Pichia fermenting strain and application thereof in preparing fermented sour meat

Abstract

The present application belongs to the field of microbial technology, and relates to a fermentation acid meat microorganism, in particular to a Pichia fermentans and application thereof in preparation of fermentation acid meat. The present application discloses a fermentation Pichia fermentans ZY-1, the preservation number of which is CGMCC NO.24770, and the preservation date is April 26, 2022, the present application can significantly increase the relative content of esters with fruity aroma, aldehydes with green grass flavor, improve and enrich the flavor characteristics of fermentation acid meat, and has a broad application prospect when applied to other fermented meat products as a fermentation agent.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to microorganisms for fermenting sauerkraut, specifically to a strain of Pichia pastoris for fermentation and its application in the preparation of fermented sauerkraut. Background Technology

[0002] Fermented sauerkraut is a traditional naturally fermented meat product. During fermentation, the proteins and fats in the meat produce flavor precursors such as amino acids and fatty acids under the action of microorganisms and enzymes, giving the product a unique flavor and nutrition. On the other hand, a large number of beneficial microorganisms in the meat grow and multiply during the fermentation process, becoming the dominant microorganisms in fermented meat products. This limits the growth and reproduction of spoilage microorganisms and the production of harmful substances such as nitrites in fermented meat products, thus greatly improving the safety of fermented meat products.

[0003] The high salt concentration (low water activity) and low pH conditions in fermented sauerkraut create favorable conditions for yeast growth and reproduction. Yeast promotes the production of volatile flavor compounds such as esters, ketones, and alcohols in sauerkraut through glycolysis, proteolysis, and fat decomposition. It also slows down rancidity and protects red nitrosomyoglobin from decomposition by degrading peroxides and consuming oxygen, thus stabilizing the color of the sauerkraut and improving its sensory properties.

[0004] Currently, there is limited research in China on using native yeasts as starter cultures in fermented meat, and commercially available starter cultures do not significantly contribute to product flavor. Research on yeast aroma production mainly focuses on spirits, with few reports on its application in fermented sour meat products. The aroma-producing yeasts screened from products not only possess strong growth and metabolic capabilities but also promote the release of large amounts of volatile substances such as esters and aldehydes during their growth and reproduction. Therefore, utilizing this characteristic of yeast as a starter culture for inoculating meat for a certain period is of great significance for improving and enhancing the quality stability of fermented sour meat and imparting its unique flavor characteristics. Summary of the Invention

[0005] The purpose of this invention is to provide a Pichia pastoris that can produce volatile flavor compounds and its application in meat fermentation, based on the problems existing in the prior art.

[0006] This invention is achieved through the following technical solution:

[0007] A fermenting Pichia fermentans ZY-1 strain capable of producing volatile flavor compounds is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.24770, deposited on April 26, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its 5.8S rRNA nucleotide sequence is shown in SEQ ID NO.1.

[0008] The Pichia fermentans ZY-1 provided by this invention was isolated from a fermented yogurt sample.

[0009] The Pichia fermentans ZY-1 provided by this invention grows well on PDA medium. The colony morphology is milky white, flat, opaque, wrinkled and dull, with irregular edges and elliptical cell morphology.

[0010] The Pichia fermentans ZY-1 provided by this invention, after total DNA extraction and PCR amplification, had its gene sequence compared with known sequences in the NCBI online gene database. The results showed that it had 99.75% similarity to the known standard strain, Pichia fermentans HR-1DS 1308, numbered KM029994.1.

[0011] The Pichia fermentans ZY-1 provided by this invention was applied to fermented sauerkraut. Solid-phase microextraction combined with gas chromatography-mass spectrometry (GC-MS) revealed a total of 38 volatile substances in sauerkraut inoculated with Pichia fermentans ZY-1: 25 esters, 5 acids, 2 alcohols, 5 aldehydes, and 1 ketone. The key volatile flavor compounds (ROAV greater than 1) included ethyl butyrate, ethyl heptanoate, ethyl octanoate, ethyl decanoate, ethyl hexanoate, propyl 2-methylbutyrate, ethyl valerate, n-hexanol, 1-octen-3-ol, decanal, phenylacetaldehyde, and nonanal. Compared with uninoculated Pichia fermentans, inoculation with fermented Pichia fermentans ZY-1 significantly increased the relative content of esters in the fermented meat from 36.83% to 41.33% (p<0.01) and the relative content of aldehydes from 0.77% to 1.93% (p<0.01).

[0012] The Pichia fermentans ZY-1 provided by this invention can significantly increase the relative content of fruity esters and grassy aldehydes, improve and enrich the flavor characteristics of fermented sour meat, and has broad application prospects as a starter for other fermented meat products. Attached Figure Description

[0013] Figure 1 This is a colony morphology diagram of Pichia fermentans ZY-1 fermented in this invention on PDA medium;

[0014] Figure 2 This is a cell morphology diagram of Pichia fermentans ZY-1 fermented according to the present invention;

[0015] Figure 3 This is a phylogenetic tree diagram of the Pichia fermentans ZY-1 identified in this invention.

[0016] Figure 4 This is a graph showing the number and relative content of volatile substances in fermented sour meat enhanced by Pichia fermentans ZY-1 according to the present invention.

[0017] Figure 5 This is a heat map showing the composition of all volatile substances in fermented sour meat after fermentation with Pichia fermentans ZY-1, as described in this invention. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] Example 1: Isolation and screening of strains

[0020] Weigh 25.0 g of the fermented sour meat sample and add it to 225 mL of sterile physiological saline. Shake at 28°C and 150 rpm for 30 minutes. Then, perform serial dilutions to obtain 10... -2 10 -3 10 -4 The bacterial suspension. Pipette 10... -1 -10 -40.2 mL of bacterial suspension was spread onto potato dextrose agar (PDA) medium, with two plates for each dilution. The plates were incubated at 28°C for 5 days. Then, starting from 10... -1 -10 -4 Pick 15-20 strains of different morphology but with typical Pichia pastoris colony characteristics from each dilution plate, and further streak them for isolation and purification. Repeat 2-3 times, observing the colony purification under a microscope. Based on the colony morphology on solid medium and the odor of the culture dish, screen the target Pichia fermentans ZY-1 strain from the strains isolated and purified from the fermented yogurt sample, and observe the colony morphology and cell morphology characteristics of this strain. Streak a single purified colony on yeast peptone dextrose (YPD) agar, then incubate the slant at 28°C. After the colonies have fully grown on the slant, store the medium at 4°C. Figure 1 , 2 As shown in the attached figure, the colony morphology of Pichia fermentans ZY-1 on PDA medium and its cell morphology under a microscope are as follows. Figure 1 and Figure 2 As shown: the colonies are milky white, flat, opaque, wrinkled and dull, with irregular edges, oval cell shape, and reproduce by budding.

[0021] Example 2: Identification of the strain

[0022] Accurately pipette 1 mL of Pichia fermentans ZY-1 bacterial suspension into a plate, pour carbon source and nitrogen source assimilation medium into it, and then add 50 mmol / L carbon source (glucose, lactose, sucrose, maltose, citric acid) and nitrogen source (potassium nitrate, ammonium sulfate) filtered through a 0.22 μm membrane onto the surface of the medium. Incubate at 30 °C for 24 hours. Use a carbon (nitrogen) source-free medium as a control experiment and observe the growth of each colony on the medium.

[0023] Carbon source assimilation medium: ammonium sulfate 5g; potassium hydrogen phosphate 1g; magnesium sulfate heptahydrate 0.5g; yeast extract 0.2g; agar 20g; water 1000mL. Sterilization conditions: 115℃, 15min.

[0024] Nitrogen source assimilation medium: glucose 20g; potassium dihydrogen phosphate 1g; magnesium sulfate heptahydrate 0.5g; yeast extract 0.2g; agar 20g; water 1000mL; pH: 6.4-6.6; sterilization conditions: 115℃, 15min.

[0025] The results of the carbon and nitrogen source assimilation experiments of Pichia fermentans ZY-1 are shown in Table 1. Pichia fermentans ZY-1 can utilize glucose and citric acid as carbon sources and potassium nitrate as a nitrogen source.

[0026] Table 1 Results of carbon and nitrogen source assimilation experiments in Pichia pastoris fermentation

[0027] glucose + lactose - maltose - compound Z compound Z compound Z sucrose - Citric acid + Cellobiose - compound Z compound Z ammonium sulfate - potassium nitrate +

[0028] Note: + indicates a positive reaction, and - indicates a negative reaction.

[0029] Total DNA was extracted from *Pichia fermentans* ZY-1 using a DNA extraction kit, and then PCR amplification was performed using the universal fungal primers ITS1 / ITS4. The PCR product was sequenced, and the 5.8S rRNA nucleotide sequence of *Pichia fermentans* ZY-1 was obtained, as shown in SEQ ID NO.1. The gene sequence of *Pichia fermentans* ZY-1 was analyzed using BLAST (Basic Local Alignment Search Tool) on the NCBI website. Sequences with homology higher than 99% were compared with the determined sequence in the database. The comparison results showed that *Pichia fermentans* ZY-1 had a 99.75% similarity to the known standard strain, *Pichiafermentans* HR-1 DS 1308 (number KM029994.1). Furthermore, the comparison results were used to construct a phylogenetic tree using the Neighbor-joining algorithm in MEGA software (see attached). Figure 3 The target strain was identified as *Pichia fermentans*. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.24770, and the deposit date was April 26, 2022.

[0030] Example 3: Application of the strain

[0031] 1. Pichia fermentans ZY-1 from a test tube was streaked onto PDA medium and incubated at 28°C for 5 days to activate the strain. The activated Pichia fermentans ZY-1 was then used as the starter culture (1.0 × 10⁻⁶ cells / mL). 7The fermented meat was added at 1% of the raw meat (cfu / g), with a control group of unfermented meat. Both groups were placed in a well-ventilated room and allowed to ferment naturally for 30 days. After fermentation, GC-MS was used to determine the volatile flavor compounds in the two groups of fermented meat, thus obtaining the effect of enhanced fermentation with Pichia fermentans ZY-1 on the overall flavor of the fermented meat.

[0032] 2. Determination of volatile flavor compounds in sour meat (SPME-GC-MS):

[0033] ①.SPME conditions: Weigh 3g of sample into a 10mL extraction bottle, insert the extraction head into the extraction bottle and place it on a magnetic stirrer (temperature set to 60℃) at a distance of 1cm from the sample for 5 minutes to equilibrate. Then remove the fiber head and extract the volatile components from the sample for 40 minutes. After the volatile components are extracted, desorb at 250℃ in non-split mode at the injection port for 5 minutes, and start the gas chromatography-mass spectrometry for detection.

[0034] ②. GC conditions: An HP-5MS capillary column (30m × 0.25mm, 0.25μm) was used as the chromatographic column. The injection method was splitless, with helium of ≥99.999% purity as the carrier gas. The injection port temperature was 250℃, and the flow rate was controlled at 1.0mL / min. The column temperature was set as follows: 50℃ for 1min, then increased to 130℃ at 8℃ / min and held for 1min, and finally increased to 280℃ at 15℃ / min and held for 2min. The subsequent program was 280℃ for 3min.

[0035] ③. MS conditions: electron energy 70 eV, ion source temperature 230℃, m / z 30~450, ionization mode: electron ionization source.

[0036] ④. Qualitative and quantitative analysis of volatile components: The ion fragments, mass-to-charge ratio, and other relevant parameters in the mass spectrometry of the identified volatile substances were compared to obtain compounds with a matching degree greater than 80% with the NIST17.L mass spectrometry database for analysis. The relative content (%) of each volatile component detected during the fermentation of sour meat was obtained by peak area normalization. The relative odor activity (ROAV) value was obtained based on the relative content of volatile substances and their corresponding sensory thresholds. When ROAV ≥ 1, the substance was considered to contribute to the overall flavor of the sample.

[0037] 3. Results and Analysis

[0038] GC-MS detected 38 volatile compounds in both the control group and the fermented sour meat samples from Pichia fermentans ZY-1. Esters were the most numerous and had the highest relative content, with 25 species (41.39%); acids had 5 species (2.95%); alcohols had 2 species (2.19%); aldehydes had 5 species (1.93%); and ketones had 1 species (0.23%). (See attached image.) Figure 4 As shown, compared with uninoculated fermented meat (NF group), fermentation enhanced with Pichia fermentans ZY-1 (PF group) significantly increased the relative content of esters and aldehydes (p < 0.01) and significantly decreased the relative content of acids and ketones (p < 0.01), while having no significant effect on alcohols. Esters and aldehydes are the main volatile substances in fermented fermented meat, which impart fruity and grassy aromas. Pichia fermentans promotes esterification, thus significantly reducing the relative content of acids while not significantly reducing the relative content of alcohols. The relative content of all volatile substances and relative odor activity (ROAV) values ​​in inoculated and uninoculated fermented Pichia fermentans ZY-1 fermented meat are shown in Table 2. Therefore, the abundant esters, acids, alcohols, and aldehydes in fermented meat give the product its unique flavor characteristics.

[0039] Table 2. Relative contents and relative odor activity (ROAV) of each volatile substance in the samples.

[0040]

[0041]

[0042]

[0043] Note: " / " indicates that no detection was detected.

[0044] After 30 days of enhanced fermentation with Pichia fermentans ZY-1, 16 new volatile compounds, mainly esters, were produced: isoamyl formate, ethyl oleate, octyl formate, vinyl crotonate, S-methyl-2-methylthiopropionate, ethyl hexadecenoate, ethyl glycolate, hexyl formate, valeric acid, 2-methylbutyric acid, 2-methylvaleric acid, 1-octen-3-ol, decanal, 2-phenyl-2-butenal, hexadecaldehyde, and 4-methyl-1,2,4-triazolidine-3,5-dione. Simultaneously, the relative contents of 11 other volatile compounds, including isoamyl 4-methylvalerate, ethyl 5-methylhexanoate, ethyl octanoate, ethyl laurate, ethyl decanoate, ethyl hexanoate, ethyl 3-phenylpropionate, ethyl palmitate, 4-methylvaleric acid, phenylacetaldehyde, and nonanal, were significantly increased. Among them, 1-octen-3-ol, phenylacetaldehyde, nonanal, ethyl decanoate, and ethyl hexanoate have relative odor activities greater than 1, contributing significantly to the overall flavor of the sour meat. Ethyl hexanoate has a relative content of 20.71%, exhibiting a pineapple aroma; ethyl decanoate has a weak fruity aroma, similar to a nutty aroma, with a content of 0.39%; phenylacetaldehyde has a floral aroma with a relative content of 1.53%; nonanal has a relative content of 0.3%; and 1-octen-3-ol has a mushroom aroma with a relative content of 0.18%. Standardized heatmaps more intuitively display the distribution of each volatile substance in the two groups of sour meat; the darker the color, the higher the relative content of that volatile substance in the sour meat. Figure 5 It can be seen that Pichia fermentans ZY-1 significantly affects the composition of volatile substances in sour meat.

[0045] The differential volatile substances induced by inoculation with Pichia fermentans ZY-1 result in a richer and more unique flavor profile in fermented sour meat. Its application as an aroma-producing fermenting agent has broad prospects and is of great significance for the formation of good product quality.

[0046] The above description is merely an embodiment of the present invention, but the scope of the present invention is not limited thereto. It should be noted that improvements can be made to the technical solutions of the present invention without departing from the scope of the present invention, and the improved content shall also be within the protection scope of the present invention.

Claims

1. A strain of Pichia fermentans ZY-1, characterized in that, The Pichiafermentans ZY-1 has the accession number CGMCC NO.24770 and is deposited at the China General Microbiological Culture Collection Center on April 26, 2022.

2. A fermenting agent, characterized in that, The fermenting agent contains Pichia fermentans ZY-1 as described in claim 1.

3. The fermenting agent according to claim 2, characterized in that, The concentration of the fermented Pichia fermentans ZY-1 was 1.0 × 10⁻⁶. 7 cfu / g.

4. The application of the Pichia fermentans ZY-1 as described in claim 1 or the starter culture as described in claim 2 or 3 in the preparation of fermented sauerkraut.

5. A method for preparing fermented sour meat, characterized in that, The method includes the following steps: (1) Activate the Pichia fermentans ZY-1 as described in claim 1 to obtain a fermentation agent; (2) The fermentation agent described in step (1) is inoculated into the raw meat and fermented to obtain the fermented sour meat.

6. The preparation method according to claim 5, characterized in that, The concentration of Pichia fermentans ZY-1 in the fermentation spawn was 1.0 × 10⁻⁶. 7 cfu / g.

7. The preparation method according to claim 6, characterized in that, In step (2), the fermentation agent is added to the raw meat at 1% of the raw meat, and the meat is naturally fermented for 30 days.

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