Method for fermenting air-dried sausages by completely replacing nitrite

Abstract

The invention belongs to the technical field of food, and relates to a method for fermenting air-dried sausages by completely replacing nitrite. According to the invention, lactobacillus fermentum CGMCC 1.1880, lactobacillus brevis CHOL1 and carrot powder are mixed to completely replace nitrite for food fermentation. The method can completely replace nitrite to promote the color development of the fermented meat product, so that the chromaticity can reach the effect similar to that of adding 140 mg / kg of sodium nitrite in meat, the residual quantity of nitrite in the fermented meat product is reduced, and the possibility that secondary amine in the nitrite and the meat reacts to generate a cancerogenic substance N-nitrosamine is further reduced; the residual quantity of nitrite in the air-dried sausage does not exceed 2mg / kg of meat, and the content of nitrosamine does not exceed 2.5 mu g / kg of meat. The method is of great significance in improving the safety of fermented food.

Description

Technical Field

[0001] The invention relates to the technical field of food, and is a method for completely replacing nitrite fermented meat products by adding a mixed starter and carrot powder, and an application of the method in air-dried sausage fermentation. Background Art

[0002] Air-dried sausage is a special fermented meat product in northern my country and Europe and the United States. It is deeply favored by consumers because of its unique flavor, rich nutrition, beautiful color and long shelf life.

[0003] Nitrite is a key food additive in meat production. It can react with myoglobin (Mb) to form nitrosomyoglobin (MbFe II NO), which promotes the color of meat products, inhibits harmful bacteria, delays lipid oxidation and forms the unique flavor of meat products. However, the residual nitrite will react with secondary amines such as secondary amines in meat to form carcinogenic N-nitrosamines, which seriously threatens people's health. Therefore, it is necessary to reduce the residual amount of nitrite. Finding a substitute for nitrite in meat products is of great significance.

[0004] Nowadays, there are only a handful of substances that can be used as nitrite substitutes in meat products, and some nitrite substitutes have been reported to have problems. For example, red yeast rice is a pigment that has been widely used. It is a natural pigment extracted from Monascus purpurogenum, and it can also play a good coloring role. However, there are reports that some health products containing red yeast rice ingredients can cause kidney diseases and harm human health. Therefore, red yeast rice pigment has safety hazards, so the addition of red yeast rice pigment to food needs to be more cautious. Therefore, it is urgent to screen new and healthy nitrite substitutes in order to completely avoid food safety risks and provide more options for the use of alternatives to nitrite. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for completely replacing nitrite fermented air-dried sausage, with the aim of solving the problem of the need to reduce nitrite addition in the prior art, and also solving the problem of potential safety hazards when using red yeast rice pigment as a nitrite substitute, thereby enriching the types of safe nitrite substitutes.

[0006] The first technical solution provided by the present invention is a mixed fermentation agent, which contains fermented Lactobacillus CGMCC 1.1880 and Lactobacillus brevis CHOL1. The Lactobacillus brevis CHOL1 has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 20, 2024, with a preservation number of GDMCC NO.64656.

[0007] In certain embodiments, the ratio of the viable counts of Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 is 1:5 to 5:1.

[0008] In certain embodiments, in the mixed fermentation agent, the mixed bacteria concentration of the fermented Lactobacillus CGMCC 1.1880 and the Lactobacillus brevis CHOL1 is not less than 1×10 5 CFU / mL or 1×10 5 CFU / g. Furthermore, the bacterial concentrations of the fermented Lactobacillus CGMCC 1.1880 and the Lactobacillus brevis CHOL1 are not less than 1.67×10 4 CFU / mL or 1.67×10 4 CFU / g.

[0009] The second technical solution provided by the present invention is a nitrite substitute, which comprises the fermentation agent described in the first technical solution and carrot powder.

[0010] In certain embodiments, the total amount of the mixed bacteria of Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 added to the nitrite substitute is 1×10 5 ~1×10 8 CFU / mL or 1×10 5 ~1×10 8 CFU / g.

[0011] In certain embodiments, the carrot powder is present in the nitrite substitute at a concentration of 5-50 mg / mL or 5-50 mg / g.

[0012] The third technical solution provided by the present invention is a method for preparing air-dried sausage, comprising the following steps:

[0013] (1) Prepare the raw materials for air-dried sausage;

[0014] (2) Adding auxiliary materials and the nitrite substitute described in the second technical solution to the raw materials of step (1) to carry out pickling, enema, fermentation, drying, packaging, and finished products.

[0015] In certain embodiments, the raw material of the air-dried sausage is obtained by mincing tenderloin with a fat-to-lean meat ratio of 1:9-3:7.

[0016] In certain embodiments, in step (2), the auxiliary materials include 1-2wt% salt, 0.3-0.5wt% monosodium glutamate, 1-1.5wt% liquor, 1-5wt% sugar and 1-1.5wt% soy sauce.

[0017] In certain embodiments, in step (2), the total amount of the fermented Lactobacillus CGMCC 1.1880 and the Lactobacillus brevis CHOL1 mixed is 1×10 5 ~1×10 8 CFU / g 肉 , and the ratio of the number of live bacteria between the two is 1:5 to 5:1, and the amount of carrot powder added is 0.5-5wt%.

[0018] In certain embodiments, in step (2), the fermentation and drying conditions are: temperature of 10-15° C., relative humidity of 70%-75%, and time of 10-12 days.

[0019] The fourth technical solution provided by the present invention is a method for reducing nitrite and / or nitrosamine in air-dried sausage, which comprises adding the substitute described in the second technical solution together with auxiliary materials to the raw materials of the air-dried sausage for pickling, enema, fermentation and drying, packaging, and finished product.

[0020] In certain embodiments, the raw material of the air-dried sausage is obtained by mincing tenderloin with a fat-to-lean meat ratio of 1:9-3:7.

[0021] In certain embodiments, the auxiliary materials include 1-2wt% salt, 0.3-0.5wt% monosodium glutamate, 1-1.5wt% liquor, 1-5wt% sugar and 1-1.5wt% soy sauce.

[0022] In certain embodiments, the total amount of the fermented Lactobacillus CGMCC 1.1880 and the Lactobacillus brevis CHOL1 mixed is 1×10 5 ~1×10 8 CFU / g 肉 , and the ratio of the number of live bacteria between the two is 1:5-5:1, and the concentration of carrot powder is 0.5-5wt%.

[0023] In certain embodiments, the fermentation and drying conditions are: temperature of 10-15° C., relative humidity of 70%-75%, and time of 10-12 days.

[0024] The fifth technical solution provided by the present invention is the use of the fermentation agent described in the first technical solution or the nitrite substitute described in the second technical solution in reducing nitrite and / or nitrosamines in air-dried intestines.

[0025] The technical effects of the present invention are as follows:

[0026] The present invention provides a method for using a mixture of fermented lactobacillus CGMCC 1.1880, Lactobacillus brevis CHOL1 and carrot powder to completely replace nitrite for fermented food. This method can completely replace nitrite to promote the color development of fermented meat products, so that the color can reach the same level as adding 140 mg / kg肉 It has a similar effect to sodium nitrite, reducing the residual nitrite in fermented meat products, thereby reducing the possibility of nitrite reacting with secondary amines in meat to produce carcinogenic N-nitrosamines, so that the residual nitrite in air-dried sausage does not exceed 2 mg / kg 肉 , the content of nitrosamines shall not exceed 2.5μg / kg 肉 This is of great significance in improving the safety of fermented foods.

[0027] Biomaterial Deposit

[0028] A strain of Lactobacillus brevis CHOL1, classified and named Levilactobacillus brevis, has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO.64656, a deposit date of May 20, 2024, and a deposit address of 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0030] The raw materials used in the examples are:

[0031] 1. Staphylococcus aureus CICC 24064, Lactobacillus plantarum CICC 10345, and Lactobacillus plantarum AS1.555 were purchased from Beijing Center for Biological Collection; Staphylococcus aureus CICC 11117s, Staphylococcus carnosus CICC 25173, and Staphylococcus calf CICC10850 were purchased from China Industrial Microbiological Culture Collection Administration Center; Staphylococcus carnosus GIM 1.955 was purchased from Guangdong Microbiological Culture Collection Center; Staphylococcus xylosus CS300 was purchased from Chr. Hansen, Germany; Micrococcus variant GIMJC031 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.; Italian Sacco VBL-97 and Italian Sacco VBL-45 were purchased from Italian Sacco Co., Ltd.; Lyocarni WBL-45 composite strain was purchased from the agent of Shanghai Haoyue Industrial Co., Ltd., and the manufacturer was ClericiSacco, Italy.

[0032] 2. MRS liquid medium: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7±0.2.

[0033] 3. MRS solid medium: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5±0.2.

[0034] 4. Carrot powder was purchased from Xinghua Tiannong Fruit and Vegetable Food Co., Ltd.

[0035] Example 1: Screening of starter cultures

[0036] Sources of alternative fermentation agents: We searched for existing lactic acid bacteria strains in the laboratory, all of which were derived from fermented foods, and obtained a total of 18 available lactic acid bacteria strains.

[0037] Among them, Lactobacillus brevis CHOL1: isolated from homemade sauerkraut. The genome of Lactobacillus brevis CHOL1 was extracted for 16srDNA identification. The genome extraction method was extracted according to the glass bead method of the "Concise Molecular Biology Experiment Guide", and Blast n analysis was performed. It was found that the bacteria had the highest homology with Levilactobacillus brevis, reaching 99.93%. Therefore, the strain was identified as Levilactobacillus brevis and named as Lactobacillus brevis (Levilactobacillusbrevis) CHOL1. The Lactobacillus brevis CHOL1 grew well in MRS solid culture medium and was cultured at 37°C for 24 hours to form colonies of Lactobacillus brevis that were round, smooth on the surface, and milky yellow.

[0038] Based on morphological and 16s rDNA identification, the strain CHOL1 was tentatively identified as Levilactobacillus brevis and was deposited in Guangdong Microbiological Culture Collection Center on May 20, 2024, with the deposit number: GDMCCNO.64656. The deposit address is: Guangdong Microbiological Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0039] Fermentation culture screening method: 18 strains of lactic acid bacteria were subjected to a simulated system colorimetric test. They were cultured in a nitrosomyoglobin simulated system for 24 hours and their colorimetric conditions were measured.

[0040] Myoglobin simulation system verifies fermentation performance of starter: Preparation of 2 mg / mL MbFe III solution, MbFe III The original solution (20 mg / mL) was incubated in a 50°C water bath for 30 min, and then the MbFe IIIThe stock solution was centrifuged (10000g, 5min, 4℃), the supernatant was filtered through a 0.22μm membrane and added to MRS liquid medium to make a 2.0mL final system (0.2mL myoglobin stock solution + 1.76mL MRS liquid medium). The simulated system was inoculated with 40μl of bacterial solution and anaerobically cultured at 37℃ for 18h, and the color of the supernatant was measured after centrifugation (10000g, 5min, 4℃).

[0041] Table 1 Myoglobin simulation system color test results

[0042]

[0043]

[0044] Note: Chroma is represented by a* / b* value. The larger the a* / b* value, the brighter the red color of the product.

[0045] From the experimental results, it can be seen that the CHOL1 and CGMCC1.1880 strains have the best color development effect, so these two strains are used as fermentation agents in air-dried sausages.

[0046] Example 2: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 1:1 with a total amount of 10 7 CFU / g 肉 , application of carrot powder with an addition amount of 1wt% in air-dried sausage

[0047] Step 1: Preparation of starter, fermentation lactobacillus CGMCC 1.1880 and brevis lactobacillus CHOL1 are stored at -80℃, strain activation is performed before use, and subculture is performed for two generations. Single colonies are picked in MRS liquid culture medium and cultured overnight on a shaker (37℃, 200rpm, 18h). Then 10% of the inoculum is inoculated into MRS liquid culture medium and cultured overnight on a shaker (37℃, 200rpm, 18h). Collect the cells by centrifugation and precipitate, wash the cells twice with sterile saline, centrifuge again to collect the cells by precipitating, suspend with an appropriate amount of sterile water and prepare 10 7 CFU / ml, and obtain the bacterial solution of Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 for use.

[0048] Step 2: Preparation of air-dried sausage, select tenderloin (fat-to-lean meat ratio 2:8), cut into dices, add auxiliary materials and nitrite substitutes for marinating, sausage filling, fermentation and drying, packaging, and finished product.

[0049] The auxiliary materials include salt: 1wt%, monosodium glutamate: 0.5wt%, liquor: 1.5wt%, sugar: 1wt%, soy sauce: 1.5wt%;

[0050] The nitrite substitute comprises fermented lactobacillus CGMCC 1.1880 and brevis lactobacillus CHOL1, and the total addition amount is: 10 7 CFU / g 肉 , and the ratio of the two is 1:1, carrot powder: 1wt%.

[0051] The fermentation and drying conditions are as follows: the temperature is generally set at (12±2)°C, the relative humidity is 70% to 75%, and the time is 12 days.

[0052] The above product is named as Example 2.

[0053] Example 3: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 1:1 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 0.5wt% in the application of air-dried sausage.

[0054] The concentration of carrot powder in Example 2 was replaced with 0.5 wt %, and other conditions or parameters were consistent with those in Example 2.

[0055] The above product is named as Example 3.

[0056] Example 4: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 1:1 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 5wt% in the application of air-dried sausage.

[0057] The concentration of carrot powder in Example 2 was replaced with 5 wt %, and other conditions or parameters were consistent with those in Example 2.

[0058] The above product is named as Example 4.

[0059] Example 5: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 1:2 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 1wt% in the application of air-dried sausage.

[0060] The ratio of Lactobacillus fermentum CGMCC 1.1880 to Lactobacillus brevis CHOL1 in Example 2 was replaced with 1:2, and other conditions or parameters were consistent with Example 2.

[0061] The above product is named as Example 5.

[0062] Example 6: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 2:1 with a total amount of 107 CFU / g 肉 The addition amount of carrot powder is 1wt% in the application of air-dried sausage.

[0063] The ratio of Lactobacillus fermentum CGMCC 1.1880 to Lactobacillus brevis CHOL1 in Example 2 was replaced with 2:1, and other conditions or parameters were consistent with Example 2.

[0064] The above product is named as Example 6.

[0065] Example 7: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 1:5 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 1wt% in the application of air-dried sausage.

[0066] The ratio of Lactobacillus fermentum CGMCC 1.1880 to Lactobacillus brevis CHOL1 in Example 2 was replaced with 1:5, and other conditions or parameters were consistent with Example 2.

[0067] The above product is named as Example 7.

[0068] Example 8: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 5:1 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 1wt% in the application of air-dried sausage.

[0069] The ratio of Lactobacillus fermentum CGMCC 1.1880 to Lactobacillus brevis CHOL1 in Example 2 was replaced with 5:1, and other conditions or parameters were consistent with Example 2.

[0070] The above product is named as Example 8.

[0071] Comparative Example 1: Add 140 mg / kg 肉 Naturally fermented air-dried sausage with sodium nitrite.

[0072] Step 1: Preparation of air-dried sausage, select tenderloin (fat-to-lean meat ratio 2:8) and cut into cubes.

[0073] Step 2: Add auxiliary materials and marinate for 1-2 hours, wherein the auxiliary materials include salt: 1wt%, monosodium glutamate: 0.5wt%, white wine: 1.5wt%, white sugar: 1wt%, soy sauce: 1.5wt%, sodium nitrite: 140mg / kg 肉 .

[0074] Step 3: sausage filling, fermentation and drying, packaging, and finished product. The fermentation and drying conditions are: the temperature is generally set at (12±2)°C, the relative humidity is 70% to 75%, and the time is 12 days.

[0075] The above product is named Comparative Example 1.

[0076] Comparative Example 2: Fermented Lactobacillus CGMCC 1.1880 single bacteria fermented air-dried sausage, inoculation amount is 10 7 CFU / g 肉 .

[0077] Step 1: Preparation of starter: The preparation of Lactobacillus fermentum CGMCC 1.1880 bacterial solution is as in step 1 of Example 2.

[0078] Step 2: Preparation of air-dried sausage, as in Example 2.

[0079] The auxiliary materials described are as in Example 2;

[0080] The nitrite substitute is a fermented Lactobacillus CGMCC 1.1880 bacterial solution, added in an amount of 10 7 CFU / g 肉 .

[0081] The fermentation and drying conditions are as in Example 2.

[0082] The above product is named Comparative Example 2.

[0083] Comparative Example 3: Lactobacillus brevis CHOL1 single-bacterium fermented air-dried sausage, inoculation amount is 10 7 CFU / g 肉 .

[0084] Step 1: Preparation of starter culture, such as the preparation of Lactobacillus brevis CHOL1 bacterial solution in step 1 of Example 2.

[0085] Step 2: Preparation of air-dried sausage, as in Example 2.

[0086] The auxiliary materials described are as in Example 2;

[0087] The nitrite substitute is Lactobacillus brevis CHOL1 bacterial solution, added in an amount of 10 7 CFU / g 肉 .

[0088] The fermentation and drying conditions are as in Example 2.

[0089] The above product is named Comparative Example 3.

[0090] Comparative Example 4: Only carrot powder was added to ferment the air-dried sausage, and the amount of carrot powder added was 1 wt%.

[0091] Step 1: Preparation of air-dried sausage, select tenderloin (fat-to-lean meat ratio 2:8) and cut into dices.

[0092] Step 2: Add auxiliary materials and nitrite substitute and marinate for 1-2 hours, wherein the auxiliary materials are as in Example 2; wherein the nitrite substitute is carrot powder, and the addition amount is 1wt%.

[0093] Step 3: as step 3 in comparative example 1.

[0094] The above product is named Comparative Example 4.

[0095] Comparative Example 5: The inoculation amount of Lactobacillus fermentum CGMCC 1.1880 is 10 7 CFU / g 肉 , the addition amount of carrot powder is 1% in the application of air-dried sausage.

[0096] The nitrite substitute in Comparative Example 2 was replaced as follows: the addition amount of Lactobacillus fermentum CGMCC 1.1880 was: 10 7 CFU / g 肉 , carrot powder: 1wt%, and other conditions or parameters are the same as those in Comparative Example 2.

[0097] The above product is named Comparative Example 5.

[0098] Comparative Example 6: Lactobacillus brevis CHOL1 inoculation amount is 10 7 CFU / g 肉 , the addition amount of carrot powder is 1% in the application of air-dried sausage.

[0099] The nitrite substitute in Comparative Example 2 was replaced as follows: the addition amount of Lactobacillus brevis CHOL1 was: 10 7 CFU / g 肉 , carrot powder: 1wt%, and other conditions or parameters are the same as those in Comparative Example 2.

[0100] The above product is named Comparative Example 6.

[0101] Comparative Example 7: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 1:1 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 0.3wt% in the application of air-dried sausage.

[0102] The concentration of carrot powder in Example 2 was replaced with 0.3 wt %, and other conditions or parameters were consistent with those in Example 2.

[0103] The above product is named Comparative Example 7.

[0104] Comparative Example 8: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 1:1 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 6wt% in the application of air-dried sausage.

[0105] The concentration of carrot powder in Example 2 was replaced with 6 wt %, and other conditions or parameters were consistent with those in Example 2.

[0106] The above product is named Comparative Example 8.

[0107] Comparative Example 9: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 1:6 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 1wt% in the application of air-dried sausage.

[0108] The ratio of Lactobacillus fermentum CGMCC 1.1880 to Lactobacillus brevis CHOL1 in Example 2 was replaced with 1:6, and other conditions or parameters were consistent with Example 2.

[0109] The above product is named Comparative Example 9.

[0110] Comparative Example 10: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 were mixed and inoculated in a ratio of 6:1 with a total amount of 10 7 CFU / g 肉 The addition amount of carrot powder is 1wt% in the application of air-dried sausage.

[0111] The ratio of Lactobacillus fermentum CGMCC 1.1880 to Lactobacillus brevis CHOL1 in Example 2 was replaced with 6:1, and other conditions or parameters were consistent with Example 2.

[0112] The above product is named Comparative Example 10.

[0113] Comparative Example 11: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus plantarum CICC 10345 were mixed and inoculated in a ratio of 2:1 with a total amount of 10 7 CFU / g 肉 , the amount of red yeast rice added is 0.2g / kg 肉 , the amount of beetroot red added is 24mg / kg 肉 The amount of vitamin E added is 0.03wt%, and the amount of nisin sprayed is 0.4g / kg 肉 Application in air-dried sausage.

[0114] Step 1: Preparation of starter, fermentation Lactobacillus CGMCC 1.1880 bacterial solution is prepared as in step 1 of Example 2. The preparation method of Lactobacillus plantarum CICC 10345 bacterial solution is as follows: Lactobacillus plantarum CICC 10345 is activated before use and subcultured for two generations. Pick a single colony in MRS liquid culture medium and culture it on a shaker overnight (37°C, 200rpm, 18h). Then inoculate it into MRS liquid culture medium with an inoculum size of 10%, and culture it on a shaker overnight (37°C, 200rpm, 18h). Collect the bacteria by centrifugation and precipitate, wash the bacteria twice with sterile saline, centrifuge again to collect the bacteria by precipitating, suspend with an appropriate amount of sterile water and modulate 10 7 CFU / ml, and obtain Lactobacillus plantarum CICC 10345 bacterial liquid.

[0115] Step 2: Preparation of air-dried sausage, as in Example 2.

[0116] The auxiliary materials described are as in Example 2;

[0117] The nitrite substitute is a mixture of Lactobacillus fermentum 1.1880 and Lactobacillus plantarum CICC 10345, and the total addition amount is: 10 7 CFU / g 肉 , the ratio of the two is 2:1, red yeast rice: 0.2g / kg 肉 , beetroot red: 24 mg / kg 肉 , Vitamin E: 0.03wt%, nisin spraying amount is 0.4g / kg 肉 .

[0118] The fermentation and drying conditions are as in Example 2.

[0119] The above product is named Comparative Example 11.

[0120] Comparative Example 12: Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus plantarum AS1.555 were mixed and inoculated in a ratio of 2:1 with a total amount of 10 7 CFU / g 肉 , the amount of red yeast rice added is 0.2g / kg meat, the amount of beetroot red added is 24mg / kg meat, the amount of vitamin E added is 0.03wt%, and the amount of nisin sprayed is 0.4g / kg 肉 Application in air-dried sausage.

[0121] The Lactobacillus plantarum CICC 10345 in Example 11 was replaced by Lactobacillus plantarum AS1.555, and other conditions or parameters were consistent with those in Example 11.

[0122] The above product is named Comparative Example 12.

[0123] Comparative Example 13: The inoculation amount of Staphylococcus aureus CICC 25173 was 2×10 9 CFU / g 肉 The inoculum size of Staphylococcus aureus CICC10850 was 1×10 9 CFU / g 肉 , application of yam powder 1wt%, lotus root powder 1wt%, celery powder 1wt% and D-isoascorbic acid 1wt% in air-dried sausage.

[0124] Step 1: Preparation of starter culture, Staphylococcus aureus CICC 25173 and Staphylococcus calf CICC 10850 were stored at -80°C, and the strains were activated before use and subcultured for two generations. A single colony was picked in LB broth medium and cultured overnight on a shaker (37°C, 200rpm, 48h). Then, 10% of the inoculum was inoculated into LB broth medium and cultured overnight on a shaker (37°C, 200rpm, 48h). The cells were collected by centrifugation and precipitated, and the cells were washed twice with sterile saline, and the cells were collected by centrifugation again to precipitate and suspend with an appropriate amount of sterile water and prepare 2×10 9 CFU / ml and 1×10 9 CFU / ml were used to obtain Staphylococcus aureus CICC 25173 and Staphylococcus calf CICC 10850 bacterial liquids for use.

[0125] Step 2: Preparation of air-dried sausage, as in Example 2.

[0126] The auxiliary materials described are as in Example 2;

[0127] The nitrite substitute is Staphylococcus carnosus CICC 25173 with an inoculum of 2×10 9 CFU / g 肉 and Staphylococcus aureus CICC 10850 with an inoculum size of 1 × 10 9 CFU / g 肉 , yam powder 1wt%, lotus root powder 1wt%, celery powder 1wt%, D-isoascorbic acid 1wt%

[0128] The fermentation and drying conditions are as in Example 2.

[0129] The above product is named Comparative Example 13.

[0130] Comparative Example 14: The inoculum size of Staphylococcus carnosus GIM 1.955 was 2×10 9 CFU / g 肉 The inoculum size of Staphylococcus aureus CICC10850 was 1×10 9 CFU / g 肉 , application of yam powder 1wt%, lotus root powder 1wt%, celery powder 1wt% and D-isoascorbic acid 1wt% in air-dried sausage.

[0131] The Staphylococcus carnosus CICC 25173 in Example 13 was replaced by Staphylococcus carnosus GIM 1.955, and other conditions or parameters were consistent with those in Example 13.

[0132] The above product is named Comparative Example 14.

[0133] Comparative Example 15: Staphylococcus aureus CICC 25173 and Staphylococcus calf CICC 10850 were mixed in a ratio of 2:1 and the total amount was 10 7 CFU / g 肉 , application of yam powder 1wt%, lotus root powder 1wt%, celery powder 1wt% and D-isoascorbic acid 1wt% in air-dried sausage.

[0134] The ratio of Staphylococcus carnosus CICC 25173 to Staphylococcus calf CICC 10850 in Example 13 was replaced with 2:1, and other conditions or parameters were consistent with Example 13.

[0135] The above product is named Comparative Example 15.

[0136] Comparative Example 16: Application of vegetable juice fermented by Staphylococcus xylosus CS300 and Staphylococcus carnosus GIM 1.955 in air-dried sausage.

[0137] Step 1: Enzyme hydrolysis: wash and mince the vegetables (including cabbage, spinach, celery, lettuce, white radish, Chinese cabbage, and rapeseed), add half the weight of the vegetables in water, homogenize, add 0.01% of enzyme preparation A and 0.01% of enzyme preparation B by weight of the vegetables, keep at 50°C for 0.5h, and obtain vegetable enzymatic hydrolyzate.

[0138] Wherein, the enzyme preparation A is a mixture of cellulase, hemicellulase and pectinase in a mass ratio of 1:1:0.5; the enzyme preparation B is a mixture of one or more of papain, neutral protease, composite protease and bromelain and flavor protease in a mass ratio of 1:1;

[0139] Step 2: fermentation, adding 1% salt, 0.5% glucose and 2% liquid seeds to the vegetable enzymatic hydrolysate obtained in step 1, fermenting at 25-35° C. for 2 days to obtain vegetable fermented liquid, and detecting the nitrite content of the fermented liquid to be above 2500 mg / kg, the fermentation is terminated;

[0140] The preparation method of the liquid seed is as follows: picking bacterial strains from the inclined surface of a test tube, inoculating the strains with seed culture medium, culturing at 25-35°C, measuring the OD600 value every 2 hours, that is, measuring the bacterial density at a wavelength of 600nm, and selecting the bacterial strains with an OD600 value of 0.4 as liquid seeds;

[0141] The seed culture medium comprises: 0.5 g glucose, 0.5 g soy peptone, 0.5 g dipotassium hydrogen phosphate, and 100 ml water.

[0142] The bacterial species are Staphylococcus xylosus CS300 and Staphylococcus carnosus GIM 1.955.

[0143] Step 3: Filtering: filtering the vegetable fermented liquid obtained in step 2 using a filter cloth with a mesh size of 200 to obtain a filtrate;

[0144] Step 4: Decolorization, add 1% by weight of activated carbon to the filtrate of step 3, keep at 20°C for 10 minutes, and stir continuously during the process to obtain a decolorized solution.

[0145] Step 5: Concentration: Use a vacuum concentrating device to concentrate the decolorized liquid obtained in step 4 at a concentration temperature of 40° C. Distill off 80% of the water to make the weight of the concentrate 20% of the original weight. The nitrite content in the concentrate is detected to be above 10,000 mg / kg, thereby obtaining a fermented vegetable juice rich in natural nitrite.

[0146] Step 6: Preparation of air-dried sausage: select tenderloin (fat-to-lean meat ratio 2:8), cut into dices, add auxiliary materials and fermented vegetable juice for marinating, sausage filling, fermentation and drying, packaging, and finished product.

[0147] The auxiliary materials described are as in Example 2;

[0148] Wherein the fermented vegetable juice is added in an amount of 1wt%.

[0149] The fermentation and drying conditions are as in Example 2.

[0150] The above product is named Comparative Example 16.

[0151] Comparative Example 17: Application of vegetable juice fermented by Micrococcus variant GIM JC031 and Staphylococcus carnosus GIM 1.955 in air-dried sausage.

[0152] The Staphylococcus xylosus CS300 in Example 16 was replaced by Micrococcus variants GIM JC031, and other conditions or parameters were consistent with those in Example 16.

[0153] The above product is named Comparative Example 17.

[0154] Comparative Example 18: Application of vegetable juice fermented by Staphylococcus xylosus CS300, Staphylococcus carnosus GIM 1.955 and Micrococcus variant GIMJC031 in air-dried sausage.

[0155] The bacterial species in Example 16 were replaced by Staphylococcus xylosus CS300 and Staphylococcus carnosus GIM 1.955 with Staphylococcus xylosus CS300, Staphylococcus carnosus GIM 1.955 and Micrococcus variants GIMJC031, and other conditions or parameters were consistent with those in Example 16.

[0156] The above product is named Comparative Example 18.

[0157] Comparative Example 19: Adding a fermentation agent Lyocarni WBL-45 composite strain (Staphylococcus carnosus, Staphylococcus xylosus and Lactobacillus sake, with a total bacterial count of 4.25×10 10 CFU / g) 0.1wt% and vegetable juice (spinach juice, celery juice and onion juice in a ratio of 1:1:1) 30wt% were used in air-dried sausage.

[0158] Step 1: Remove the roots of fresh spinach, leaves of celery, and skin of onion bought from the market. After washing, cut the celery and spinach into 1 cm lengths, dice the onion, use a blender to make a slurry, and place in a 4°C cold storage for later use.

[0159] Step 2: Preparation of air-dried sausage, select tenderloin (fat-to-lean meat ratio 2:8), cut into dices, add auxiliary materials, fermentation agent and vegetable juice for marinating, sausage filling, fermentation and drying, packaging, and finished product.

[0160] The auxiliary materials described are as in Example 2;

[0161] The fermentation agent is a Lyocarni WBL-45 composite strain, which is composed of Staphylococcus carnosus, Staphylococcus xylosus and Lactobacillus sake, with a total bacterial count of 4.25×10 10 CFU / g, the manufacturer is Clerici Sacco of Italy, the agent is Shanghai Haoyue Industrial Co., Ltd., and the addition amount is 0.1wt%.

[0162] The vegetable juice comprises 10 wt % of spinach juice, 10 wt % of celery juice and 10 wt % of onion juice.

[0163] The fermentation and drying conditions are as in Example 2.

[0164] The above product is named Comparative Example 19.

[0165] Comparative Example 20: Application of celery powder fermented by Italian Sacco VBL-97 in air-dried sausage.

[0166] Step 1: Preparation of celery juice: fresh celery is washed, leafed, chopped, juiced and filtered to obtain celery juice.

[0167] Step 2: Preparation of celery fermentation broth: Glucose was first prepared into a 50% concentrated solution, and then added to a conical flask at a ratio of 8% (final concentration). The amount of celery juice added was 50%, and the inoculum of VBL-97 strain was 1.25×10 6 CFU / ml (i.e., dissolve 1 g of bacterial powder in 10 ml of sterile water and then take 100 μl into a conical flask), and make up the total weight to 300 g with sterile water.

[0168] The VBL-97 strain was provided by Sacco Ltd., Italy.

[0169] Step 3: Place the prepared celery fermentation liquid in a 38°C constant temperature oscillator for fermentation for 24 hours, and freeze-dry to obtain the fermented celery powder for later use. 1wt%

[0170] Step 4: Preparation of air-dried sausage: select tenderloin (fat-to-lean meat ratio 2:8), cut into dices, add auxiliary materials and fermented celery powder for marinating, sausage filling, fermentation and drying, packaging, and finished product.

[0171] The auxiliary materials described are as in Example 2;

[0172] The fermented celery powder is added in an amount of 1wt%;

[0173] The fermentation and drying conditions are as in Example 2.

[0174] The above product is named Comparative Example 20.

[0175] Comparative Example 21: Application of celery powder fermented by Italian Sacco VBL-45 in air-dried sausage.

[0176] The Italian Sacco VBL-97 in Example 20 was replaced by the Italian Sacco VBL-45, and the other conditions or parameters were the same as those in Example 20.

[0177] The above product is named Comparative Example 21.

[0178] Comparative Example 22: Imitation Staphylococcus aureus CICC 24064 added in an amount of 10 7 CFU / g 肉 , the application of celery powder in air-dried sausage is 0.3wt%.

[0179] Step 1: Preparation of starter culture, imitating Staphylococcus aureus CICC 24064 stored at -80 ℃, strain activation before use, subculture two generations. Pick a single colony into MSA medium and culture overnight on a shaker (37 ℃, 200 rpm, 48h). Then inoculate 10% of the inoculum into MRA liquid medium and culture overnight on a shaker (37 ℃, 200 rpm, 48h). Centrifuge to collect the precipitate, wash the cells twice with sterile saline, centrifuge again to collect the precipitate, suspend with an appropriate amount of sterile water and prepare 10 7 CFU / ml, and the Staphylococcus aureus CICC 24064 bacterial solution must be simulated for use.

[0180] Step 2: Preparation of air-dried sausage, select tenderloin (fat-to-lean meat ratio 2:8), cut into dices, add auxiliary materials and celery powder for marinating, stuffing, fermentation and drying, packaging, and finished product.

[0181] The auxiliary materials described are as in Example 2;

[0182] The celery powder is added in an amount of 0.3 wt %.

[0183] The fermentation and drying conditions are as in Example 2.

[0184] The above product is named Comparative Example 22.

[0185] Comparative Example 23: Imitation Staphylococcus CICC 24064 added in an amount of 10 7 CFU / g 肉 , the application of celery powder in air-dried sausage is 1wt%.

[0186] The amount of celery powder added in Example 22 is replaced with 1 wt %, and other conditions or parameters are consistent with Example 22.

[0187] The above product is named Comparative Example 23.

[0188] Comparative Example 24: Imitation Staphylococcus CICC 11117s added in an amount of 10 7 CFU / g 肉 , the application of celery powder in air-dried sausage is 0.3wt%.

[0189] The imitation Staphylococcus aureus CICC 24064 in Example 22 was replaced by the imitation Staphylococcus aureus CICC 11117s, and other conditions or parameters were consistent with Example 22.

[0190] The above product is named Comparative Example 24.

[0191] Test Case

[0192] The embodiments of the present invention and the comparative examples were tested:

[0193] 1. Colorimetry.

[0194] According to the results of the determination of nitrosamine residues, the group that can reduce nitrosamine residues was screened and its color was further determined. The specific determination method is as follows:

[0195] The color of the air-dried sausage and smelly mandarin fish was measured using an UltraScan Pro colorimeter. The instrument was set to reflectance mode and calibrated using a white standard plate. The a* / b* value was used as the color index for evaluating the examples and comparative examples. Each sample was measured 6 times in parallel and the average value was calculated. The results are shown in Table 2.

[0196] Table 2 Chromaticity values ​​of air-dried sausage (a* / b*)

[0197]

[0198]

[0199] In practical applications, the a* / b* value can more accurately reflect the results of human visual observation. The larger the a* / b* value, the brighter the product color. The chromaticity results show that the mixture of fermented lactobacillus 1.1880 and brevis lactobacillus CHOL1 with carrot powder can play a role in color development, and the use of this natural fermentation agent to completely replace nitrite in air-dried sausage (Examples 2 to 8) can achieve the same color as adding 140 mg / kg 肉 Nitrite (Comparative Example 1) had a similar effect.

[0200] 2. Determination of residual nitrite.

[0201] To determine the residual nitrite in the examples and comparative examples, the spectrophotometric method was used according to GB 5009.33-2016 "National Food Safety Standard Determination of Nitrite and Nitrate in Food". The specific method is as follows:

[0202] (1) Nitrite extraction: Weigh 5 g (accurate to 0.001 g) of homogenized sample (if water is added during the preparation process, it should be converted according to the amount of water added), place it in a 250 mL stoppered conical flask, add 12.5 mL of 50 g / L saturated borax solution, add about 150 mL of water at about 70°C, mix well, heat in a boiling water bath for 15 min, take out and cool in a cold water bath, and let it stand at room temperature. Quantitatively transfer the above extract to a 200 mL volumetric flask, add 5 mL of 106 g / L potassium ferrocyanide solution, shake well, and then add 5 mL of 220 g / L zinc acetate solution to precipitate protein. Add water to the scale, shake well, let it stand for 30 min, remove the upper fat layer, filter the supernatant with filter paper, discard 30 mL of the initial filtrate, and set aside the filtrate.

[0203] (2) Determination of nitrite: Pipette 40.0mL of the above filtrate into a 50mL stoppered colorimetric tube, and plute 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL, 1.50mL, 2.00mL, and 2.50mL of sodium nitrite standard solution (5ug / mL) into 50mL stoppered colorimetric tubes, respectively. Add 2mL of 4g / L p-aminobenzenesulfonic acid solution into the standard tube and sample tube, mix well, let stand for 3-5min, then add 1mL of 2g / L naphthaleneethylenediamine hydrochloride solution into each tube, add water to the scale, mix well, let stand for 15min, use a 1cm colorimetric cup, adjust the zero point with a zero tube, measure the absorbance at a wavelength of 538nm, and draw a standard curve for comparison. Simultaneously make a reagent blank.

[0204] The residual amounts of nitrite in the examples and comparative examples are shown in Table 3.

[0205] Table 3 Residual nitrite in air-dried sausage (mg / kg)

[0206]

[0207]

[0208] It was found through experiments that compared with the comparative example, the residual amount of nitrite in the group (embodiment) in which Lactobacillus fermentum 1.1880 and Lactobacillus brevis CHOL1 were mixed with carrot powder to completely replace nitrite for air-dried sausage was significantly lower, and even in Examples 2, 3, 5, and 6, the residual amount of nitrite was so low that it could not be detected, and the highest residual amount of nitrite in the embodiment group did not exceed 2 mg / kg 肉 .

[0209] 3. Determination of nitrosamine content.

[0210] According to the results of the determination of nitrite residue, the experimental group that can degrade nitrite was screened to further determine its nitrosamine residue. The specific determination method is as follows:

[0211] Sample pretreatment: weigh 10g of homogenized sample, add 10uL NDPA-d14 internal standard (2ug / L), add 15mL acetonitrile, vortex mix, freeze in a -20℃ refrigerator for 30min, add 4g magnesium sulfate and 1g sodium chloride, oscillate for 30s, and centrifuge at 8000rpm / min at 4℃ for 10min; take 6mL of supernatant and add it to a 15mL centrifuge tube containing 50mg PSA, 150mg C18E and 900mg anhydrous sodium sulfate, vortex thoroughly for 1min, centrifuge at 4℃ 8000rpm / min for 10min, take 5mL of supernatant and concentrate it to 1mL by nitrogen blowing, pass it through a 0.22um organic filter membrane, and wait for detection.

[0212] GC parameters: carrier gas is helium; constant flow rate: 1.0 mL / min; temperature program: hold at 40 °C for 0.5 min, increase to 115 °C at a rate of 6 °C / min, hold for 1 min, then increase to 150 °C at a rate of 15 °C / min, hold for 0.5 min, then increase to 180 °C at a rate of 2 °C / min, hold for 2 min; solvent delay: 8.5 min;

[0213] MS parameters: ionization mode: EI; ion source temperature: 240°C; injection port temperature: 240°C; quadrupole temperature: 150°C; interface temperature: 240°C; detection mode: MRM; gain factor: 1.

[0214] The results of the determination of the nitrosamine content in the examples and comparative examples are shown in Table 4.

[0215] Table 4 Nitrosamine content in air-dried sausage (μg / kg)

[0216]

[0217]

[0218] As can be seen from Table 4, compared with the group to which nitrite was added (Comparative Example 1), the group to which nitrite was completely replaced by a mixture of fermented Lactobacillus 1.1880 and Lactobacillus brevis CHOL1 and carrot powder (Example) could significantly reduce the residual amount of nitrosamines in the air-dried intestines, and the residual amount of nitrosamines in the air-dried intestines was no more than 2.5 μg / kg. 肉 Although the groups (Comparative Examples 2 to 24) that also used natural fermentation agents to completely replace nitrites can also reduce the residual amount of nitrosamines in the air-dried sausages compared with Comparative Example 1, the effect is far less than that of the Example group.

[0219] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A mixed fermentation agent, characterized in that: The mixed fermentation agent contains Lactobacillus fermentum CGMCC 1.1880 and Levilactobacillus brevis CHOL1, and the Lactobacillus brevis CHOL1 has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 20, 2024, with the collection number GDMCCNO.64656.

2. The leavening agent according to claim 1, characterized in that The ratio of the live bacteria counts of the fermented lactobacillus CGMCC 1.1880 and the lactobacillus brevis CHOL1 is 1:5 to 5:

1.

3. The leavening agent according to claim 1, characterized in that In the mixed fermentation agent, the mixed bacteria concentration of the fermented lactobacillus CGMCC 1.1880 and the brevis lactobacillus CHOL1 is not less than 1×10 5 CFU / mL or 1×10 5 CFU / g.

4. A nitrite substitute, characterized in that: The nitrite substitute comprises the fermentation agent according to any one of claims 1 to 3 and carrot powder.

5. The nitrite substitute according to claim 4, characterized in that The total amount of the mixed bacteria of Lactobacillus fermentum CGMCC 1.1880 and Lactobacillus brevis CHOL1 added to the nitrite substitute is 1×10 5 ~1×10 8 CFU / mL or 1×10 5 ~1×10 8 CFU / g.

6. The nitrite substitute according to claim 4, characterized in that The concentration of the carrot powder in the nitrite substitute is 5-50 mg / mL or 5-50 mg / g.

7. A method for preparing air-dried sausage, characterized in that: The steps include: (1) Prepare the raw materials for air-dried sausage; (2) Adding auxiliary materials and the nitrite substitute according to any one of claims 4 to 6 to the raw materials of step (1) to carry out pickling, enema, fermentation, drying, packaging, and finished products.

8. The method according to claim 7, characterized in that In step (2), the auxiliary materials include 1-2wt% salt, 0.3-0.5wt% monosodium glutamate, 1-1.5wt% liquor, 1-5wt% sugar and 1-1.5wt% soy sauce; The total amount of the fermented lactobacillus CGMCC 1.1880 and the brevis lactobacillus CHOL1 mixed is 1×10 5 ~1×10 8 CFU / g 肉 , and the ratio of the number of live bacteria between the two is 1:5 to 5:1, and the amount of carrot powder added is 0.5-5wt%; The fermentation and drying conditions are as follows: temperature of 10-15° C., relative humidity of 70%-75%, and time of 10-12 days.

9. A method for reducing nitrite and / or nitrosamine in air-dried intestines, characterized in that: The method comprises adding the nitrite substitute according to any one of claims 4 to 6 together with auxiliary materials into the raw materials of air-dried sausages for pickling, enema, fermentation, drying, packaging and finished products.

10. Use of the fermentation agent according to any one of claims 1 to 3 or the nitrite substitute according to any one of claims 4 to 6 in reducing nitrite and / or nitrosamine in air-dried intestines.

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