A strain of williopsis sp. and its application

Abstract

The application discloses a strain of producing Xiang Feibien Ceberlindnera yeast and application thereof, the classification and naming of the strain is Feibien Ceberlindnera yeast JGM9-1, the scientific name is Cyberlindnera fabianii JGM9-1, has been preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO: M 20221401, and the preservation date is September 9, 2022.Feibien Ceberlindnera yeast JGM9-1 can be used as excellent microorganisms special for tuna cooking liquid deodorization, the strain can effectively reduce the unpleasant flavor of tuna cooking liquid, and produce unique fermentation fragrance, and the strain has important significance for research and development of high value-added tuna cooking liquid processing products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a fragrance-producing strain of *Fibrians cerebralis* and its applications. Background Technology

[0002] Tuna, also known as tuna or mackerel, is a large, pelagic, and important commercially valuable edible fish, often called "ocean gold." It is one of the three most nutritious fish recommended by the World Nutrition Organization, containing all eight essential amino acids, as well as DHA, EPA, and other nutrients and functional substances. It is low in fat and rich in nutrients. Tuna can be processed into sashimi, canned tuna, and tuna floss. During the processing of tuna fillets and canned tuna, approximately 300-500 kg of tuna cooking liquid is produced per 1000 kg of tuna. This cooking liquid is rich in easily absorbed amino acids, soluble proteins, nucleotides, and unsaturated fatty acids. It has a delicious flavor with the unique seafood taste of tuna and is a high-quality raw material for producing condiments and protein fortifiers. However, its strong fishy smell can significantly affect the taste of further processed products, limiting the high-value comprehensive development and utilization of high-quality tuna cooking liquid. Currently, the cooking liquid from tuna in my country has not been well developed and utilized. Most of it is discharged as wastewater without treatment, which not only wastes protein resources greatly but also increases wastewater treatment costs and poses environmental pollution risks.

[0003] Currently, the main methods for deodorizing seafood include physical, chemical, and biological methods. Physical deodorization typically involves adding a certain amount of adsorbent to the raw material, allowing the odor-causing substances to be adsorbed and thus achieving a deodorizing effect. However, this method suffers from incomplete deodorization and is difficult to apply industrially. Chemical deodorization utilizes compounds in aquatic products that exhibit unpleasant flavors to react with chemical substances, but this method is also prone to producing off-flavors and is difficult to apply industrially. Biological deodorization does not involve the addition of other additives or chemical substances, posing no harm to the human body. Microbial fermentation not only effectively reduces unpleasant flavors in fermented products but also infuses them with unique and pleasant flavors, thereby improving the overall flavor value of the food. Gu Saiqi et al. used yeast to deodorize kelp, achieving a deodorization rate as high as 39.75%, demonstrating excellent deodorization results. Zhang Haiyan et al. used yeast extract to deodorize sea bass, resulting in fish fillets without any fishy smell, and without affecting the color, texture, or freshness of the fillets. Yeast increases the content of aromatic compounds, giving the fermentation broth a special flavor and aroma.

[0004] Different aquatic products contain different substances responsible for their fishy odor, and therefore require different deodorizing microorganisms. Chinese patent "A Method for Deodorizing Kelp Using Compound Microorganisms ZL201710981772.1" discloses a kelp microbial deodorizing strain and its method; Chinese patent "A Fermented Algae Cake ZL201810898673.1" discloses a Gracilaria microbial deodorizing strain and its method; and Chinese patent application "A Method for Deodorizing Shellfish Meat Using Ozone Combined with Oxygen-Absorbing Microbial Fermentation" discloses a scallop microbial deodorizing agent and its method. However, these strains have not been very effective in deodorizing tuna cooking liquid. Summary of the Invention

[0005] The purpose of this invention is to provide a fragrance-producing strain of *Fénix ferrienceberlindnerella* and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A fragrance-producing yeast strain, classified as *Cyberlindnera fabianii* JGM9-1, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221401 and deposit date of September 9, 2022.

[0008] The flavor compounds in tuna cooking liquid mainly consist of decanal, hexanal, and 1-octen-3-ol, which have a fishy smell; 2-nonanone, which has an oily smell; and heptanal, trimethylamine, and dimethyl trisulfide, which have a sour and rancid smell. These are the main components of the fishy odor in tuna cooking liquid. Currently, the market lacks excellent microorganisms for removing the fishy smell from tuna.

[0009] The *Saccharomyces cerevisiae* JGM9-1 provided by this invention can be used to deodorize tuna cooking liquid. This strain can effectively remove fishy odor substances such as decanal, hexanal, 1-octen-3-ol, trimethylamine, and dimethyl trisulfide from tuna cooking liquid, producing flavor substances with floral and fruity aromas such as β-phenylethanol, octanol, ethyl pyruvate, ethyl propionate, ethyl octanoate, ethyl acetate, isoamyl alcohol, ethyl phenylacetate, and ethyl myristate. The fermented liquid has no obvious fishy odor and has a combination of floral, fruity, and tuna-specific flavors.

[0010] The excellent deodorizing microorganism for tuna cooking liquid selected in this invention, *Saccharomyces cerevisiae* JGM9-1, can degrade fishy substances in tuna cooking liquid, reduce the unpleasant flavor of tuna cooking liquid, and produce a unique fermented aroma. This is of great significance for the research and development of high-value-added tuna cooking liquid processed products. Attached Figure Description

[0011] Figure 1 This is a colony morphology diagram of strain JGM9-1.

[0012] Figure 2 This is a microscopic morphological diagram of strain JGM9-1.

[0013] Figure 3 Sequencing results for 26S rDNA detection of strain JGM9-1.

[0014] Figure 4 Phylogenetic tree of 26S rDNA D1 / D2 sequences of strain JGM9-1 and related species.

[0015] Figure 5 Flavor wheel for tuna fermented cooking liquid.

[0016] Figure 6 Average variable importance projection (VIP) value; where red bars indicate VIP values ​​>1 and green bars indicate VIP values ​​<1; (A) VIP-CK is the VIP value of the tuna cooking liquid stock solution group (CK treatment), and (B) VIP-J corresponds to the VIP value of the yeast single-strain fermentation group J (J treatment).

[0017] Figure 7 Correlation analysis of characteristic flavor compounds in yeast. Detailed Implementation

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0019] Example 1: Isolation, Screening, Identification and Preservation of Strains

[0020] Yeast strains were isolated from the fermentation broth of honeycomb samples with excellent flavor using the streak plate method; and superior yeast strains with unique fermentation flavors were screened out through fermentation flavor and brewing characteristic tests.

[0021] 1. Isolation and purification of bacterial strains

[0022] Using honeycomb samples with excellent flavor as the source of inoculum, serial dilutions were performed and spread onto yeast isolation and purification medium plates. After repeated streak separation and microscopic examination, relatively pure strains were obtained. Based on the TTC color change test, 12 strains with typical yeast colony characteristics were selected and numbered JGM8-1, JGM8-2, JGM8-3, JGM8-4, JGM9-1, JGM9-2, JGM9-3, JGM9-4, JGM9-5, JGM9-6, JGM9-7, and JGM9-8. The obtained strains were inoculated onto malt extract slant medium, cultured at 28℃ for 1 day, and stored at 4℃ for later use.

[0023] 2. Screening of strains

[0024] The isolated strains were added to rice saccharification liquid culture medium at an inoculum of 2%, and fermented at 20℃ for 5 days. The aroma-producing yeast strains were screened based on the fermentation flavor.

[0025] Fermented samples were categorized into five levels based on their flavor characteristics. Level 1: Strong fermented aroma, marked with "++++"; Level 2: Relatively strong fermented aroma, marked with "++++"; Level 3: Moderate fermentation, marked with "++"; Level 4: Light fermented aroma, marked with "++"; Level 5: No fermented aroma, marked with "-". Twenty trained professionals were asked to conduct independent evaluations, and the average value was used for statistical analysis.

[0026] The fermentation flavor test results of 12 yeast strains in rice saccharification liquid medium are shown in Table 1. Yeast JGM9-1 had a rich fermentation aroma, rated "++++". Yeasts JGM9-5, JGM9-8, JGM9-2, JGM9-3, JGM9-4, and JGM9-7 also had fermentation aromas, rated "++++" and "+++" respectively. Yeasts JGM8-2, JGM8-3, and JGM9-6 had a milder fermentation aroma, while yeasts JGM8-1 and JGM8-4 did not produce any fermentation aroma, rated "-". The results indicate that JGM9-1 is the superior yeast strain with a unique fermentation flavor.

[0027] Table 1. Evaluation of fermentation flavor of 12 yeast strains

[0028]

[0029] 3. Identification of strains

[0030] Strain JGM9-1 was inoculated onto MEA medium and incubated at 28°C for 72 hours. Colonies were observed to be flat, white, creamy, viscous, reflective, and with irregular edges. Figure 1 As shown. Figure 2 Under a microscope, the cells were observed to be nearly spherical or elliptical, budding, solitary or paired, and measuring 1.5–6.5 μm × 1–6 μm.

[0031] Table 2 Physiological and biochemical characteristics of strain JGM9-1

[0032]

[0033]

[0034] Note: + indicates a positive reaction, and - indicates a negative reaction.

[0035] As can be seen from Table 2, strain JGM9-1 can use D-glucose, D-xylose, cellobiose, trehalose, glycerol, sorbitol, D-melatoninose, xylitol, D-maltose, D-raffinose, and D-sucrose as carbon sources.

[0036] The 26S rDNA of strain JGM9-1 was detected, and the sequencing results are as follows: Figure 3 As shown. The sequence alignment results are as follows:

[0037] Alignment: JGM9-1

[0038] 100.0% Cyberlindnera fabianii CBS 5640T(KY107353)

[0039] 99.3% Cyberlindnera bimundalis NRRL Y-5343T(EF550329)

[0040] 99.1% Cyberlindnera veronae NRRL Y-7818T(EF550322)

[0041] 98.9% Cyberlindnera americana NRRL Y-2156T(EF550328)

[0042] 98.9% Candida stauntonica ATCC MYA-4699T(JQ812698)

[0043] 98.7% Cyberlindnera mississippiensis CBS 7023T (KY107376)

[0044] 98.5% Cyberlindnera xishuangbannaensis NYNU 16752T(KY213813)

[0045] 98.4% Candida pattaniensis JCM 12475T(NG 059424)

[0046] 98.0% Cyberlindnera amylophila CBS 7020T (KY107350)

[0047] 98.0% Cyberlindnera xylosilytica NRRL YB-2097T(NG 064313)

[0048] A phylogenetic tree was constructed using MEGA software and the neighbor-joining method, and similarity calculations were repeated 1000 times. Figure 4 The growth tree nodes only show values ​​where the Bootstrap value is greater than 70%, and the superscript "T" indicates the type strain.

[0049] Based on the high homology between the strain's gene sequencing results and yeast, combined with the identification results such as the strain's physiological and biochemical characteristics, strain JGM9-1 was identified as *Cyberlindnera fabianii* JGM9-1, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20221401 and deposit date of September 9, 2022.

[0050] Example 2: Application of Fibrian Seberlindnerella vaginalis JGM9-1

[0051] This embodiment examines the effect of Febian Seberlindnerella vaginalis JGM9-1 on deodorizing tuna cooking liquid.

[0052] I. Selection and breeding of superior strains for deodorizing tuna cooking liquid

[0053] 1. Materials and Methods

[0054] 1.1 Materials

[0055] 1.1.1 Strains

[0056] Yeasts: *Saccharomyces cerevisiae* JGM9-1, *Saccharomyces cerevisiae* J4 (an excellent strain for deodorizing kelp, disclosed in application number ZL201010534961.2), *Pichia pastoris* 31485 (CICC 31485), *Saccharomyces cerevisiae* JJ4 (an excellent strain for deodorizing scallops and seaweed, disclosed in application number ZL201810898674.6), and *Saccharomyces cerevisiae* JH301 (disclosed in application number ZL201510411228.4).

[0057] 1.1.2 Tuna cooking liquid

[0058] Tuna cooking broth: Provided by Fujian Haihui Biotechnology Industry Co., Ltd., filtered, packaged, and stored at -20℃ for later use. The cooking broth has the following characteristics: pH 6.67; soluble solids 3.75%; total acid 0.238 g / 100 g (calculated as lactic acid 0.090); amino acid nitrogen 0.07 g / kg; reducing sugar 0.032 g / 100 g; total sugar 0.089 g / 100 g; protein 2.84 g / 100 g; crude polysaccharide 0.0184 g / 100 g; hydroxyproline 0.236 g / 100 g.

[0059] 1.2 Test Methods

[0060] 1.2.1 Preparation of culture medium

[0061] YPD liquid culture medium: for yeast culture; sterilize at 121℃ for 30 min, cool, and use later.

[0062] 1.2.2 Seed liquid preparation

[0063] After activation, the bacterial strains were inoculated into suitable culture media and cultured until the bacterial count reached 10⁻⁶. 8 After obtaining the cfu / mL concentration, centrifuge at 5500 r / min and 4℃ for 10 min, then add an equal volume of sterile water to the bacterial sample and set aside for later use.

[0064] 1.2.3 Fermentation Process

[0065] Take 100 mL of the cooking liquid and place it in a 250 mL Erlenmeyer flask. Add 2% (w / w) white granulated sugar and 3 g / L malic acid. Sterilize in a 100°C water bath for 10 min and then cool. (The process is repeated 1×10⁻⁶ times.) 7 Inoculate the selected strains at a CFU / mL level and ferment at 30℃ for 72 h. Collect the fermentation broth and centrifuge for later use. Each treatment was repeated 3 times.

[0066] 1.2.4 Sensory evaluation of fermentation flavor of different strains

[0067] Using the fermentation broth obtained in 1.2.3 as the object and the original tuna cooking broth without added bacteria as the control (CK group), sensory evaluation was used as the test index to screen suitable yeast strains for deodorizing tuna cooking broth.

[0068] 1.2.5 Evaluation of the deodorizing effect of JGM9-1

[0069] Take 100 mL of the cooking liquid and place it in a 250 mL Erlenmeyer flask. Add 20 g / L white sugar and 3 g / L malic acid. Sterilize in a 100°C water bath for 10 min and then cool. (The process is repeated in the original text.) 7 Yeast JGM9-1 was inoculated at CFU / mL and fermented at 30±1℃ for 72 h. The fermentation broth was centrifuged at 8000 r / min for 5 min, and the supernatant was collected. The characteristic volatile flavor compounds and their contents for each treatment were detected by HS-SPME-GC-MS, and the flavor sensory evaluation was performed. Each treatment was repeated 3 times.

[0070] 1.2.6 Sensory Evaluation Methods

[0071] Thirteen trained professionals conducted a cross-sectional description of the tuna cooking liquid according to the "General Methodology of Sensory Analysis" in GB / T 10220. They selected fishy, ​​sour, oily, green, floral, fruity, alcoholic, and lactic acid fermentation aromas as flavor sensory descriptors. The evaluation team used a scale of "0-5" to quantitatively describe the flavor intensity and drew sensory profile diagrams to compare the deodorization effects of different bacteria after fermentation.

[0072] 1.2.7 Data Processing

[0073] SPSS 20.0 software was used to perform analysis of variance on the data.

[0074] 2 Results and Analysis

[0075] 2.1 Screening of suitable bacterial strains for deodorizing tuna cooking liquid

[0076] Flavor intensity was quantitatively described using a scale of 0-5. The tuna cooking liquid was scored after 72 hours of fermentation with a single strain. (See below) Figure 5 Y1 (Fibrian Seberlindnerella vaginalis JGM9-1) produces a more pronounced floral and fruity aroma, while Y2 (J4 Saccharomyces cerevisiae) produces a distinct alcoholic aroma; Y5 (31485 Pichia pastoris), Y6 (JJ4 Saccharomyces cerevisiae), and Y7 (JH301 Saccharomyces cerevisiae) produce unpleasant off-flavors.

[0077] Considering deodorization, aroma production, and stability, Y1 (Fibrian Seberlindnerella vaginalis JGM9-1) is the optimal strain for deodorization during tuna cooking broth fermentation.

[0078] 2.2 Analysis of the consumption and production of characteristic flavor compounds in *Saccharomyces cerevisiae* JGM9-1 (Fibrian sacchariformis).

[0079] Based on the thresholds reported in existing technologies, the OAV values ​​of flavor components in tuna fermentation broth were calculated, and the results are shown in Table 2. The contents of major volatile flavor compounds in the original tuna cooking broth and JGM9-1 fermentation broth are shown in Table 1, and the VIP value analysis results are shown in [Table 1]. Figure 6Based on the combined VIP and OAV analyses (OAV value ≥ 1 and VIP value > 1), and combined with the description of flavor compounds in Table 2, the main flavor compounds in the tuna cooking liquid CK group (CK treatment) are decanal (6.35), hexanal (3.62), and 1-octen-3-ol (3.18), which have a fishy smell; 2-nonanone (26.10), which has an oily smell; and heptanal (2.30), trimethylamine (6.62), and dimethyl trisulfide (3.41), which have a sour and rancid smell. These are the main components of the fishy smell in the tuna cooking liquid. In addition, the CK group also contains some flavor compounds, such as 2-ethyl-furan (11.03) and 2-pentyl-furan (14.91), which have a fishy smell. The main flavor compounds in the yeast fermentation group (J treatment) were: fruity aroma compounds such as isoamyl alcohol (1.28), octanol (2.74), ethyl pyruvate (24.74), ethyl propionate (1.76), ethyl octanoate (6.37), and ethyl acetate (2.86); floral aroma compounds such as β-phenylethanol (11.85), ethyl phenylacetate (5.57), and ethyl tetradecanoate (1.08); and fishy aroma compounds such as 2-ethyl-furan (1.50) and 2-pentyl-furan (3.23).

[0080] Table 1. Changes in flavor compounds before and after fermentation of JGM9-1

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Table 2 Volatile flavor compounds with OAV>1

[0087]

[0088] 2.2 Contribution of Yeast to the Succession of Characteristic Flavor Compounds in Tuna Cooking Broth

[0089] Sperman correlation analysis was used to analyze the characteristic flavor compounds of *Saccharomyces cerevisiae* JGM9-1 fermentation (P < 0.05 and correlation |r| > 0.5). The results are shown in [Figure number missing]. Figure 7Based on the combined Opls-DA analysis, Spearman correlation analysis, OAV analysis, and the flavor descriptions of related substances in Table 2, flavor substances with VIP values ​​> 1.0, correlation |r| > 0.5, and OAV > 1 were identified as characteristic flavor substances of microbial fermentation in tuna cooking broth. The results showed that the flavor characteristic substances consumed by the metabolic process of *Saccharomyces cerevisiae* JGM9-1 were decanal, hexanal, and 1-octen-3-ol, which have a fishy odor; trimethylamine and dimethyl trisulfide, which have a sour odor; and the characteristic flavor substances produced were β-phenylethanol, ethyl myristate, and ethyl phenylacetate, which have a floral aroma; and ethyl pyruvate, ethyl propionate, ethyl laurate, ethyl octanoate, octanol, ethyl acetate, and isoamyl alcohol, which have a fruity aroma.

Claims

1. An aroma-producing yeast strain, characterized in that, The aroma-producing yeast is classified and named *Saccharomyces ferbens* (Fibrians sericultures). Cyberlindnera fabianii JGM9-1 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20221401 and deposit date of September 9, 2022.

2. The application of Febian Seberlindnerella vaginalis JGM9-1 as described in claim 1 in the deodorization of tuna cooking liquid.

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