention has higher proteolytic activity and can effectively promote amino acid production. This result is consistent with the TCA-soluble peptide analysis results, further verifying the promoting effect of yeast in protein degradation and amino acid release. Bolumar et al. (Bolumar T, Sanz Y, Flores M, Aristoy MC, Toldrá F, Flores J. Sensory improvement of dry-fermented sausages by the addition of cell-free extracts from...) Debaryomyces hansenii and Lactobacillus sakei [J]. Meat Science, 2006, 72(3): 457-66. ) indicates that D. hansenii The total amino acid content in the CECT 12487 inoculation group was 2.698 mg / g. (Yoo et al. Isolation of...) Debaryomyces hansenii and selection of an optimal strain to improve the quality of low-grade beef rump( middle gluteal (during dry aging[J]. Animal Bioscience. 2023, 36(9): 1426-1434.) It was found that inoculation... D. hansenii The total amino acid content in the beef of SMFM201905-5 was 4.27 mg / g, which is far lower than the effect of the bacteria used in this invention.

[0050] Table 2. Content of free amino acids (mg / g) in different fermented sausages at the end of fermentation. ; Note: nd: Not detected. Results are expressed as mean ± standard error (SE). Different letters (a–c) indicate significant differences between groups. P < 0.05).

[0051] Asp: Aspartic acid; Glu: Glutamic acid; His: Histidine; Ser: Serine; Arg: Arginine; Gly: Glycine; Thr: Threonine; Pro: Proline; Ala: Alanine; Val: Valine; Met: Methionine; Cys: Cystine; Ile: Isoleucine; Leu: Leucine; Phe: Phenylalanine; It indicates a pleasant flavor. It indicates an unpleasant flavor.

[0052] ∑Umami amino acids: The total number of amino acids responsible for umami flavor (Asp and Glu); ∑Sweet amino acids: The total number of amino acids responsible for sweetness (Thr, Ser, Gly, Ala, and Pro); ∑ Bitter amino acids: The total number of amino acids responsible for bitterness (Val, Met, Ile, Leu, Phe, Tyr, Lys, and His).

[0053] Based on their taste characteristics, free amino acids were classified into three categories: umami (Asp, Glu), sweet (Thr, Ser, Gly, Ala, Pro), and bitter (Val, Met, Ile, Leu, Phe, Tyr, Lys, His). The total amount of umami amino acids in the 1.50% NaCl-DH groups was significantly higher than that in the 2.50% NaCl and 1.50% NaCl groups. P < 0.05, indicating that reduced-salt inoculation with *Hansophila haematocephala* HYYAKS44 can increase the content of umami amino acids in fermented sausages. The total amount of sweet amino acids in the 1.50% NaCl and 1.50% NaCl-DH groups was significantly higher than that in the 2.50% NaCl group. P < 0.05, indicating that simply reducing salt or inoculating with Hansenula d'Barry yeast AKS44 after salt reduction both increased the content of sweet amino acids. There was no significant difference in the total amount of bitter amino acids among the three groups of sausages. P The value > 0.05 indicates that salt reduction and yeast inoculation have no significant effect on bitter amino acids. In conclusion, salt reduction inoculation with Hansenula d'Barry yeast HYYAKS44 can enhance the umami and sweetness of salt-reduced fermented sausages.

[0054] 4. TAV ​​Analysis like Figure 2 As shown, the TAVs of glutamic acid (Glu), arginine (Arg), glycine (Gly), and alanine (Ala) are all greater than 1, indicating that they are key amino acids for the formation of flavor in fermented sausages. Among them, the TAV of Glu in the 1.50% NaCl-DH group was significantly higher than that in the 2.50% NaCl group and the 1.50% NaCl group. P< 0.05), indicating that inoculation with Hansenula d'Barry yeast AKS44 significantly enhances the umami perception of sausages. The TAV of Arg in the 1.50% NaCl-DH group was significantly lower than that in the 2.50% NaCl group and the 1.50% NaCl group ( P <0.05, indicating that inoculation with *Hansenula haematocephala* HYYAKS44 under reduced salt conditions can reduce the bitterness caused by Arg. Meanwhile, the TAV of Ala in the 1.50% NaCl-DH group was significantly higher than that in the 1.50% NaCl group and the 2.50% NaCl group ( P The value < 0.05 indicates that inoculating with *Hansenula baiji* HYYAKS44 in reduced-salt fermented sausages can further enhance sweetness perception. In conclusion, the application of *Hansenula baiji* HYYAKS44 in reduced-salt fermented sausages can synergistically enhance umami and sweetness while reducing bitterness, thereby significantly optimizing the overall flavor characteristics.

[0055] 5. Peptide analysis Based on the above results, inoculation with *Hansenula d'Barry* HYYAKS44 significantly increased the content of nucleotides, TCA-soluble peptides, and free amino acids, thereby enhancing the overall flavor of the low-salt fermented sausage. This indicates that yeast inoculation promotes protein hydrolysis through microbial protease activity. Previous studies have shown that peptides with molecular weights below 3 kDa can impart more pronounced umami characteristics. Therefore, this invention further compared the distribution and content of <3 kDa peptides in sausages inoculated with *Hansenula d'Barry* HYYAKS44 with uninoculated sausages treated with 2.50% NaCl and 1.50% NaCl.

[0056] like Figure 3 As shown, 280, 216, and 327 peptides (<3 kDa) were identified in the 2.50% NaCl, 1.50% NaCl, and 1.50% NaCl-DH treatment groups, respectively. Of these, 133 peptides were common to all three treatments, while the 1.50% NaCl-DH group contained 104 peptides unique to it. Figure 4-6 As shown, the peptide chain lengths are mainly distributed between 11–15 amino acids, followed by 16–20, 6–10, and 21–25 amino acids. Previous studies have indicated that short peptides (<10 amino acids) play an important role in the flavor formation of fermented meat products. Therefore, this invention further screened peptides with a length of 6–10 amino acids as potential umami peptides. Notably, the number of short peptides with 6–10 amino acids in the 1.50% NaCl-DH group was higher than in other treatment groups. Durá et al. (Durá MA, Flores M, Toldrá F. Effect of Debaryomyces(Reference: spp. on the proteolysis of dry-fermented sausages[J]. Meat Science, 2004, 68(2): 319-28.). The Hansenula polymorpha strain used in the experiment contributed little to the proteolysis of fermented sausages; however, this invention shows that after inoculation with Hansenula polymorpha strain HYYAKS44, the number of peptides smaller than 3 kDa increased significantly, indicating that this specific strain has a stronger proteolysis potential.

[0057] The flavor characteristics of peptides are closely related to their amino acid composition and sequence. Aspartic acid (Asp) and glutamic acid (Glu) are considered to impart umami and contribute to umami perception. To further explore potential peptides related to umami, this invention utilizes multiple prediction tools, including BIOPEP-UWM, Umami-YYDS, Umami-MRNN, and the TastePeptides Database, to screen peptides specific to the 1.50% NaCl-DH group. Ultimately, peptides with an umami prediction probability greater than 95% and a PeptideRanker score greater than 0.5 were selected as potential umami peptides.

[0058] As shown in Table 3, six peptides were detected and met all screening criteria only in the 1.50% NaCl-DH group. These peptides are DDHFLFDKP, DTPSDGKL, FPPDVGGN, ISEQFTAMFR, PETPPP, and VDNPGHPFI (sequences shown in SEQ ID NO: 1-6), indicating their potential to enhance the umami flavor of low-salt fermented sausages. This is a first discovery in this invention and has not been addressed in existing technologies (such as CN114634881A).

[0059] Table 3. Umami peptides in sausages inoculated with Hansen de Bali .

[0060] 6. Analysis of volatile flavor compounds The flavor compounds in fermented sausages originate from a series of physical and biochemical changes in fats, proteins, and carbohydrates during sausage production, as well as added flavorings. They are mainly related to the presence of volatile substances such as aldehydes, ketones, acids, alcohols, and esters. As shown in Table 4, a total of 67 volatile substances were identified. In the *Saccharomyces hansenii* HYYAKS44 inoculation group, 36 volatile chemical substances were detected, including 4 aldehydes, 4 ketones, 2 acids, 3 alcohols, 4 alkanes, and 19 esters. Compared with the 1.5% NaCl group, the *Saccharomyces hansenii* HYYAKS44 inoculation group had a richer variety of volatile chemical substances, which significantly contributes to the richness and complexity of the sausage flavor. Among them, ketones such as 1-cyclopropyl-ethyl ketone, 3,3-dimethyl-2-oxetane, and 3-hepten-2-one; alcohols such as 2-methyl-2-propanol and 2,4-dimethyl-3-pentanol; and esters such as methyl 2-oxobutyrate, methyl propionate, 3-methyl butyl valerate, methyl 4-methyl-2-oxovalerate, ethyl valerate, ethyl propionate, and propyl propionate are flavor compounds unique to the Hansenula de Barry yeast HYYAKS44 group.

[0061] The inoculated group had the lowest total aldehyde content (313.50 μg / kg), indicating that the yeast may have converted some aldehydes into alcohols or esters through metabolism, reducing undesirable flavors that could result from oxidation. However, key aldehydes such as heptanal and nonanal were still present, indicating that the basic meaty aroma was not destroyed. The inoculated group had the lowest total ketone content (34.81 μg / kg), but it also contained characteristic ketone compounds such as 1-cyclopropyl ethyl ketone (19.21 μg / kg), 3,3-dimethyl-2-oxetane (1.97 μg / kg), and 3-hepten-2-one (4.21 μg / kg). These substances may be produced by yeast metabolism, contributing unique floral, fruity, or grassy aromas. The inoculated group had extremely low total acid content (10.78 μg / kg), far lower than other groups, suggesting that the yeast may have used organic acids for esterification reactions to synthesize esters, reducing the risk of rancidity.

[0062] The inoculated group had the lowest total alcohol content (33.47 μg / kg), but it contained unique alcohols such as 2-methyl-2-propanol and 2,4-dimethyl-3-pentanol, which may serve as precursors for ester synthesis. The inoculated group had the highest total alkane content (88.51 μg / kg), especially methylcyclobutane and undecane, which may have originated from lipid degradation or yeast metabolism. These compounds had little impact on flavor but reflected differences in metabolic activity.

[0063] Aldehydes and ketones were present in higher amounts in the uninoculated group, possibly related to lipid oxidation. However, the inoculated group converted them into more stable esters through esterification, optimizing the flavor balance. Although the inoculated group did not necessarily have the highest total amounts of aldehydes, ketones, acids, and alcohols, its ester composition and content were significantly increased, which was key to its flavor optimization. The total ester content (2362.33 μg / kg) in the 1.5% NaCl-DH (inoculated yeast group) was significantly higher than the other two groups, indicating that *Hansenula d'Barry* HYYAKS44 significantly promoted ester synthesis. The three most abundant esters in the inoculated group were ethyl hexanoate, methyl hexanoate, and ethyl butyrate, with significantly higher contents than in the uninoculated group. Esters were the most prominent flavor category in the inoculated group, with a much higher total amount than other groups and a greater variety (19 types), contributing most to pleasant flavors such as fruitiness and sweetness.

[0064] In summary, *Hansophila barleyi* strain HYYAKS44 significantly enhances the variety and content of ester flavor compounds in low-salt fermented sausages, while promoting the conversion of aldehydes, ketones, and acids into esters. This enhances the complexity and stability of the sausage aroma and reduces the risk of oxidative off-flavors and rancidity. Furthermore, the formation of characteristic ketones and alcohols in the inoculated group further enriches the aroma profile, indicating that this strain possesses significant technical advantages in flavor regulation of low-salt fermented sausages.

[0065] The *Hanson's d'Barry* strain HYYAKS044 of this invention differs fundamentally in function from previously disclosed similar strains (such as SH4 in CN114634881A). While the SH4 strain primarily contributes to increasing the total amount of volatile flavor compounds and improving physical texture, the core contribution of the HYYAKS044 strain lies in its ability to directionally promote protein hydrolysis, producing unique umami peptides, and systematically increasing the levels of key flavor compounds such as umami nucleotides and amino acids. This achieves targeted and potent compensation for the core flavor of "umami" in low-salt fermented sausages. This strain provides a novel and irreplaceable microbial resource for developing high-quality low-salt fermented meat products.

[0066] Table 4. Characteristic volatile flavor compounds of fermented sausages inoculated with different combinations of starter cultures. ; ;

[0067] Note: Results are expressed as mean ± standard error (SE). Different letters (a–c) indicate significant differences between groups. P <0.05).

[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 Debaryomyces hansenii with high yield of umami peptides, characterized in that: A Debaryomyces hansenii (HYYAKS044) was isolated from Ankang sausage Debaryomyces hansenii and deposited with the China General Microbiological Culture Collection Center on September 30, 2025, and given the accession number CGMCC NO. 35995.

2. The yeast of claim 1 for use in enhancing the umami taste of low-salt fermented sausages.

3. The use of the yeast strain of claim 2 in improving the umami taste of low-salt fermented sausages, characterized in that: The number of peptide fragments <3 kDa is increased in fermented sausages inoculated with the yeast.

4. The use of the yeast strain of claim 2 in improving the umami taste of low-salt fermented sausages, characterized in that: The <3 kDa peptide fragments include at least one of the umami peptides shown in SEQ ID NO: 1-6.

5. Use of the yeast strain according to claim 2 for the preparation of low-salt fermented sausages, characterized in that: The content of umami nucleotides and / or umami free amino acids is significantly increased in fermented sausages inoculated with the yeast.

6. The use of the yeast strain of claim 2 in enhancing the umami taste of low-salt fermented sausages, characterized in that: The total content of ester volatile flavor substances is significantly increased in fermented sausages inoculated with the yeast.

7. A method of enhancing the umami taste of low-salt fermented sausages, characterized by: Inoculating the yeast of claim 1 during the fermentation process of sausages.

8. A umami peptide composition, characterized by: A umami peptide composition comprising at least one umami peptide selected from the group consisting of the amino acid sequences shown in SEQ ID NO: 1-6; the umami peptide composition is derived from the process of fermenting low-salt meat products by Debaryomyces hansenii HYYAKS044.

9. The umami peptide composition of claim 8 for use in enhancing the umami taste of low-salt fermented sausages.

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