Preparation method of fermented sausage and bacterial strain used by same

Abstract

The invention relates to a preparation method of a fermented sausage and a bacterial strain used by the fermented sausage, the staphylococcus xylosus Y-18 used in the invention is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is April 30, 2024, and the strain preservation number is CCTCCNO: M 2024844. Compared with traditional fermented sausages, the staphylococcus xylosus Y-18 leavening agent is inoculated, so that the TBARS value of the sausages can be remarkably reduced, and the redness value and the overall color of the sausages are effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for preparing a fermented sausage and a strain used therein. Background Art

[0002] Fermented sausage refers to a type of meat product that produces a unique flavor by using microbial fermentation under artificial control or natural conditions. Most of my country's traditional fermented sausages are produced by natural fermentation. The source of the microorganisms in the product is unclear, the product quality is greatly affected by the environment, the production is unstable, and the safety and quality of the product are difficult to guarantee.

[0003] Coagulase-negative Staphylococci play an important role in the flavor and color quality of fermented meat products. They generally have protease, lipase and nitrate reductase activities, which affect the flavor characteristics of the product, promote the drying process of meat products in the mature stage, and improve the texture. At the same time, they promote the formation of nitrosylmyoglobin, which is conducive to the formation and stability of good color of meat products. Pereira et al. found that by inoculating coagulase-negative Staphylococci, the content of malondialdehyde in fermented sausages can be effectively reduced and its flavor can be improved. Therefore, the screening research of coagulase-negative Staphylococci is of great significance to the characteristic development of fermented meat products.

[0004] Fermented meat products with targeted inoculation strains can solve the instability of natural fermentation while ensuring taste and texture, greatly improving product safety and quality reliability, and inoculation of coagulase-negative Staphylococcus aureus has a significant effect on improving sausage quality. Therefore, screening coagulase-negative Staphylococcus aureus with good fermentation and growth performance and applying it to fermented sausages is of great significance to sausage processing. Summary of the invention

[0005] The object of the present invention is to provide a starter for reducing the degree of oxidation corruption of sausages and improving the flavor of sausages and a method for preparing sausages by fermentation.

[0006] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a strain of Staphylococcus xylosus Y-18, which is preserved in the China Center for Type Culture Collection (CCTCC), the preservation date is April 30, 2024, and the strain preservation number is CCTCCNO: M 2024844.

[0007] In order to achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: the use of the Staphylococcus xylosus Y-18 in the preparation of fermented meat products.

[0008] The preferred technical solution is: the fermented meat product is fermented sausage.

[0009] The preferred technical solution is: the Staphylococcus xylosus Y-18 is transferred from the slant to the MSA agar medium with an inoculation loop for activation, and cultured at 30-37° C. for 36-48 hours. The obtained culture is then transferred to the MSA agar medium with an inoculation loop, and cultured at 30-37° C. for 28-36 hours to obtain a second inoculum.

[0010] To achieve the above-mentioned object and other related objects, the technical solution provided by the present invention is: a method for preparing fermented sausage, characterized in that it comprises the following steps:

[0011] S1: adding seasonings and auxiliary materials to the raw meat, and then marinating to obtain pre-fermented meat;

[0012] S2: inoculating the starter into the fermented meat and mixing well, and then making sausages;

[0013] S3: Fermentation: Fermentation at 22-30°C and relative humidity of 80-90%;

[0014] S4: Drying: Fermentation at 10-18°C and relative humidity 75-85%;

[0015] S5: Drying: Fermentation at 10-15°C and relative humidity of 70-80%;

[0016] The fermentation agent contains Staphylococcus xylosus Y-18, which is deposited in the China Center for Type Culture Collection (CCTCC) with a preservation date of April 30, 2024 and a strain preservation number of CCTCCNO: M 2024844.

[0017] The preferred technical scheme is: the fermentation agent is a liquid bacterial agent, and after the Staphylococcus xylosus Y-18 is activated, it is inoculated into a liquid culture medium for culture (the culture medium components are 30 g / L sucrose, 20 g / L yeast extract, 5 g / L sodium chloride, and the initial pH is 6.0-7.0; the culture is shaken at 35°C, and the shaking speed is 120-150 r / min) for 12 hours, 8000×g, 4°C centrifuged for 5 minutes, the bacterial precipitate is collected, washed with sterile saline for 3 times, and then resuspended in sterile water for use.

[0018] The preferred technical solution is: in S1, the raw meat is fresh pork hind leg lean meat and pork back fat, the fascia and tendon parts on the surface are removed and then rinsed, the meat is minced and mixed according to the mass ratio of pork hind leg lean meat to back fat of 8:2.

[0019] The preferred technical solution is: in S1, the pickling temperature condition is 3-6°C.

[0020] The preferred technical solution is: the seasoning and auxiliary materials are added according to the following mass percentages with the raw meat: 2% salt, 1% glucose, 0.01% sodium nitrite, 1% white sugar, 0.1% ginger powder, 1% cooking wine, 0.1% white pepper powder, 0.1% five-spice powder, 0.1% thirteen spices, and the prepared starter is added at 10 7 cfu / g inoculated into sausages.

[0021] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0022] 1. Compared with traditional fermented sausages, the inoculation of the Staphylococcus xylosus Y-18 starter culture of the present invention can significantly reduce the TBARS value of the sausages and effectively improve the redness value and overall color of the sausages.

[0023] 2. The sausage fermented with the Staphylococcus xylosus Y-18 starter of the present invention significantly improves the flavor quality of the fermented sausage and reduces the degree of oxidation corruption of the sausage. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the cell morphology of Staphylococcus xylosus Y-18 under an optical microscope (magnification 10х100);

[0025] Figure 2 It is the change in pH during sausage processing;

[0026] Figure 3 It is the change in color during sausage processing;

[0027] Figure 4 is the abundance of biomarkers in sausage samples at the end of fermentation. DETAILED DESCRIPTION

[0028] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0029] See also Figure 1-4. It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0030] Deposit of biological material:

[0031] Staphylococcus xylosus Y-18 was deposited in China Center for Type Culture Collection on April 30, 2024, with the deposit number CCTCC M 2024844. The deposit address is: China Center for Type Culture Collection (Wuhan, China).

[0032] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0033] Example 1: A method for preparing fermented sausage and strains used therein

[0034] 1. Collection of traditional fermented meat products and isolation, purification and screening of coagulase-negative Staphylococci

[0035] 1. Isolation of Coagulase-Negative Staphylococci

[0036] Sour cabbage, sour meat, pickled fish (Guizhou), sausage (Sichuan), smoked fish, and bacon (Hunan and Anhui) were collected from all over the country. 10.0 g of the sample was weighed under sterile conditions, added with 90 mL of sterile saline, homogenized and then diluted.

[0037] Take 0.1 mL of the sample dilution and spread it on MSA solid culture medium, culture it at 37°C for 24 h, pick colonies with different shapes and continue to streak culture on MSA solid culture medium, repeat 3-4 times until pure colonies are obtained.

[0038] 2. Screening of coagulase-negative staphylococci

[0039] The purified strains were subjected to Gram staining microscopy and catalase test, and Gram staining-positive and catalase-positive strains were screened out. The obtained strains were subjected to protein, lipase test, nitrite reductase test, mucus and pigment test, hemolysis test, glucose gas production test, H2S production test, arginine ammonia production test, and amino acid decarboxylase test. The results are shown in Table 1. A strain of Staphylococcus aureus Y-18 was screened out. The strain was isolated from the sausage produced by Anhui Huangshan Quanjiang Ecological Agriculture Technology Co., Ltd., and its Gram staining results are shown in Table 1. Figure 1 shown.

[0040] Table 1: Physiological and biochemical screening results of Staphylococcus aureus

[0041]

[0042]

[0043] 2. Identification of coagulase-negative staphylococci:

[0044] The identified strain was sent to a biological company for sequence identification after extracting bacterial DNA and obtaining amplified products through PCR amplification. The sequence results showed that SEQ No. 1 strain Y-18 was identified as Staphylococcus xylosus.

[0045] 3. Preparation of Fermented Sausage

[0046] 1. Preparation of Starter Culture

[0047] After activating Staphylococcus aureus Y-18, 1% was inoculated into liquid culture medium and cultured for 12 h. The precipitate was collected and washed three times with sterile saline and then resuspended for later use.

[0048] 2. Preparation of Fermented Sausages

[0049] Fresh lean pork and pig back fat were blended at a mass ratio of 8:2. The auxiliary materials were added in the following proportions: 2% salt, 1% glucose, 0.01% sodium nitrite, 1% white sugar, 0.1% ginger powder, 1% cooking wine, 0.1% white pepper powder, 0.1% five-spice powder, and 0.1% thirteen spices. The prepared starter was inoculated into the sausage at 107 cfu / g. The intestine was ensured to be full and free of bubbles during enema. It was then hung in a constant temperature and humidity chamber, fermented at 25°C and 85% relative humidity for 72h (fermentation), moved to 15°C and 80% relative humidity for 6d to slow down fermentation (drying), and finally dried at 13°C and 75% relative humidity for 3d to obtain the finished product (maturation). The experimental groups were blank control group (CK group) and starter inoculation group (S group).

[0050] 4. Determination of pH value of fermented sausages:

[0051] The pH values ​​of sausage samples of CK group and S group were measured at three stages of fermentation 0d, 3d, air-dried 3d, 6d and matured 3d after enema. 10.0g sample was accurately weighed, chopped, and mixed with 100mL distilled water. The sample was homogenized at 8000×g for 30s, filtered, and the supernatant was measured with a pH meter. The average value was obtained by repeating three times.

[0052] The results are as follows Figure 2 As shown in the figure, the pH of the CK group and the S group were similar before fermentation. After 3 days of fermentation, the pH of the S group had dropped to about 5.2, and it dropped to the lowest point on the 6th day of fermentation. As the fermentation time prolonged, the pH values ​​of each group slowly recovered, and the pH change trends of the CK group and the S group were similar. The pH of the CK group was always higher than that of the S group inoculated with Staphylococcus xylosus, indicating that the inoculated starter strain had the ability to produce acid, shorten the fermentation time of the sausage, and inhibit the growth of miscellaneous bacteria.

[0053] Five: Color analysis

[0054] The chromaticity of the sausages of the CK group and the S group was measured on the 0th, 3rd, 6th, 9th, and 12th day. The sausage samples were chopped, mixed, and pressed into thin slices with a thickness of 1.5 cm and a diameter of 2.5 cm. The brightness value (L*), yellowness value (b*), and redness value (a*) at 6 different positions were measured with a colorimeter, and the average value was taken.

[0055] The results are as follows Figure 3 As shown in the figure, the brightness and yellowness of the sausages in the CK group and the S group showed a downward trend as the fermentation progressed. On the third day of fermentation, the redness values ​​of each group increased rapidly. Compared with the CK group, the inoculation of Staphylococcus xylosus starter culture could significantly increase the redness of the fermented sausages and effectively improve the color quality of the product.

[0056] VI: Determination of volatile compounds in fermented sausages

[0057] The volatile compound content of the finished sausages of group CK and group S was determined. The volatile flavor substances in the fermented sausages were analyzed by headspace solid phase microextraction-gas chromatography-mass spectrometry. The chopped sausage sample (2.0 g) was placed in a 20 mL headspace sample bottle, and 10 μL of 2,4,6-trimethylpyridine (concentration of 0.1 g / L) was added as an internal standard. After the headspace bottle was equilibrated at 60 ° C for 5 minutes, a pre-aged 75 μm composite CAR / PDMS extraction needle was inserted for adsorption for 30 minutes, and then the extraction head was immediately inserted into the gas injection port for 5 minutes.

[0058] The volatile compounds were determined using a gas chromatography mass spectrometry (GC MS) system (5977B, Agilent). The gas chromatography conditions were: DB-WAX (30m×0.25mm×0.25μm); injection port temperature was 250℃; injection in splitless mode; helium was used as the carrier, and the flow rate was 1mL / min; program temperature rise: column initial temperature was 40℃, maintained for 5min; then heated to 90℃ at 2℃ / min without dwelling; then heated to 180℃ at 5℃ / min without dwelling, then heated to 230℃ at 10℃ / min, and retained for 8min. Mass spectrometry conditions: ion source temperature was 230℃; mass scanning range was 30-550m / z.

[0059] The identification of volatile compounds was performed by comparing with the NIST 20 mass spectral library, taking the similarity greater than 80% as the identification result, and comparing with the results reported in the literature for auxiliary identification. The quantification of volatile compounds was performed by the internal standard method, using 2,4,6-trimethylpyridine solution dissolved in n-hexane as the internal standard substance, and the quantitative results were expressed in μg / kg.

[0060] The results are shown in Table 2. The content of aldehydes in the S group was significantly higher than that in the CK group. The content of alcohols in the S group was also higher than that in the CK group, especially 2,3-butanediol, which is a flavor substance with a creamy aroma and has an outstanding contribution to the flavor of the sausage samples. For esters and ketones, the content of L(-)-ethyl lactate in the inoculated starter group was significantly improved, and the content of ketones also increased. Compared with the blank control group, the content of acids in the starter group was significantly higher. Therefore, the inoculation of starter has an improving effect on the flavor and safety of fermented sausages.

[0061] Table 2: Volatile compound content of fermented sausages

[0062]

[0063]

[0064] Note: Different lowercase letters in the same line indicate significant differences among samples (P<0.05), and ND means that the corresponding compound was not detected.

[0065] The fermented sausage samples of the CK and S groups on the last day were taken, and the malondialdehyde content in the fish meat fermentation process was determined by spectrophotometry according to the second method in GB 5009.181-2016 "National Food Safety Standard Determination of Malondialdehyde in Food", and the results are as follows. It can be clearly found that the TBARS value of the group inoculated with Staphylococcus xylosus fermentation culture is significantly lower than that of the blank control CK group, indicating that the inoculation of the fermentation culture inhibits the oxidative corruption of the sausage and improves the safety quality of the sausage.

[0066] Table 3: Malondialdehyde content in fermented sausages

[0067] CK group Group S TBARS (mg / kg) 1.04±0.05b 0.55±0.03a

[0068] Note: Different lowercase letters in the same row indicate significant differences among samples (P<0.05).

[0069] IX: Determination of biomarkers in fermented sausages

[0070] Take 50mg of refrigerated sausage sample and put it into a 2mL centrifuge tube containing 1mL (75% 9:1 methanol:chloroform, 25% H2O2) extraction solution. Place the centrifuge tube in a tissue grinder and grind it at a frequency of 50Hz for 60s. After repeated operation, ultrasonicate for 30min at room temperature and place it in an ice water bath for 30min. The treated sample is centrifuged at 12000rpm for 10min at 4℃ and the supernatant is collected and concentrated to dryness. After drying, add 0.2mL of ethyl acetate:ethanol (1:1, v / v) mixture to dry again, and finally dissolve the dried sample with (acetonitrile:water 1:1, v / v). The reconstituted sample is filtered with a 0.22um filter membrane, and the filtered supernatant is transferred to a sample bottle for testing. Take 20ul of solution from each sample and mix it as a quality control (QC) sample.

[0071] The extracted samples were analyzed by LC using a Vanquish UHPLC System (Thermo Fisher Scientific, USA). Chromatographic separation was performed using an ACQUITY UPLC HSS T3 (150×2.1 mm, 1.8 μm) (Waters, Milford, MA, USA). The LC-ESI(+)-MS analysis conditions were as follows: the mobile phase was 0.1% (v / v) formic acid in acetonitrile and 0.1% (v / v) formic acid in aqueous solution. The LC-ESI(-)-MS analysis conditions were acetonitrile and ammonium formate (5 mM). The column temperature was 40°C, the flow rate was 0.25 mL / min, and the injection volume was 2 uL. The mass spectrometry detection of metabolites was performed on an Orbitrap Exploris 120 (Thermo Fisher Scientific, USA) using an ESI ion source.

[0072] The raw data obtained by LC MS / MS were converted into .mzXML format by ProteoWizard, and then peak alignment, retention time correction and peak area extraction were performed using XCMS software. The data extracted by XCMS were first subjected to metabolite structure identification and data preprocessing, and then experimental data quality evaluation was performed, and finally data analysis was performed. The results are shown in the figure. Figure 4As shown. Four potential fermentation biomarkers related to sausage fermentation were identified: Subaphylline, Naringenin, beta-Alanyl-L-lysine, and 3'-AMP. Subaphylline is a putrescine-like substance and its relative abundance in the blank control group (CK group) was significantly higher than that in the inoculated group (S group), while Naringenin, which has antibacterial, antioxidant, anti-inflammatory, free radical scavenging and lipid-lowering effects, was higher in the inoculated group (S group). The abundance of beta-Alanyl-L-lysine in the inoculated group (S group) was significantly higher than that in the control group (CK group). Beta-Alanyl-L-lysine is a dipeptide substance composed of β-alanine and L-lysine units connected by peptide bonds. It is a tautomer of β-alanyl-L-lysine zwitterion. Peptides may contribute directly or indirectly to food and have a significant effect on the taste of fermented meat. 3'-AMP is a substance that plays a role in the prevention and treatment of vascular diseases. It can be clearly found that the relative abundance of 3'-AMP in the CK group is significantly lower than that in the inoculated starter group (S group). The changes in the relative abundance of the above substances indicate that inoculation helps reduce the production of oxidative spoilage substances in fermented sausages and also has a certain impact on the flavor of the sausages.

[0073] SEQ No.1 Staphylococcus xylosus Y-18 molecular sequence:

[0074] .

[0075] The above description is only used to explain the preferred embodiments of the present invention, and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A strain of Staphylococcus xylosus Y-18, deposited in the China Center for Type Culture Collection (CCTCC), with a deposit date of April 30, 2024, and a strain deposit number of CCTCCNO: M 2024844.

2. Use of Staphylococcus xylosus Y-18 according to claim 1 in the production of fermented meat products.

3. The use of Staphylococcus xylosus Y-18 in the production of fermented meat products according to claim 1, characterized in that: The fermented meat product is fermented sausage.

4. A method for preparing fermented sausage, characterized in that: The following steps are involved: S1: adding seasonings and auxiliary materials to the raw meat, and then marinating to obtain pre-fermented meat; S2: inoculating the starter into the fermented meat and mixing well, and then making sausages; S3: Fermentation: Fermentation at 22-30°C and relative humidity of 80-90%; S4: Drying: Fermentation at 10-18°C and relative humidity of 75-85%; S5: Drying: Fermentation at 10-15°C and relative humidity of 70-80%; The fermentation agent contains Staphylococcus xylosus Y-18, which is deposited in the China Center for Type Culture Collection (CCTCC) on April 30, 2024, and the strain deposit number is CCTCCNO: M 2024844.

5. The method for preparing fermented sausage according to claim 4, characterized in that: The fermentation agent is a liquid bacterial agent. After activating Staphylococcus xylosus Y-18, 1% is inoculated into a liquid culture medium and cultured for 12 h. The culture medium is centrifuged at 8000×g and 4°C for 5 min, and the precipitate is collected, washed with sterile saline for 3 times, and then resuspended for later use.

6. The method for preparing fermented sausage according to claim 4, characterized in that: In S1, the raw meat is fresh pork hind leg lean meat and pork back fat, the fascia and tendon parts on the surface are removed and then rinsed, the meat is minced and mixed in a mass ratio of pork hind leg lean meat to back fat of 8:

2.

7. The method for preparing fermented sausage according to claim 4, characterized in that: In S1, the pickling temperature condition is 3-6°C.

8. The method for preparing fermented sausage according to claim 4, characterized in that: The seasoning and auxiliary materials are added according to the following mass percentages with the raw meat: 2% salt, 1% glucose, 0.01% sodium nitrite, 1% white sugar, 0.1% ginger powder, 1% cooking wine, 0.1% white pepper powder, 0.1% five-spice powder, and 0.1% thirteen spices. 7 cfu / g inoculated into sausages.

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