Fermented lactobacillus mucilaginosus and application thereof in fermented sausages

Abstract

The invention discloses lactobacillus mucilaginosus and application thereof in fermented sausages, and belongs to the technical field of microorganisms. The strain is lactobacillus mucilaginosus XW6 which is submitted to the China Center for Type Culture Collection on December 30, 2024, the preservation address is Wuhan University Center, Wuchang District, Wuhan City, Hubei Province, the preservation number is CCTCC NO: M 20242942, and the classification name is Limosilicilla fermentum. The lactobacillus mucilaginosus XW6 has been submitted to the China Center for Type Culture Collection on December 30, 2024, and the preservation number is CCTCC NO: M 20242942. The strain disclosed by the invention can degrade nitrite, the degradation rate is as high as 93.96%, and meanwhile, the strain is used as a food leavening agent for fermenting meat products, so that the nitrite in fermented sausages is effectively degraded, and the safety of the fermented sausages is improved.

Description

Technical Field

[0001] The invention relates to a fermented mucus lactobacillus and application thereof in fermented sausages, belonging to the technical field of microorganisms. Background Art

[0002] Traditional Chinese sausage is a non-ready-to-eat meat product made from fresh or frozen livestock and poultry meat with other auxiliary materials, and processed by processes such as mincing, mixing, pickling, stuffing, sun-drying or air-drying), smoking or not smoking. Sausage has been eaten for thousands of years. Traditionally, it is fermented and matured for several weeks by naturally existing microorganisms in the environment at natural ambient temperature, eventually forming the unique rich flavor and firm texture of sausage. However, cured meat products are inevitably infected by harmful microorganisms during the long-term fermentation process in a complex natural environment, resulting in the deterioration of product quality and even harm to people's health after consumption.

[0003] At present, the food industry generally uses additives such as nitrites or nitrates to enhance the color of cured meats and inhibit the proliferation of pathogenic bacteria such as Clostridium botulinum and Staphylococcus aureus, as well as environmental spoilage bacteria, to give cured meats a unique flavor and inhibit protein oxidation and fat oxidation and rancidity during the fermentation and storage of the final product. However, the added chemical food additive nitrite is usually not completely degraded during the maturation and storage of sausages. A normal adult will be poisoned if he ingests 0.2g to 0.5g, and will die if he ingests 3g. The strong toxic effect of nitrite on the human body is manifested in that it can oxidize ferrous ions in the blood into high-valent iron ions, causing hypoxia in the body, leading to poisoning or even death; it reacts with amines produced by protein metabolism to form nitrosamine carcinogens, leading to cancers such as gastric cancer and esophageal cancer; nitrites can interfere with the body's metabolism of iodine and cause thyroid enlargement; and ingestion by pregnant women can cause fetal malformations.

[0004] At present, there are mainly physical methods (high pressure method and adsorption method), chemical methods (additive degradation method) and nitrite replacement additive method to reduce nitrite. Physical methods and chemical methods cannot fundamentally reduce the content of nitrite in food. They can only control the amount of nitrite generated under certain conditions, affect its flavor, and increase food safety risks. The additive replacement method cannot fully play the role of nitrite in meat processing; although some natural additives have certain antibacterial effects, their effects are not as significant as nitrite. At the same time, substitutes such as natural spices and condiments are usually more expensive, which will increase the production cost of food. Summary of the invention

[0005] In order to solve the deficiencies and shortcomings of the prior art, one of the objects of the present invention is to provide a fermentative mucus lactobacillus XW6, which has been deposited in the China Type Culture Collection on December 30, 2024, with the preservation address being Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the preservation number being CCTCC NO: M20242942, and the taxonomic name being: Limosilactobacillusfermentum.

[0006] Another object of the present invention is to use the fermented Lactobacillus mucilaginosus XW6 as a starter in fermenting sausages.

[0007] Preferably, the preparation process of the Chinese sausage is as follows: fresh pork is skinned and tendons are removed, the pork is cleaned with clean water, and minced into meat fillings with a meat grinder; 2-2.5% (mass) salt, 1-3% (mass) white sugar, 0.5-2% (mass) five-spice powder, 2-4% (mass) fermented mucus lactobacillus XW6, and 28-30 mg / kg nitrite are added according to the mass of the meat fillings, and the meat fillings are mixed evenly; after the mixing is completed, the mixed meat fillings are filled and formed using a sausage stuffer and pig small intestine casings; the mass of each Chinese sausage is 140-160 g; the prepared Chinese sausages are placed at 12-18° C. for fermentation for 15-20 days; after the fermentation is completed, the Chinese sausages are vacuum packed and stored at -18° C. to -20° C.

[0008] Technical effects of the present invention:

[0009] (1) The present invention screened and obtained a new fermentative mucus Lactobacillus XW6.

[0010] (2) When the fermented Lactobacillus mucilaginosus XW6 of the present invention is used to ferment sausages, it not only degrades nitrite, but also decomposes macromolecular substances in meat products, which is not only beneficial to human digestion and absorption but also gives the meat products a unique fermented flavor. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Colony diagram of fermentation Lactobacillus mucilaginosus XW6 strain

[0012] Figure 2 This is the morphological image of fermenting mucus Lactobacillus XW6 under an electron microscope.

[0013] Figure 3 This is the pH value of sausage after adding fermented Lactobacillus mucilaginosus XW6.

[0014] Figure 4 This is the degradation of nitrite in sausage after adding fermented Lactobacillus mucilaginosus XW6. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the described contents.

[0016] Unless otherwise specified, the reagents used in the present invention are conventional commercially available reagents.

[0017] Example 1

[0018] Screening of fermentative Lactobacillus mucilaginosus XW6

[0019] A method for screening and fermenting mucus lactobacillus from dry-cured ham. The dry-cured ham used in this embodiment is Xuanwei ham, which can be purchased from the market. The specific preparation method is as follows:

[0020] (1) Take 10 g of Xuanwei ham, cut it into minced meat with a knife, and place it in a conical flask filled with 100 mL of physiological saline, seal it, and place the conical flask on a shaker for sufficient shaking (25°C, 150 rpm / min, 2 h) for later use.

[0021] (2) Pipette 1 mL of the shaken liquid and add it to 9 mL of sterile MRS medium. Vortex the mixture and incubate at 37°C until the test tube becomes turbid and there is no wrinkled material covering the surface of the medium. This is the first-generation bacterial solution. Pipette 1 mL of the first-generation bacterial solution and add it to 9 mL of sterile MRS medium. Incubate at 37°C for 12 h to obtain the second-generation bacterial solution. Pipette 1 mL of the second-generation bacterial solution and add it to 9 mL of sterile MRS medium. Incubate at 37°C for 12 h to obtain the third-generation bacterial solution.

[0022] (3) Dilute the third generation bacterial solution by 10 -6 Spread on a solid MRS plate, Figure 1 For the results of lactic acid bacteria coating, after the colonies with calcium-soluble circles grow, pick the colonies that meet the morphology of lactic acid bacteria (milky white round colonies) and culture them in liquid MRS at 37°C for 12 hours for bacterial enrichment. Take 1 mL of the centrifuged and remove the supernatant, wash twice with physiological saline, and then resuspend for Gram staining microscopy. Figure 2 These are the results of microscopic examination of lactic acid bacteria.

[0023] (4) Take another 1 mL of bacterial solution for PCR detection. The primers used for PCR detection are as follows:

[0024] Upstream primer: AGAGTTTGATCMTGGCTCAG

[0025] Downstream primer: GGYTACCTTGTTACGACTT;

[0026] The PCR product was subjected to 1% agarose electrophoresis at 150 V and 100 mA. The electrophoresis bands were observed after 20 min. If the electrophoresis results were consistent with the expectations, the PCR product was sequenced. The spliced ​​sequencing results were input into the official website of NCBI for BLAST comparison. It was determined that the homology of the screened strain with the fermentative mucus Lactobacillus reached up to 100%, and the 16SrDNA gene sequence result was shown in SEQ ID NO.1.

[0027] Based on the above physiological and biochemical characteristics and 16SrDNA gene sequence results, the bacteria screened by the present invention should belong to fermented mucus lactobacillus and named as fermented mucus lactobacillus (Limosilactobacillusfermentum) XW6. It was deposited in the China Center for Type Culture Collection on December 30, 2024, with the deposit address at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20242942.

[0028] Example 2

[0029] Nitrite degradation ability of fermented Lactobacillus mucilaginosus XW6

[0030] Nitrite was added to 10 mL of MRS to make the final concentration of nitrite 150 μg / mL, and 100 μL of fermented Lactobacillus mucilaginosus XW6 was added. After fermentation at 37°C for 12 h, the nitrite content was determined according to the national standard GB 5009.33-2016 method. The degradation rate of nitrite is shown in Table 1.

[0031] Table 1 Nitrite degradation rate

[0032] strain Nitrite (μg / mL) Degradation rate (%) Fermented Lactobacillus mucilaginosus XW6 9.06±0.21 93.96±2.10

[0033] As shown in Table 1, after fermentation of Lactobacillus mucilaginosus XW6 was inoculated at 37°C and grown for 12 hours, the nitrite content of 150 μg / mL was degraded to 9.06±0.21 μg / mL, and the degradation rate was as high as 93.96%±2.10%.

[0034] Example 3

[0035] Application of fermented Lactobacillus mucilaginosus XW6 in fermented sausage

[0036] (1) Preparation of fermented sausages by fermentation of Lactobacillus mucilaginosus XW6

[0037] Sausage formula: Mix fresh pork lean meat and fat in a mass ratio of 7:3 and use a meat grinder to make meat filling. Add 2.5% (mass) salt, 1% (mass) white sugar, 0.5% (mass) five-spice powder, 2% (mass) fermented mucus lactobacillus XW6, and 30mg / kg nitrite according to the mass of the meat filling. Among them, the blank group sausage does not add fermented mucus lactobacillus XW6. Use a sausage stuffer to fill the mixed meat filling into the pig intestine, and the mass of each sausage is 145-155g. Hang the sausage in a clean processing room and ferment at 15℃ for 20 days. Take samples every 5 days to measure the color difference, pH and nitrite content of the sausage.

[0038] (2) Color difference measurement of sausage

[0039] The color of the sausage was measured using a CR-400 portable colorimeter (Konica Minolta Investment Co., Ltd., Shanghai, China). The light source was D65, the observer standard was 10°, and the field of view was 8 mm. Three points on the sausage surface were randomly selected and the color of the sausage was measured immediately. Each point was measured 3 times, and the L* (brightness), a* (redness) and b* (yellowness) of the sausage sample were recorded. The data of the sausage with fermented mucus lactobacillus added are shown in Table 3, and the data of the sausage without fermented mucus lactobacillus added are shown in Table 4.

[0040] Table 3 Sausage color difference value (fermented mucus lactobacillus)

[0041] Chromatic Aberration 0 5 10 15 20 L* <![CDATA[51.43±1.31 a ]]> <![CDATA[48.63±1.25 b ]]> <![CDATA[47.75±1.48 bc ]]> <![CDATA[46.23±1.04 cd ]]> <![CDATA[45.14±0.99 d ]]> a* <![CDATA[17.59±0.77 c ]]> <![CDATA[19.62±0.67 b ]]> <![CDATA[21.63±0.68 a ]]> <![CDATA[19.31±0.44 b ]]> <![CDATA[18.36±0.56 c ]]> b* <![CDATA[12.64±0.43 a ]]> <![CDATA[10.57±0.45 b ]]> <![CDATA[9.14±0.33 c ]]> <![CDATA[6.38±0.26 d ]]> <![CDATA[4.82±0.17 e ]]>

[0042] Note: Different superscripts in the same row represent statistically significant differences (P < 0.05).

[0043] Table 4 Sausage color difference value (blank)

[0044] Chromatic Aberration 0 5 10 15 20 L* <![CDATA[50.54±1.38 a ]]> <![CDATA[48.15±1.44 b ]]> <![CDATA[46.35±1.36 bc ]]> <![CDATA[45.17±1.43 cd ]]> <![CDATA[44.32±1.09 d ]]> a* <![CDATA[17.59±0.54 c ]]> <![CDATA[22.72±0.62 bc ]]> <![CDATA[24.58±0.71 a ]]> <![CDATA[23.60±0.54 b ]]> <![CDATA[22.14±0.66 bc ]]> b* <![CDATA[12.18±0.32 a ]]> <![CDATA[10.01±0.31 b ]]> <![CDATA[8.36±0.21 c ]]> <![CDATA[6.51±0.24 d ]]> <![CDATA[4.77±0.14 e ]]>

[0045] Note: Different superscripts in the same row represent statistically significant differences (P < 0.05).

[0046] As shown in Tables 3 and 4, the L* value of the sausage fermented with Lactobacillus mucilaginosus group showed a downward trend, from 51.43±1.31 at 0d to 45.14±0.99 at 20d, while the blank group decreased from 50.54±1.38 at 0d to 44.32±1.09. The a* value of the lactic acid bacteria group increased from 17.59±0.77 at 0d to 21.63±0.68 at 10d and then decreased to 18.36 at 20d, while the blank group increased from 17.59±0.54 at 0d to 24.58 at 10d and then decreased to 22.14±0.66 at 20d. This is because nitrite increases the redness (a*) of the sausage, and after fermentation with Lactobacillus mucilaginosus, nitrite is degraded by the fermented Lactobacillus mucilaginosus, and a* shows a downward trend in the later stage of fermentation. The b* value of the fermented mucus Lactobacillus group showed a downward trend during the fermentation period. From 0d to 20d, the b value decreased from 12.64±0.43 to 4.82±0.17, while that of the blank group decreased from 12.18±0.32 on 0d to 4.77±0.14 on 20d.

[0047] (3) Determination of pH value of sausage

[0048] 5 g of Chinese sausage was mixed with 50 mL of distilled water in a beaker, and then homogenized by a homogenizer (8000 rpm / min, 5 min) and then measured with a pH meter.

[0049] Figure 3 is the pH value of the sausage fermentation process. The pH value of the fermentation Lactobacillus mucus group decreased from 6.61±0.11 on day 0 to 4.82±0.14 after 20 days of fermentation, and the pH value of the blank group decreased from 6.58±0.29 on day 0 to 5.23±0.12.

[0050] (4) Determination of nitrite content in sausage

[0051] The nitrite content of sausage was determined according to GB 5009.33-2016. 5 g of sausage sample was collected and placed in a conical flask filled with 80 mL of distilled water, and then 1 mL of KOH (1 mol / L) was added. After ultrasonic treatment (800 W, 10 s of work and 5 s interval) for 30 min, the mixture was incubated at 80 ° C for 6 min to cool, filtered with filter paper, 40 mL of filtrate was reacted with 2 mL of sulfonamide (4 g / L) for 5 min, and then 1 mL of N-(1-naphthyl)ethylenediamine dihydrochloride (2 g / L) was added, and the distilled water without sample was used as a blank, and the absorbance was measured at 538 nm. Finally, the nitrite content in the sausage sample was calculated according to the nitrite standard curve.

[0052] from Figure 4It can be seen that after 5 days of fermentation of sausages with 30 mg / kg nitrite added, the nitrite content of the fermented Lactobacillus mucus group dropped significantly to 10.15±1.77 mg / kg, while that of the blank group was 27.52±1.21 mg / kg. After 20 days of fermentation, it dropped to 2.14±0.38 mg / kg, and the blank group dropped to 14.38±0.51 mg / kg. After 20 days of fermentation, the nitrite content of the sausages in the fermented Lactobacillus mucus group and the blank group was lower than the limit of 30 mg / kg for fermented meat products in GB 2762-2022, but the nitrite content of the sausages in the fermented Lactobacillus mucus group was much lower than that of the blank group, which shows that the fermented Lactobacillus mucus XW6 has a good ability to degrade nitrite in sausage applications.

[0053] The above results show that the fermented mucus Lactobacillus XW6 screened by the patent of the present invention has significant nitrite degradation activity, can effectively degrade the nitrite content in sausages, and is expected to be used to solve the safety problem of nitrite in fermented foods.

Claims

1. A strain of Lactobacillus fermentum XW6, with a deposit number of CCTCCNO: M 20242942.

2. Use of the fermented mucus lactobacillus XW6 according to claim 1 as a starter in fermented sausages.

Images