Use of weissella cibaria in fermented meat products
By using Enterobacter SL7 as a starter culture in fermented meat products, the problems of dark color and nitrite accumulation in fermented meat products were solved, resulting in improved color and flavor as well as enhanced safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHENGDU UNIV
- Filing Date
- 2022-09-15
- Publication Date
- 2026-06-02
AI Technical Summary
Existing fermented meat products have a dark color and nitrates are converted into nitrites during the preparation process, leading to nitrite accumulation. Existing alternative methods have problems such as low color quality, poor taste, short shelf life, and excessive nitrite residue.
Weissella paramesenteroides SL7 is used as a starter culture to ferment meat products, replacing nitrites and utilizing it to degrade nitrites and convert them into metmyoglobin, thereby improving color and flavor.
It achieves beautiful color and rich flavor in fermented meat products, reduces nitrite residue, extends shelf life, and has natural safety.
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Figure CN116396885B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial isolation, screening, and application technology. Specifically, it relates to the application of *Weisseria enterica* in fermented meat products. Background Technology
[0002] Nitrates and nitrites are widely present in food, especially in fermented meat products. However, during the preparation of fermented meat products, the color always gradually becomes dark and dull, and during fermentation, nitrate-reducing bacteria convert nitrates into nitrites, leading to a large accumulation of nitrites in the early stages of fermentation.
[0003] Currently, improving product color mainly involves adding nitrites. Although some studies have shown that adding nitrites can be reduced or replaced with red amaranth powder or certain microorganisms, drawbacks such as low color quality, poor taste, short shelf life, and excessive nitrite residues still exist. Therefore, it is essential to develop a preparation process that can completely replace the addition of nitrites and effectively maintain the color and flavor of meat products.
[0004] In view of this, the present invention is proposed. Summary of the Invention
[0005] To address the problems in the background art, the present invention provides the application of Enterobacter vesiculosus in fermented meat products.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0007] The application of Weissella paramesenteroides in fermented meat products. The Weissella paramesenteroides SL7 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 23, 2022, with accession number GDMCC No:62429.
[0008] Preferably, the Enterobacter SL7 is prepared as a starter culture and then used to ferment meat products, replacing nitrite in achieving color development in meat products.
[0009] Preferably, the method for preparing the starter culture from *Westernella enterica* comprises:
[0010] Enterobacter spp. SL7 was inoculated into MRS broth and cultured at 35–37°C for 18–24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 3–5°C and 4500–5500 r / min for 6–8 min, and the supernatant was discarded to obtain the bacterial cells.
[0011] A lyophilization protectant was added to the bacterial cells and then freeze-dried to obtain the fermentation agent.
[0012] Preferably, the cell density of the starter culture obtained by freeze-drying is 1.0 × 10⁻⁶. 10 ~6.0×10 10 CFU / g.
[0013] Preferably, the freeze-drying protectant comprises 10-15% skim milk powder and 1-2% sucrose, based on the cell volume.
[0014] Preferably, the inoculum amount of Enterobacter SL7 in fermented meat products is 2-5%.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] The *Enterobacter sieboldii* starter culture provided by this invention can be used as a starter culture to replace nitrite in fermented meat products, enhancing product color, achieving color development and protection effects, and improving product quality. The *Enterobacter sieboldii* starter culture provided by this invention has advantages such as being natural and safe, containing no added nitrite, and possessing a beautiful color and rich flavor, and has broad application prospects in the food industry. Attached Figure Description
[0017] Figure 1 A phylogenetic tree constructed based on the 16S rRNA sequence of Enterobacter sieboldii SL7;
[0018] Figure 2 Ultraviolet scan image of Enterobacter SL7 cultured in MRS liquid medium containing metmyoglobin for 20 h;
[0019] Figure 3 The change in metmyoglobin content of Enterobacter SL7 cultured in MRS liquid medium containing metmyoglobin over 20 h.
[0020] Figure 4 The transmittance colorimetric value of Enterobacter sieboldii SL7 cultured in MRS liquid medium containing metmyoglobin for 20 h.
[0021] Figure 5 The growth curve and pH changes of Enterobacter SL7;
[0022] Figure 6 The growth of Enterobacter SL7 at different NaCl concentrations;
[0023] Figure 7 For the determination of color value of fermented sausage;
[0024] Figure 8 This shows the pH changes during the fermentation process of sausages. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.
[0026] Example 1: Isolation, screening, and identification of strains
[0027] 1.1 Isolation and screening of strains
[0028] The strain required for this invention was isolated from Sichuan cured pork. Under aseptic conditions, 25g of the internal components of the cured pork were taken, chopped, placed in a sterile homogenizing bag, and 225mL of sterile physiological saline was added. The mixture was then homogenized in a beater for 3 minutes. A 10-fold serial dilution was performed, resulting in a total of 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 Seven concentrations were prepared, and 200 μL of sample was spread on MRS medium containing 1.5% calcium carbonate. Each concentration was replicated in triplicate. After standing for 30 min, the samples were incubated upside down at 37°C for 48 h.
[0029] Colonies with obvious calcium dissolution zones were inoculated into MRS medium and streaked for purification until single colonies appeared and the morphology of the colonies was consistent throughout the plate and under the microscope. Then, the physiological and biochemical indicators of the strains were measured. Gram-positive strains that do not produce H2S, do not produce gas or mucus when fermenting glucose, and myoglobin-positive strains were selected. The selected strains were stored as slant culture at -4℃ for later use.
[0030] 2) Screening of strains that can degrade nitrite
[0031] The isolated strains were inoculated at 1% in MRS broth medium containing 150 μg / mL NaNO2 and pH 6.0. After incubation at 35 °C for 24 h, the NaNO2 content in the medium was detected, and the ability of the strains to degrade nitrite was expressed as the nitrite degradation rate.
[0032] Morphological characteristics of lactic acid bacteria on MRS medium containing calcium carbonate: milky white colonies, varying in size and shape, moist, with calcium-dissolving zones and clear zones. Twelve strains with different morphologies and large clear zones were selected from the strains on MRS calcium carbonate medium. The strains were purified until single colonies appeared and the morphology of the colonies was consistent throughout the plate and under the microscope. All strains were then transferred to slant culture and stored at -4℃, simultaneously sealed with glycerol for later use. Determination of nitrite and nitrate in food according to GB5009.33—2016 "National Food Safety Standard - Determination of Nitrite and Nitrate in Food" showed that strain SL7 had the best nitrite degradation effect, reaching a degradation rate of 98%, significantly higher than other strains. Therefore, strain SL7 was selected as the superior strain.
[0033] 1.2 Molecular identification of the strain
[0034] Bacterial genomic DNA extraction: Bacterial DNA was extracted using the TSINGKE Plant DNA Extraction Kit (Universal).
[0035] The extracted product was amplified by PCR. The amplification system was: 45 μL of 1×TSE101 Gold Mix, 2 μL of 27 F, 2 μL of 1492R, and 1 μL of Template.
[0036] PCR amplification reaction conditions: 98℃ for 4 min pre-denaturation; 98℃ for 10 s, 55℃ for 15 s, 72℃ for 15 s, 38 cycles; 72℃ for 10 min for retrieval extension, 4℃ for termination.
[0037] The PCR products were sequenced by the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. Homology analysis was performed between the sequencing results and the 16S rDNA sequences in the GenBank database to identify the microbial species. Strain SL7 was thus identified as... Weissella paramesenteroides The nucleotide sequence of this strain is shown in SEQ ID NO: 1. The phylogenetic tree constructed from the 16 rRNA sequence of this strain is shown below. Figure 1 As shown.
[0038] The strain Weissella paramesenteroides SL7 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 23, 2022, with accession number GDMCC No:62429.
[0039] Example 2: Assay for the conversion of methemoglobin by Enterobacter sieboldii
[0040] Enterobacter spp. SL7 was inoculated into MRS liquid medium containing methemoglobin, and a layer of paraffin oil was added on the surface. The medium was incubated at 35°C for 18-20 h. The culture medium was centrifuged (10000×g, 5 min, 4°C), and the supernatant was collected for testing. MRS medium containing methemoglobin but without Enterobacter spp. SL7 was used as a blank control group.
[0041] (1) Spectroscopic analysis: The color of myoglobin mainly depends on the binding state of the sixth ligand of the heme iron atom. Different sixth ligands result in different absorbance values of different myoglobin derivatives at various wavelengths. Therefore, the content of methemoglobin in the strain and the transformation process can be analyzed by measuring the characteristic absorption peak of the methemoglobin solution.
[0042] The supernatant obtained from centrifuging *Westernella enterica* SL7 and the control sample were respectively subjected to UV-Vis spectral scanning in the wavelength range of 400-700 nm using a UV-1800 UV-Vis spectrophotometer. The results are as follows. Figure 2 .
[0043] (2) Determination of metmyoglobin content: The supernatant obtained by centrifuging the above-mentioned Enterobacter spp. SL7 and the control group sample were taken respectively, and their absorbance values were measured at wavelengths of 525 nm, 545 nm, 565 nm and 572 nm using a 722-S type visible spectrophotometer. The calculation formula is as follows:
[0044] Methemoglobin (Met-Mb)% = (-2.541R1 + 0.777R2 + 0.800R3 + 1.098) × 100
[0045] In the formula, R1, R2, and R3 are respectively A 572nm / A 525nm A 565nm / A 525nm With A 545nm / A 525nm Perform the calculation.
[0046] The results were repeated three times and the average was taken. Figure 3 .
[0047] (3) Determination of transmission colorimetric values of the culture medium: The supernatant obtained by centrifuging the above-mentioned Enterobacter spp. SL7 and the control sample were respectively placed in quartz cuvettes, and the L* and a* values were measured using a CR-400 colorimeter. The results are as follows: Figure 4 .
[0048] Results analysis:
[0049] Depend on Figure 2It was found that the supernatant obtained from centrifugation of *Enterobacter sieboldii* SL7 showed absorption peaks at 421 nm, 540 nm, and 579 nm, which are typical absorption peaks of nitrosomyoglobin. In contrast, the control group showed peaks at 410 nm, 505 nm, and 634 nm, which are typical absorption peaks of methemoglobin. It is inferred that *Enterobacter sieboldii* SL7 has the ability to convert methemoglobin into deoxymyoglobin. The NO produced by the decomposition of nitrite combines with myoglobin to form nitrosomyoglobin, a phenomenon not observed in the control group.
[0050] Depend on Figure 3 It can be seen that the metmyoglobin content of Enterobacter SL7 cultured in MRS liquid medium containing metmyoglobin changed within 20 h. The metmyoglobin content in the Enterobacter SL7 culture medium was significantly lower than that in the control group, decreasing to 33.27% at 20 h, while the metmyoglobin content in the control group was 70.95% at 20 h.
[0051] Depend on Figure 4 It can be seen that the a* value of the SL7 group was 29.76 after 20 hours, which was significantly higher than that of the control group (12.83). Furthermore, during the experiment, it was observed by the naked eye that the culture medium containing methemoglobin and inoculated with Enterobacter spp. SL7 gradually changed from brownish-red to bright red within 5 to 20 hours, while the culture medium without SL7 strain maintained its original brownish-red color within 20 hours.
[0052] Example 3: Bioperformance test of Enterobacter sieboldii
[0053] (1) Growth curve of strain and acid production determination
[0054] Enterobacter spp. SL7, activated for 18 h, was inoculated at 1% in MRS liquid medium and cultured at 35°C on a shaker at 150 r / min. Appropriate amounts of bacterial suspension were taken and diluted to suitable concentrations at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 28, and 32 h of culture, and OD values were measured. 600 The absorbance and pH values are as follows: Figure 5 .
[0055] (2) Salt tolerance curve of the strain
[0056] Enterobacter spp. SL7, activated for 18 hours, was inoculated at 1% in MRS culture medium containing 0.0%, 5.0%, 7.0%, 9.0%, and 11.0% of the culture medium and cultured at 35°C for 24 hours. The bacterial OD was then measured. 600 The result is as follows Figure 6 .
[0057] Results analysis:
[0058] Depend on Figure 5 It can be seen that *Enterobacter sieboldii* SL7 is in the logarithmic growth phase of the strain during 8-18 hours, during which OD... 600 The value increased from 0.578 to 1.119, and after 18 hours, the strain entered the stationary phase of growth. OD 600 The pH value remained between 1.100 and 1.200. This indicates that the strain grew rapidly in MRS liquid medium. Meanwhile, *Enterobacter esculenta* SL7 produced acid rapidly after 4 hours, causing a significant drop in pH, reaching 3.39 by 18 hours. The pH then gradually stabilized, eventually settling at around 3.33.
[0059] Depend on Figure 6 It can be seen that strain SL7 exhibits good growth performance in the high salt concentration range of 7.0% to 11.0%, with OD at 7.0% salt concentration being [missing data]. 600 The highest value was 0.9873; at a high salt concentration of 11%, the OD value was [value missing]. 600 The value is still 0.7748, indicating that Enterobacter SL7 has strong salt tolerance and is more advantageous in the processing of certain high-salt foods.
[0060] Example 4: Application of Enterobacter SL7 in Sausage Preparation (Experimental Group)
[0061] (1) Preparation of freeze-dried starter culture: Enterobacter vesiculosus was inoculated at 1% into MRS broth medium and cultured at 35-37℃ for 18h to obtain fermentation broth; the fermentation broth was centrifuged at 4℃ and 5500 r / min under aseptic conditions for 8 min, and the supernatant was discarded to obtain bacterial cells; freeze-drying was carried out on the centrifuged bacterial cells to obtain starter culture. The freeze-drying protectant was 15% skim milk powder and 1% sucrose by volume of bacterial cells;
[0062] (2) Raw meat processing: Remove the skin from the fresh pig hind leg, wash and drain the surface water, mince it with a meat grinder, and weigh out 30 parts and 70 parts of fat.
[0063] (3) Marinating with auxiliary ingredients: Weigh out 2 parts of granulated sugar, 3 parts of salt, 1.5 parts of thirteen spices, 1.5 parts of chili powder, 1 part of Sichuan pepper powder, and 2.5 parts of MSG. Mix them with the prepared meat and roll for 10 minutes to ensure thorough mixing. Marinate at 4℃ for 8 hours.
[0064] (4) Inoculation with fermentation agent: Take the freeze-dried powder prepared above and prepare a bacterial suspension to achieve a viable count of 1×10⁻⁶. 8 CFU / mL; based on meat weight, the inoculum amount of Enterobacter sieboldii starter in sausages is 3%;
[0065] (5) Sausage filling: Wash the sausage filling equipment with boiling water before filling, stuff the meat prepared in the previous step into the casing, tie it tightly with cotton thread after filling, and use a needle to release the air;
[0066] (6) Fermentation: Fermentation temperature 37℃, fermentation humidity 85%, 30h;
[0067] (7) Baking: Set the temperature to 35℃ and the relative humidity to 45% for 6 hours;
[0068] (8) Mature air drying: The sausages are air dried alternately at 12℃ during the day and 8℃ at night, with a wind speed of 1m / s and a relative humidity of 70% for 4 days;
[0069] (9) After the product matures, it is vacuum-packed to obtain the finished product, which is then stored at 4°C.
[0070] Example 5: Application of Enterobacter SL7 in Sausage Preparation
[0071] (1) Preparation of freeze-dried starter culture: 1% of *Westernella plantarum* SL7 was inoculated into MRS broth medium and cultured at 35℃ for 18 h to obtain fermentation broth; the fermentation broth was centrifuged at 4℃ and 5500 r / min under aseptic conditions for 8 min, and the supernatant was discarded to obtain bacterial cells; a freeze-drying protectant was added to the centrifuged bacterial cells and freeze-dried to obtain the starter culture. The freeze-drying protectant was 12% skim milk powder and 1.5% sucrose, based on the bacterial cell volume.
[0072] (2) Raw meat processing: Remove the skin from the fresh pork hind leg, wash and drain the surface water, mince it with a meat grinder, and weigh out 250g of fat and 750g of pork.
[0073] (3) Marinating with auxiliary ingredients: Weigh out 25g of granulated sugar, 30g of salt, 15g of thirteen spices, 15g of chili powder, 10g of Sichuan pepper powder, and 20g of MSG. Mix them with the prepared meat and roll and knead for 10 minutes to ensure thorough mixing. Marinate at 4℃ for 8 hours.
[0074] (4) Inoculation with fermentation agent: Take the freeze-dried powder prepared above and prepare a bacterial suspension to achieve a viable count of 1×10⁻⁶. 8 CFU / mL; based on meat weight, the inoculum amount of Enterobacter sieboldii starter in sausages was 4%;
[0075] (5) Sausage filling: Wash the sausage filling equipment with boiling water before filling, stuff the meat prepared in the previous step into the casing, tie it tightly with cotton thread after filling, and use a needle to release the air;
[0076] (6) Fermentation: Fermentation temperature 37℃, fermentation humidity 85%, 30h;
[0077] (7) Baking: Set the temperature to 35℃ and the relative humidity to 45% for 6 hours;
[0078] (8) Mature air drying: The sausages are air dried alternately at 12℃ during the day and 8℃ at night, with a wind speed of 1m / s and a relative humidity of 70% for 4 days;
[0079] (9) After the product matures, it is vacuum-packed to obtain the finished product, which is then stored at 4°C.
[0080] Comparative Example 1: Sausage preparation without leavening agent (Comparative Example 1 group)
[0081] Instead of inoculating with a fermenting agent, the bacterial suspension in step (4) of Example 4 was replaced with an equal volume of sterile physiological saline, and the remaining steps were the same as in Example 4.
[0082] Comparative Example 2: Sausages prepared without inoculation with *Westernella enterica* and with the addition of nitrite (Comparative Example 2 group)
[0083] Instead of inoculating with a fermenting agent, the bacterial suspension in step (4) of Example 4 was replaced with an equal volume of sterile physiological saline, and 0.015% nitrite was added to the excipients in step 2. The remaining steps were the same as in Example 4.
[0084] Comparative Example 3 (Comparative Example 3 Groups)
[0085] Compared to Example 4, *Enterobacter sieboldii* SL7 was replaced with an equal amount of *Enterobacter sieboldii* CGMCC NO. 22238 (purchased from the China Microbiological Culture Collection Center) from Example CN114456979A. The viable count of *Enterobacter sieboldii* (CGMCC NO. 22238) was 1 × 10⁻⁶. 8 CFU / g, the remaining steps are the same as in Example 4.
[0086] The color value, pH value, volatile basic nitrogen, peroxide value, nitrite content, and volatile aroma substances and their relative contents were measured in Examples 4, 1, 2 and 3 above.
[0087] The color difference of sausage meat samples was measured using a CR-400 colorimeter, and the results are as follows: Figure 7 .like Figure 7As shown, the experimental group had a brightness value (L*) of 43.07 and a redness value (a*) of 17.27, significantly higher than Comparative Examples 1, 2, and 3. Comparative Example 1 had the lowest brightness value (L*) of 21.46 and redness value (a*) of 5.58, indicating a dull meat color. Comparative Example 3, with the addition of *Enterobacter vesiculosus* (CGMCC NO. 22238), produced sausages with a brightness value (L*) of 28.79 and a redness value (a*) of 8.28, slightly higher than Comparative Example 1, but less effective than Comparative Example 2 with the addition of nitrite. This indicates that Comparative Example 3 cannot replace nitrite fermentation for sausages. The fermentation with *Enterobacter vesiculosus* SL7 in the experimental group imparted a characteristic rose-red color to the sausages, with good gloss and good market value, demonstrating that *Enterobacter vesiculosus* SL7 is a fermenting agent with good color-developing and color-protecting effects.
[0088] The pH of the sample was measured using an insertion pH meter, and the results are as follows: Figure 8 .like Figure 8 As shown, the pH value of the experimental group dropped rapidly in the early stage of fermentation, reaching 4.450 on day 2. Throughout the entire processing, the pH value of the experimental group was significantly lower than that of other groups. This is because Enterobacter SL7 has a strong acid-producing ability, which causes the pH of the sausage to drop rapidly, giving the sausage its characteristic sour aroma. At the same time, the low pH inhibits the growth and reproduction of pathogenic bacteria and spoilage microorganisms in the sausage, thus extending the shelf life of the sausage.
[0089] Volatile basic nitrogen was determined according to GB 5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food", peroxide value was determined according to GB 5009.181-2016 "Determination of Malondialdehyde in Food", and nitrite content was determined according to GB5009.33-2016 "National Food Safety Standard - Determination of Nitrite and Nitrate in Food". The results were averaged and are shown in Table 1.
[0090] Table 1. Determination of volatile basic nitrogen, peroxide value, and nitrite content in fermented sausage products.
[0091] .
[0092] Note: Data are mean ± standard deviation.
[0093] As shown in Table 1, the peroxide value and nitrite residue of the experimental group were 4.29 mg / 100g and 2.15 mg / kg, respectively, which were significantly lower than those of Comparative Groups 1, 2, and 3. Furthermore, according to the national standard of nitrite residue below 30 mg / kg, the nitrite residue in the sausage of Comparative Group 2 was 31.81 mg / kg, which already exceeded the standard.
[0094] The types and quantities of volatile aroma compounds were determined using GCMS, and the results are shown in Table 2. As shown in Table 2, the addition of Enterobacter vesiculosus SL7 to the experimental group produced 38 flavor compounds, significantly increasing the content of acids and esters in the sausage, resulting in a rich, dry-cured flavor and an appealing aroma, greatly enhancing the flavor of the fermented sausage.
[0095] Table 2 Comparison of types and quantities of volatile aroma compounds in finished sausages (Unit: species)
[0096] .
[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. The application of *Weisseria gonorrhoeae* in fermented meat products, characterized in that, The type of enteroweissella is Weissella paramesenteroides SL7, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 23, 2022, with accession number GDMCC No:62429.
2. The application as described in claim 1, characterized in that, The Enterobacter SL7 strain is prepared as a starter culture and used to ferment meat products, replacing nitrite in achieving color development in meat products.
3. The application as described in claim 2, characterized in that, The method for preparing the starter culture by *Westernobacterium enterosum* includes: Enterobacter sericulture SL7 was inoculated into MRS broth medium and cultured at 35-37℃ for 18-24 h to obtain fermentation broth; the fermentation broth was centrifuged at 3-5℃ and 4500-5500 r / min for 6-8 min, and the supernatant was discarded to obtain bacterial cells; as well as A lyophilization protectant was added to the bacterial cells and then freeze-dried to obtain the fermentation agent.
4. The application as described in claim 3, characterized in that, The cell density of the starter culture obtained by freeze-drying was 1.0 × 10⁻⁶. 10 ~6.0×10 10 CFU / g.
5. The application as described in claim 4, characterized in that, Based on the volume of the bacterial cells, the freeze-drying protectant contains 10-15% skim milk powder and 1-2% sucrose.
6. The application as described in claim 1 or 2, characterized in that, The inoculation amount of the Enterobacter SL7 in fermented meat products is 2-5%.