Tetragenococcus halophilus and application thereof
By inoculating halophilic Tetragenococcus during the ham fermentation process, the food safety problem caused by nitrite was solved, the formation of zinc protoporphyrin was promoted, the uniform and stable color of the ham was achieved, and the safety and quality of the ham were improved.
Patent Information
- Application Number
- CN202510982815.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-09-30
AI Technical Summary
In the prior art, the use of nitrite in ham processing poses food safety issues, such as the formation of nitrosamines, which leads to uneven color and potential health risks, and insufficient formation of zinc protoporphyrin, which affects the red color stability of the ham.
Halophilic Tetragenococcus is inoculated during the fermentation stage of ham to promote the formation of zinc protoporphyrin, replacing nitrite for color development. The bacteria are inoculated in batches through the spraying method to control the amount of bacterial liquid and fermentation conditions to ensure safety and color uniformity.
The effective formation of zinc protoporphyrin in ham is achieved, the red color stability of the ham is improved, the production of nitrosamines is avoided, and food safety and color uniformity are ensured.
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Figure CN120718809A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of meat processing, in particular to a halophilic tetragenococcus and application thereof. Background Art
[0002] Ham is a traditional Chinese cured meat product, widely beloved for its vibrant red color, rich aroma, delicious flavor, and long shelf life. Nitrite is widely used as a key food additive in traditional meat processing. However, its use is controversial due to potential food safety concerns, such as the formation of nitrosamines. Regulations limit the maximum amount of nitrite in meat products to 150 mg / kg (calculated as sodium nitrite), with residual levels not exceeding 30 mg / kg.
[0003] The color of ham is one of the factors that measure its quality. The color of ham is mainly determined by the content of myoglobin (Mb) and hemoglobin (Hb). Changes in the chemical form and content of Mb directly affect the color of dry-cured ham. Nitrite can react with myoglobin in muscle to generate stable nitrosomyoglobin, which makes dry-cured ham present an attractive pink color for a long time. At pH 5.0-6.0, the conversion rate of nitrosomyoglobin is over 90%. The color reaches saturation when NaNO2>80mg / kg. 30-50mg / kg can meet the basic color requirements of most meat products. Considering the balance between color and safety, the added amount of nitrite used on a large commercial scale is 80-120mg / kg. However, in actual application, differences in muscle parts can lead to uneven nitrite color and color difference.
[0004] Zinc protoporphyrin IX (ZnPP) is a naturally occurring metalloporphyrin compound that is the chemical essence of the red pigment of salt-free dry-cured ham and the main contributor to the red color of salt-free dry-cured ham. 2+ When Zn is removed from the heme molecule, under suitable external conditions, 2+ Can replace Fe 2+ Inserted into the center of the porphyrin ring, the heme part is separated from the natural hemoglobin to form ZnPP. 2+ It is Cu removal 2+ ZnPP is the metal ion that is most easily inserted into porphyrin and can easily complete chelation with porphyrin without a catalyst; ZnPP is not easily affected by light and oxygen, has stable properties, and can effectively replace the nitroso group to produce color. Compared with the nitroso group color, ZnPP has no risk of nitrosamine formation and no hidden dangers of blue baby syndrome.
[0005] Therefore, promoting the formation of zinc protoporphyrin in dry-cured ham can safely and effectively improve the color of dry-cured ham. Summary of the Invention
[0006] In order to solve or partially solve the problems existing in the related art, one of the objects of the present invention is to provide a halophilic tetragenous coccus, the preservation number of the halophilic tetragenous coccus is CCTCC NO: M 20251414, the preservation time is June 18, 2025, the preservation unit is: China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the Latin name of the halophilic tetragenous coccus is: Halophilic tetragonococcus .
[0007] Another object of the present invention is to provide an application of halophilic Tetragenococcus in ham processing.
[0008] Preferably, Tetragenococcus halophilus is added during the fermentation stage of the ham.
[0009] Preferably, the dosage of halophilic Tetragenococcus is: 10-20 mL per kilogram of ham at a concentration of 10 7 ~10 8 CFU / mL of halophilic Tetragenococcus bacterial solution.
[0010] Preferably, the bacterial solution is sprayed over 2 to 5 days using a spraying method.
[0011] Preferably, the amount of bacterial solution sprayed per day is 20% to 40% of the total bacterial solution volume used.
[0012] The present invention does not limit the specific processing process of the ham, and all existing processing methods can be used. It only needs to add halophilic Tetragenococcus during the fermentation stage of ham processing.
[0013] The technical solution provided by the present invention can have the following beneficial effects: (1) The present invention discovered for the first time a bacterium that can promote the formation of zinc protoporphyrin in ham: Tetragenococcus halophilus. Using this bacterium to ferment ham can not only promote the formation of zinc protoporphyrin in ham, but also does not produce harmful substances.
[0014] (2) The halophilic Tetragenococcus provided by the present invention is isolated and screened from mature ham, and therefore has high safety in use.
[0015] (3) The selected halophilic tetragenococci have a good color-producing effect of replacing nitrite. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is the morphology of halophilic Tetragenococcus on the plate.
[0017] Figure 2This is a Gram stain of Tetragenococcus halophilus. DETAILED DESCRIPTION
[0018] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the contents described above.
[0019] The halophilic tetragenococcus described in the present invention is separated and purified from the biceps femoris and semimembranosus of two-year-old mature Xuanwei ham.
[0020] The colony morphology of halophilic tetragenococci is as follows Figure 1 As shown, from Figure 1 It can be seen that the halophilic tetragenous cocci are dense and small colonies with smooth surface and light yellow dots. The Gram staining results of the halophilic tetragenous cocci are as follows: Figure 2 As shown, from Figure 2 It can be seen that Tetragenococcus halophilus is a Gram-positive bacterium, and the 16S rDNA sequence of Tetragenococcus halophilus is shown in SEQ ID NO: 01.
[0021] The biochemical identification results of the halophilic tetragenococcus strain are shown in Table 1. It can be seen from Table 1 that the halophilic tetragenococcus strain does not produce viscosity, does not produce catalase, and does not have the ability to produce acid, gas, H2S and nitrate reductase.
[0022] Table 1 Biochemical identification results of strains In the embodiment, 6 hybrid pigs with black gold pig bloodline raised in the same batch, of the same age and similar weight were selected. After slaughter, 12 pig hind legs were taken, with an average leg weight of 15.26±0.91 kg. The pig hind legs were randomly divided into four groups, with 3 pigs in each group; Xuanwei Puji Ham Food Co., Ltd. was entrusted to carry out pickling and processing.
[0023] Example 1 A method for promoting the formation of zinc protoporphyrin in dry-cured ham, comprising the following steps: (1) Pretreatment of raw leg: Use a knife to trim the loose connective tissue on the meat, and remove the excess fat and leg skin on both sides to obtain the pretreated meat pieces.
[0024] (2) Curing: Remove the blood and apply salt with a mass fraction of 5% of the leg weight evenly on the pretreated pork legs from the bottom to the top, from the skin surface to the meat surface. Then, stack them in a curing room with a temperature of 4°C and a relative humidity of 80% to 90% and curing for 25 days to obtain the cured pork legs.
[0025] (3) Cleaning: Clean the pickled pork leg with water to remove surface salt and dirt to obtain a cleaned pork leg; (4) Air drying: The cleaned pig legs were hung in an air drying room at 15°C and a relative humidity of 60% for air drying for 3 days to obtain air-dried pig legs.
[0026] (5) Fermentation: The air-dried pork leg was pre-fermented in a fermentation room at 15°C and 55% relative humidity for 15 days; then, it was mid-fermented in a fermentation room at 30°C and 70% relative humidity for 30 days; and then, it was post-fermented in a fermentation room at 25°C and 60% relative humidity. When mold just grew on the surface of the ham, the ham was inoculated with halophilic tetragenous cocci by spraying at a concentration of 10 per kilogram of ham. 7 A total of 10 mL of bacterial solution with a CFU / mL was sprayed over 2 days using a spraying method, with the amount of bacterial solution sprayed each day being 50% of the total bacterial solution volume. After the inoculation, the ham was continued to ferment until mature to obtain a dry-cured meat product.
[0027] After 100 days of fermentation, the biceps femoris and semimembranosus muscle samples were taken from the ham and stored in a -80°C refrigerator for analysis.
[0028] Example 2 A method for promoting the formation of zinc protoporphyrin in dry-cured ham, comprising the following steps: (1) Pretreatment of raw meat: Use a knife to trim the loose connective tissue on the meat, and remove the excess fat and leg skin on both sides to obtain the pretreated raw meat leg.
[0029] (2) Curing: Remove the blood and evenly smear 5% salt on the pretreated pork legs from the bottom to the top, from the skin to the meat. Then, stack them in a curing room at a temperature of 4°C and a relative humidity of 80% to 90% for 3 days to obtain the cured meat blocks.
[0030] (3) Cleaning: Clean the pickled pork leg with water to remove surface salt and dirt to obtain a cleaned pork leg; (4) Air drying: The cleaned pig legs were hung in an air drying room at 15°C and a relative humidity of 60% for air drying for 3 days to obtain air-dried pig legs.
[0031] (5) Fermentation: The air-dried pork leg was pre-fermented in a fermentation room at 15°C and 55% relative humidity for 15 days; then, it was mid-fermented in a fermentation room at 30°C and 70% relative humidity for 30 days; and then, it was post-fermented in a fermentation room at 25°C and 60% relative humidity. When mold just grew on the surface of the ham, the ham was inoculated with halophilic tetragenous cocci by spraying at a concentration of 10 per kilogram of ham. 7A total of 20 mL of bacterial solution with a CFU / mL was sprayed over 5 days using a spraying method, with the amount of bacterial solution sprayed each day being 20% of the total bacterial solution volume. After the inoculation, the ham was continued to ferment until mature to obtain a dry-cured meat product.
[0032] After 100 days of fermentation, the biceps femoris and semimembranosus muscle samples were taken from the ham and stored in a -80°C refrigerator for analysis.
[0033] Example 3 A method for promoting the formation of zinc protoporphyrin in dry-cured ham, comprising the following steps: (1) Pretreatment of raw meat: Use a knife to trim the loose connective tissue on the meat, and remove the excess fat and leg skin on both sides to obtain the pretreated raw meat leg.
[0034] (2) Curing: Remove the blood and evenly smear 5% salt on the pretreated pork legs from the bottom to the top, from the skin to the meat. Then, stack them in a curing room at a temperature of 4°C and a relative humidity of 80% to 90% for 3 days to obtain the cured pork legs.
[0035] (3) Cleaning: Clean the pickled pork leg with water to remove surface salt and dirt to obtain a cleaned pork leg; (4) Air drying: Hang the cleaned pork legs in an air drying room at 15°C and a relative humidity of 60% for air drying for 3 days to obtain air-dried meat blocks.
[0036] (5) Fermentation: The air-dried pork leg was pre-fermented in a fermentation room at 15°C and 55% relative humidity for 15 days; then, it was mid-fermented in a fermentation room at 30°C and 70% relative humidity for 30 days; and then, it was post-fermented in a fermentation room at 25°C and 60% relative humidity. When mold just grew on the surface of the ham, the ham was inoculated with halophilic tetragenous cocci by spraying at a concentration of 10 per kilogram of ham. 8 A total of 15 mL of bacterial solution with a CFU / mL was sprayed over 3 days, with the amount of bacterial solution sprayed each day being 20% to 40% of the total volume of the bacterial solution used. After the inoculation, the ham continued to ferment until mature to obtain a dry-cured meat product.
[0037] After 100 days of fermentation, the biceps femoris and semimembranosus muscle samples were taken from the ham and stored in a -80°C refrigerator for analysis.
[0038] Comparative Example 1 For comparison, in this comparative example, no halophilic Tetragenococcus was added during the preparation of the ham. The specific preparation process was the same as that of Example 1, and mature dry-cured ham was finally obtained.
[0039] After 100 days of fermentation, the biceps femoris and semimembranosus muscle samples were taken from the ham and stored in a -80°C refrigerator for analysis.
[0040] Test Example 1 Determination of ZnPP content in ham ZnPP was extracted using a mixed solvent of ethyl acetate / acetic acid / dimethyl sulfoxide (10:2:1, V / V / V) under light-proof conditions. 1.5 g of ham minced meat sample was accurately weighed and placed in a 50 mL light-proof centrifuge tube. 10 mL of the mixed solvent was added and homogenized at low temperature (9000 × g , 1 min), ice bath extraction for 20 min and centrifugation for 14 min (1100× g , 4°C), collect the supernatant and filter. Repeat the extraction of the sample residue with the same volume of mixed solvent. Combine the two filtrates and shake well. By comparing the excitation and emission spectra of the ZnPP standard and sample, the excitation wavelengths were selected as 420 / 400 nm and the emission wavelengths were 590 / 630 nm. A mixed solvent of ethyl acetate / acetic acid / dimethyl sulfoxide (10:2:1, v / v / v) was used as the reagent blank. Each sample was analyzed and determined six times. 4 mg of the ZnPP standard was dissolved in 100 mL of a mixed solvent of ethyl acetate / acetic acid / dimethyl sulfoxide (10:2:1, v / v / v) and serially diluted. The standard curve was determined and the ZnPP content was calculated: Where: M ZnPP Indicates the ZnPP content per kilogram of ham, mg / kg; m ZnPP Indicates the mass of ZnPP standard, mg; m 肉 Indicates the mass of ham sample, mg, m 水 Indicates the moisture content of ham sample, mg.
[0041] The ZnPP content in the biceps femoris and semimembranosus of the mature ham in Example 1 and Comparative Example 1 is shown in Table 1 Table 1 Determination results of ZnPP content in biceps femoris and semimembranosus of ham in Examples and Comparative Examples Note: Different capital letters in the same column indicate significant differences between the control group and the inoculated group; different lowercase letters in the same column indicate significant differences between the biceps femoris and semimembranosus muscles, the same below.
[0042] Test Example 2 Determination of physical and chemical indexes of Xuanwei ham in different years The pH in different samples was measured with reference to GB 5009.237-2016 “National Food Safety Standard - Determination of pH Value of Foods”.
[0043] The moisture content of different samples was determined with reference to GB 5009.3-2016 “National Food Safety Standard - Determination of Moisture in Foods”.
[0044] The water activity in different samples was determined with reference to GB 5009.238-2016 “National Food Safety Standard - Determination of Water Activity of Foods”.
[0045] The salt content in different samples was determined with reference to GB 5009.44-2016 “National Food Safety Standard - Determination of Chloride in Foods”.
[0046] The final test results are shown in Table 2. It can be seen from the table that there is no significant difference in the moisture content, water activity and salt content of the ham after inoculation with halophilic Tetragenococcus.
[0047] Table 2 Analysis of changes in physicochemical indicators in the biceps femoris and semimembranosus muscles of hams of Examples and Comparative Examples Test Example 3 By LC-MS / MS in positive and negative ion modes, 268 and 186 small molecule metabolites were detected from the ham test samples prepared in the example and comparative example, respectively, mainly including organic acids and their derivatives, lipids and lipid molecules, amino acids, polypeptides and their derivatives, nucleosides, nucleotides and their derivatives, etc., and no harmful substances were found.
[0048] Table 2 Differential small molecule metabolites in Xuanwei ham Note: RT indicates the chromatographic retention time of metabolites; m / z represents the mass-to-charge ratio of metabolite ions.
[0049] While various embodiments of the present invention have been described above, the foregoing description is intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.
Claims
1. A strain of halophilic Tetragenococcus ( Halophilic tetragonococcus ), characterized by: The deposit number of the halophilic Tetragenococcus is CCTCC NO: M 20251414.
2. Use of the halophilic Tetragenococcus according to claim 1 in ham processing.
3. The use according to claim 2, characterized in that: Halophilic Tetragenococcus was inoculated at the end of the ham curing period and the beginning of the fermentation period.
4. The use according to claim 3, characterized in that: The inoculation amount of halophilic tetragenous cocci is: 10~20mL per kilogram of ham with a concentration of 10 7 ~10 8 CFU / mL of halophilic Tetragenococcus bacterial solution.
5. The use according to claim 4, characterized in that: Use the spraying method to spray the bacterial solution over 2 to 5 days.