A strain of Coxsackie serrata H1 and its application
By isolating and applying sausage Cockia H1, the problem of improving the flavor and texture during the fermentation of meat products without nitrite is solved, and the effect of reducing nitrite residues and inhibiting fat peroxidation is achieved, which significantly improves the quality and safety of meat products.
Patent Information
- Application Number
- CN202510053570.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-04-08
AI Technical Summary
During the fermentation process of meat products without nitrite, it is difficult to improve the flavor and texture of meat products. At the same time, it is necessary to reduce the residual amount of nitrite, inhibit fat peroxidation and promote color.
A strain of Kocuria salsicia H1 was isolated and identified and applied to meat products fermentation. Through the nitric oxide production, antioxidant and lipase activity of strain H1, the flavor and texture of meat products were improved while reducing nitrite residues.
By inoculating sausage Cockia H1, the flavor, texture and color of meat products are significantly improved, the residual amount of nitrite is reduced, fat peroxidation is inhibited, and safer and higher-quality meat products are achieved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of applied microbial technology, and specifically relates to a strain of Kocuria salsicia H1 and application thereof. Background Art
[0002] Fermented meat products are made from poultry and livestock meat, which are pickled, fermented, dried or smoked under natural or artificially controlled low temperature conditions to produce a type of meat product with special flavor, color and texture.
[0003] In the fermented meat industry, native strains existing in the natural fermentation process are often optimized to make starter cultures for standardized production. Adding exogenous starter cultures to produce fermented meat products has been widely adopted. This production method shortens the production cycle, can better control the processing process, and greatly improves the quality and food safety of meat products. Staphylococcus and Micrococcus are both native dominant strains in fermented meat products. There have been many studies on the application of Staphylococcus in meat products. At present, there are no reports on the use of Coxiella in meat fermentation. Summary of the invention
[0004] The present invention provides a Kocuria salsicia H1, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 11, 2024, with a deposit number of GDMCC NO: 64604, and a deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.
[0005] Another object of the present invention is to apply Coxiella sausage H1 in the fermentation preparation of meat products without adding nitrite. Strain H1 can improve the flavor and texture of meat products, reduce the residual nitrite, inhibit fat peroxidation, and promote the color development of meat products.
[0006] The purpose of the present invention is achieved through the following technical solutions:
[0007] 1. Sample the ham from Yunlong County, Dali Prefecture, Yunnan Province, cut it into pieces, homogenize it aseptically, and then dilute and spread it on MSA solid medium for culture; pick a single colony, streak and purify it to obtain strain H1;
[0008] 2. Identification of strain H1
[0009] (1) Morphological characteristics of strain H1
[0010] On MSA solid medium, it appears as opaque round colonies, lemon yellow, indicating smooth, neat edges; the strain is Gram-positive, arranged in pairs, chains, quadruplets, octaplexes or irregular clusters;
[0011] (2) Molecular identification
[0012] The genomic DNA of strain H1 was extracted using a bacterial genomic DNA rapid extraction kit purchased from Bioengineering. The extracted genome was used as a template to amplify the 16S rDNA gene using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplified product was recovered and purified and then sequenced. The sequencing results were analyzed by BLAST comparison using the NCBI database. Combined with the morphological characteristics and molecular identification results, the strain H1 was finally identified as Kocuria salsicia.
[0013] 3. The present invention has proved through safety evaluation experiments that strain H1 has no coagulation or hemolysis, does not contain heat-resistant deoxyribonuclease, and does not produce bioamines, and is safe for use in fermented meat products; through physiological and biochemical experiments, it has been proved that strain H1 produces nitric oxide and has strong antioxidant properties, and can effectively inhibit lipid peroxidation; it has lipoxygenase and also produces the flavor substance acetoin, which can effectively improve the flavor of fermented meat products;
[0014] 4. The strain H1 was applied to the fermentation of sausages without adding nitrite. The experimental results showed that the fermentation with the inoculated strain H1 could significantly add flavor to the sausages and enhance the umami taste; it also increased the hardness, elasticity, stickiness and chewiness of the sausages, and the nitrite content in the sausages was significantly reduced.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] 1. The present invention has isolated a strain of Coxiella sausage from Yunlong ham, which can promote the aroma and color of meat products, improve the texture and flavor of meat products, shorten the fermentation time, and improve production efficiency; the catalase produced by strain H1 can keep the color of meat products bright; the nitric oxide produced can promote the production of NO-Mb after meat products are fermented, making the meat products bright pink; it has lipase activity, can hydrolyze fat, and promote the formation of specific aromatic substances; it can produce volatile compounds such as acetoin with a special creamy aroma, which helps to improve the flavor of fermented meat products. After multiple tests such as plasma coagulation test, hemolysis test, heat-resistant deoxyribonuclease test, and production of bioamines, the results were all negative. H1 is safe and reliable for use in food fermentation;
[0017] 2. Using the starter prepared by strain H1 in meat fermentation can not only ensure the consistency of quality, but also effectively avoid the accumulation of harmful metabolites such as nitrite. In the fermented meat industry, H1 provides a strain option other than Staphylococcus and can be used for standardized production, with good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1This is a schematic diagram of the colony morphology of Coxiella sarcini H1 on MSA solid medium;
[0019] Figure 2 This is a schematic diagram of the morphology of Coxiella sarcini H1 under a microscope;
[0020] Figure 3 This is the phylogenetic tree of Coxiella sarcini H1;
[0021] Figure 4 It is the result of plasma coagulation test;
[0022] Figure 5 The results of the hemolysis test;
[0023] Figure 6 The results of the thermostable deoxyribonuclease test;
[0024] Figure 7 To produce the results of the bioamine experiment;
[0025] Figure 8 This is a schematic diagram of the detection results of lipase produced by Coxiella sarcini H1;
[0026] Fig. 9 This is the test result of acetoin production by the metabolites of Coxiella salviae H1;
[0027] Fig.10 This is the NO fluorescent probe detection diagram in Coxiella salviae H1;
[0028] Fig.11 Radar chart of effective taste indicators for fermented sausages. DETAILED DESCRIPTION
[0029] The technical scheme of the present invention is further described in detail below through examples, but the content of the present invention is not limited thereto. The methods in the present examples are conventional methods unless otherwise specified, and the materials and reagents used are obtained from commercial channels or prepared according to conventional methods unless otherwise specified;
[0030] Example 1: Isolation, screening and identification of H1 from sausage
[0031] 1. Screening, isolation and purification of strains
[0032] Samples were taken from the center of ham obtained from Yunlong County, Dali, Yunnan, cut into several small pieces, added with sterile 0.9% saline, homogenized, and appropriately diluted. After diluting with 0.9% saline, the samples were spread on MSA solid medium and cultured at 30°C for 48h; single colonies were picked and streaked on MSA solid medium for isolation, cultured at 30°C, and purified and isolated repeatedly for 3 times to obtain strain H1;
[0033] 2. Identification of strain H1
[0034] (1) Morphological identification
[0035] After purification, strain H1 appeared as opaque round colonies on MSA solid medium, with a lemon yellow color, indicating smooth and neat edges ( Figure 1 ); Gram-positive bacteria, arranged in pairs, chains, quadruplets, octaplexes or irregular clusters ( Figure 2 );
[0036] (2) Molecular identification
[0037] The genomic DNA of strain H1 was extracted using a bacterial genomic DNA rapid extraction kit purchased from Bioengineering. The extracted genome was used as a template to amplify the 16S rDNA gene fragment using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplified product was recovered and sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO: 1. The sequencing results were subjected to BLAST comparison analysis on NCBI, and the phylogenetic tree was constructed using MEGA11.0.11 software ( Figure 3 ), combined with the morphological characteristics and molecular identification results, strain H1 was determined to be Kocuria salsicia.
[0038] Example 2: Safety evaluation of sausage Koch's H1 strain
[0039] 1. Plasma coagulation test
[0040] Add 0.5 mL of sterile saline to the vial containing freeze-dried rabbit plasma to completely dissolve it, add 0.3 mL of MSA liquid culture of Coxiella salviae H1, mix thoroughly, and incubate at 30°C. Observe every half an hour for 6 hours. No coagulation occurs. Figure 4 ).
[0041] 2. Hemolysis test
[0042] Take the sausage Coxiella H1 cultured in nutrient broth (NB) medium, streak it on the blood plate, and observe it after culturing at 30℃ for 24 hours. It is observed that the sausage Coxiella H1 does not produce a hemolytic circle and has no hemolytic effect ( Figure 5 ).
[0043] 3. Thermostable deoxyribonuclease assay
[0044] The Kochella salsa H1 cultured in NB medium was inoculated on toluidine blue-DNA agar medium and cultured at 30°C for 24 h. It was observed that the surrounding of the colony did not turn into rose red, indicating that Kochella salsa H1 did not contain heat-resistant deoxyribonuclease ( Figure 6 ).
[0045] 4. Bioamine production experiment
[0046] The Kochella salsa H1 cultured in NB medium was inoculated into the liquid medium producing bioamines and cultured at 30°C for 24-48 hours. The culture medium was observed to be yellow and did not turn purple, indicating that H1 did not produce bioamines ( Figure 7 ).
[0047] Example 3: Detection of functional properties of sausage Koch's H1
[0048] 1. Catalase test
[0049] After culturing overnight at 30°C, use an inoculation loop to apply the H1 bacterial solution onto a plate containing 3% H 2 O 2 Observe the results immediately on the slide. The appearance of bubbles indicates that the catalase of Coxiella salviae H1 is positive.
[0050] 2. Lipase activity detection
[0051] The Koch strain H1 was inoculated into NB liquid medium and cultured at 200 rpm for 14-18 h. 10 μL of the bacterial solution was spotted onto tributyrin agar medium. After 72 h of culture, a hydrolysis zone ( Figure 8 ), indicating extracellular lipase activity.
[0052] 3. Acetoin production experiment
[0053] Inoculate the Koch's sausage strain H1 in the acetoin detection medium, take the culture medium and mix it with 10% NaOH in equal amounts, add a little creatine, and the culture medium will turn red, indicating that H1 produces acetoin ( Fig. 9 ).
[0054] 4. Nitric Oxide Production Experiment
[0055] NO in the cells of Coxiella salami H1 was detected by DAF-FM DA (NO-specific fluorescent probe); the third-generation cultured Coxiella salami H1 bacterial solution was centrifuged at 7000 g for 10 min to obtain the bacterial cells, and the bacteria were resuspended in NOS detection buffer to keep the bacteria at 2×10 6 -1×10 8 Then, DA-FM DA fluorescent probe was added and incubated at 37°C in the dark for 0.5-1h. Obvious green fluorescence signal was observed under a fluorescence microscope (Leica GM2500) at 100x oil immersion. Fig.10 ), the results showed that strain H1 produced nitric oxide.
[0056] 5. Antioxidant capacity test
[0057] (1) Determination of the DPPH scavenging ability of Coxiella salami H1
[0058] The strain H1 cultured overnight in NB liquid medium was centrifuged at 5000g for 10 min to collect the cells, washed 3 times with PBS, suspended in PBS, and adjusted to OD 600 nm=1.0±0.05, take 0.5mL of the bacterial suspension to be tested, add 0.5mL of 0.2mmol / L DPPH solution, mix well, react at room temperature in the dark for 30min, centrifuge at 7000rpm for 5min, measure the absorbance at 517nm, and repeat 3 times for each sample;
[0059] The clearance rate was calculated according to the following formula: Clearance rate % = [1-(A 样品 -A 空白 ) / A 对照 ]×100.
[0060] In the blank group, DPPH was replaced by an equal volume of anhydrous ethanol; in the control group, the sample solution (bacterial suspension) was replaced by an equal volume of PBS.
[0061] The results showed that the DPPH clearance rate of strain H1 was 65.56%.
[0062] (2) Determination of the ability of Coxiella salviae H1 to scavenge hydroxyl radicals (OH)
[0063] The hydroxyl radical scavenging ability detection kit (purchased from Beijing Solebow Technology Co., Ltd., catalog number: BC1320) was used for determination. The H1 bacterial suspension in step (1) was taken and the determination was performed according to the operating procedures of the kit. The measured hydroxyl radical scavenging rate of H1 was 92.34%, indicating that Coxiella sarcini H1 has strong antioxidant activity and can effectively inhibit lipid peroxidation.
[0064] Example 4: Application of sausage Koch's H1 in fermented sausage
[0065] 1. Preparation of Coxiella sausage H1 bacterial suspension
[0066] The activated Coxiella sausage H1 was inoculated into NB culture medium with an inoculation amount of 1% to 3% (v / v), placed on a shaker at 200 rpm and 30°C for 18 hours, and transferred and cultured continuously for 3 times to obtain a third-generation fermentation broth; the third-generation fermentation broth was centrifuged at 4°C and 7000g for 10 minutes, the precipitate was taken, the precipitate was washed 3 times with sterile saline, and the precipitate was resuspended to obtain an H1 bacterial suspension.
[0067] 2. Sausage production
[0068] Purchase pork hind leg meat (fat-to-lean ratio of 2:8), remove the fascia, mince and set aside; add 2.5% salt and 1% glucose to the meat and mix well; set up H1 fermentation group: inoculate H1 bacterial suspension and ensure that the total number of viable bacteria in the sausage reaches 10 7 CFU / g; the control group was not inoculated with H1 bacterial suspension, and an equal volume of sterile saline was added; the nitrite group was added with 100 mg / kg sodium nitrite and an equal volume of sterile saline.
[0069] After mixing, perform enema, ferment at 30°C and relative humidity of 75%-80% for 24 hours, and mature at 15°C and relative humidity of 60%-65% for 6-7 days.
[0070] 3. Conduct nitrite, texture, color difference and sensory evaluation on fermented mature sausages
[0071] (1) Determination of nitrite content
[0072] The determination was carried out according to the spectrophotometric method in the national standard 5009.33-2016 "National Food Safety Standard Determination of Nitrite and Nitrate in Food". The standard curve of nitrite is y=0.0372x+0.0433, R 2 =0.996 The results of nitrite detection in sausages of each experimental group are shown in Table 1. The nitrite content in the H1 fermentation group was significantly lower than that in the nitrite group and the control group;
[0073] Table 1 Nitrite content in sausages
[0074]
[0075] Note: Different lowercase letters in the same row indicate significant differences (p<0.05).
[0076] (2) Using a texture analyzer to test the texture of fermented mature sausages
[0077] The fermented mature sausage was cut into 1 cm thick slices. The force sensing element had a range of 500 N. The probe used in the experiment was a 7.5 cm diameter disc. The probe took the sample platform as the displacement zero point and moved downward at a rate of 60 mm / min. When the probe touched the sausage sample (i.e., when the trigger force reached 0.75 N), it was pressed downward at a rate of 60 mm / min. After the sample was deformed by 50%, the probe was lifted at a rate of 60 mm / min and returned to the sample surface. Then, the extrusion was repeated once. The results are shown in Table 2.
[0078] The hardness, stickiness and chewiness of the H1 fermentation group were significantly higher than those of the control group and the nitrite group. The addition of H1 fermentation increased the hardness, elasticity and stickiness of the sausage, and the chewiness also increased. The addition of Coxiella H1 could make the sausage's tissue structure tighter and effectively improve the sausage's texture.
[0079] Table 2 Effects of different treatments on sausage texture
[0080]
[0081] Note: Different lowercase letters in the same column in the table indicate significant differences (p<0.05), the same as in Table 3 and Table 5.
[0082] (3) Determination of the color of fermented sausage using a desktop colorimeter
[0083] The fermented mature sausage was cut into slices with a thickness of about 1 cm. The brightness value (L*), redness value (a*) and yellowness value (b*) of the sample were measured with a colorimeter. The reflection mode was selected and the instrument was calibrated with the built-in standard white plate to measure the L*, a* and b* values. The color difference comparison is shown in Table 3.
[0084] The a* value of sausage fermented by inoculated strain H1 was significantly higher than that of naturally fermented sausage (p<0.05), and there was no significant difference with the nitrite group (addition amount 100ppm) (P>0.05). Therefore, the color development effect of sausage inoculated with Kochella H1 replaced nitrite;
[0085] Table 3 Chromaticity of fermented sausage
[0086]
[0087] (4) 10 sensory assessors were selected to evaluate the color, flavor, taste and texture of the cooked sausages. The sensory evaluation criteria are shown in Table 4, and the sensory evaluation results are shown in Table 5.
[0088] Table 4 Sensory evaluation standards
[0089]
[0090]
[0091] When inoculated with H1 to ferment sausages, H1 can produce NO, which reacts with myoglobin to form nitrosomyoglobin, making the meat products bright pink, indirectly affecting the color of the product;
[0092] As shown in Table 5, the color, texture and taste of the inoculated H1 group were significantly higher than those of the natural fermentation group (P<0.05), and the flavor was better than that of the control group and the nitrite fermentation group (P<0.05). The inoculated H1 fermentation significantly improved the color, flavor, taste and texture of the sausage.
[0093] Table 5 Sensory evaluation results
[0094]
[0095] (5) Electronic tongue is an effective means of analyzing flavor substances. It can simulate human taste perception and detect the flavor of fermented sausages in different treatment groups using electronic tongue.
[0096] Weigh 50g of sausage sample and put it in a food processor. Add pure water at a mass ratio of 1:5 to homogenize. After filtering, take the filtrate for SA402B Plus taste analysis system test. The reference solution used in the experiment is composed of potassium chloride and tartaric acid. Tasteless represents the output (tasteless point) of the reference solution. The tasteless point of each index is 0. Based on this, when the taste value of the sample is lower than Tasteless, it means that the sample has no taste, otherwise it has.
[0097] The results are as follows Fig.11 As shown in the figure, the main taste indicators of fermented sausages are bitterness, umami, saltiness, umami aftertaste and bitter aftertaste. The bitterness of the fermentation group inoculated with H1 was the lowest; the umami and umami aftertaste of the fermentation group inoculated with H1 were significantly higher than those of the natural fermentation group and the sodium nitrite group, indicating that the fermentation inoculated with H1 can reduce bitterness and significantly increase the umami taste of sausages.
Claims
1. A strain of Kocuria salsicia H1, whose deposit number in Guangdong Microbiological Culture Collection Center is GDMCC NO:64604.
2. Use of the Koxima bacillus H1 of claim 1 in the fermentation preparation of meat products.
3. The use according to claim 1, characterized in that: It can enhance the flavor and texture of meat products, reduce residual nitrite, inhibit fat peroxidation, and promote the color development of meat products.