Smoked horse intestine leavening agent and preparation method thereof

Abstract

The invention relates to the technical field of food fermentation, in particular to a smoked horse intestine leavening agent and a preparation method thereof. The fermentation agent is composed of Weissella lactic acid bacteria MR1, lactobacillus plantarum MR2, lactobacillus plantarum MR3 and lactobacillus plantarum MC1 and MC2, whey powder, skim milk powder, trehalose and maltodextrin are used as protective agents, the fermentation agent is prepared through expanding culture, pre-freezing and vacuum freeze-drying processes, the total viable count of the obtained preparation is 1.0 * 10 < 10 >-1.0 * 10 < 12 > CFU / g, and good activity is still kept under the conditions that the pH is 4.5-6.5 and sodium chloride is 9%-12%. The salt resistance, the acid resistance, the antibacterial activity and the long-term storage stability are remarkably improved, and particularly, the salt consumption is reduced, and the product quality is improved. The invention provides a new technical approach for low-salt, standardized and industrial production of the traditional smoked horse intestines, and has wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, specifically to a smoked horse sausage fermentation agent and its preparation method. Background Technology

[0002] Smoked horse sausage is a traditional naturally fermented meat product made primarily from horse meat and sausage, and is representative of Northwest China and pastoral areas. Current processes generally rely on the natural microorganisms inherent in the horse meat and sausage for spontaneous fermentation and maturation, lacking standardized addition of fermentation strains and process control. Because the fermentation process is affected by factors such as environmental temperature, humidity, and season, the flavor, color, texture, and safety of the finished product vary considerably, making it difficult to guarantee consistency between different batches.

[0003] To extend shelf life and inhibit spoilage, traditional production methods typically involve adding high levels of salt and spices, often exceeding 3.5% salt content. While this improves microbial stability to some extent, the excessive salt content makes the finished product taste too salty, failing to meet modern consumers' demand for low-sodium, healthy foods. Furthermore, the microbial community in naturally fermented products is complex and may contain spoilage bacteria or even pathogens, posing potential food safety risks.

[0004] Lactic acid bacteria are commonly used functional bacteria in food fermentation. They can rapidly produce acid, lower the pH of the system, inhibit the growth of spoilage and pathogenic bacteria, and produce metabolites such as esters, aldehydes, and alcohols during fermentation, thereby improving the flavor and aroma of the product. Existing research shows that selecting lactic acid bacteria starter cultures with strong adaptability and a broad antibacterial spectrum can effectively improve the quality of traditional fermented meat products. However, existing lactic acid bacteria starter cultures are mostly used for products such as sausages and fermented sausages; research and development of specialized compound starter cultures for smoked horse sausage, a product with unique raw materials and processing conditions, is still lacking.

[0005] Therefore, there is an urgent need to develop a special compound lactic acid bacteria starter derived from the natural fermentation environment of smoked horse sausage, which has salt resistance, acid resistance, and antibacterial properties, in order to reduce the amount of salt used, improve the complexity of flavor, and extend the shelf life, thereby solving the shortcomings of traditional natural fermentation processes in terms of product safety, flavor stability, and health attributes. Summary of the Invention

[0006] To overcome the problems of large product quality variations, high salt content, high food safety risks, and short shelf life in the natural fermentation process of smoked horse sausage in the prior art, the present invention aims to provide a smoked horse sausage starter and its preparation method. This starter is formulated by isolating and screening *Lactobacillus weisseri* MR1, *Lactobacillus plantarum* MR2, MR3, and *Lactobacillus plantarum* MC1, MC2 from traditionally naturally fermented smoked horse sausage. These are combined with whey powder, skim milk powder, trehalose, and maltodextrin as a preservative to form a freeze-dried powder composite system. The total viable count is controlled within the range of 1.0 × 10^10 to 1.0 × 10^12 CFU / g. Even after 24 hours of cultivation under pH 4.5–6.5 and 9%–12% sodium chloride conditions, the endpoint OD600 remains between 1.2 and 3.0, thus ensuring the strain's salt and acid tolerance and high activity. This compound fermentation agent can achieve stable fermentation under low-salt conditions, rapidly reduce pH value and inhibit the growth of putrefactive and pathogenic bacteria. At the same time, it forms rich flavor substances during fermentation, improves the color and taste of smoked horse sausage, significantly extends the product shelf life, and meets the needs of low-salt, healthy and industrialized standardized production.

[0007] The objective of this invention can be achieved through the following technical solutions: A fermentation agent for smoked horse sausage comprises the following raw materials in parts by weight: 5-15 parts of *Lactobacillus weisseri* MR1; 5-15 parts of *Lactobacillus plantarum* MR2; 5-15 parts of *Lactobacillus plantarum* MR3; 13-10 parts of *Lactobacillus plantarum* MC1; 3-10 parts of *Lactobacillus plantarum* MC2; 5-15 parts of whey powder; 5-12 parts of skim milk powder; 3-10 parts of trehalose; and 5-15 parts of maltodextrin. The total viable count of the fermentation agent is 1.0 × 10¹⁰ to 1.0 × 10¹² CFU / g, and the endpoint OD600 after 24 hours of cultivation at pH 4.5-6.5 and 9%-12% sodium chloride is 1.2-3.0.

[0008] Optionally, the inhibition zone diameter of Lactobacillus weisseri MR1, Lactobacillus plantarum MR2, MR3 and Lactobacillus plantarum MC1, MC2 against Staphylococcus aureus, Escherichia coli and Salmonella in vitro is 10-20 mm.

[0009] Optionally, the viable cell retention rate of the freeze-dried starter culture is 85%–95%, and the moisture content is 2%–5%.

[0010] Optionally, the total amount of whey powder, skim milk powder, trehalose, and maltodextrin accounts for 20% to 40% of the total weight of the starter culture.

[0011] Optionally, a method for preparing a smoked horse sausage fermentation agent includes the following steps: S1, Lactic acid bacteria were isolated from traditionally naturally fermented smoked horse sausage samples to obtain Lactobacillus weisseri MR1, Lactobacillus plantarum MR2, MR3 and Lactobacillus plantarum MC1, MC2; S2 was identified by 16S rRNA sequencing, and strains that met the requirements for salt tolerance, acid tolerance and antibacterial properties were screened. S3 was cultured in MR medium until the logarithmic growth phase was reached, and the bacterial cells were collected when the OD600 at the end of the culture was between 1.5 and 2.5. S4, mix the bacterial cells with whey powder, skim milk powder, trehalose and maltodextrin, and pre-freeze at -50℃ to -60℃; S5 is subjected to vacuum freeze-drying, sieved, and sealed with nitrogen to obtain the smoked horse sausage fermentation agent.

[0012] Optionally, the culture temperature in step S3 is 20–30°C, and the culture time is 18–24 hours.

[0013] Optionally, the pre-freezing time in step S4 is 2 to 6 hours.

[0014] Optionally, the moisture content of the finished powder obtained in step S5 is 3% to 5%, and the viable bacteria count is maintained at 80% to 90% after 6 months of storage.

[0015] The beneficial effects of this invention are: The beneficial effect of this invention lies in the construction of a composite starter culture using five dominant bacterial strains derived from the natural fermentation system of smoked horse sausage. These strains have developed tolerance to high-salt and weakly acidic environments through long-term adaptation. Cultivation experiments under conditions of 9%–12% sodium chloride and pH 4.5–6.5 verified that the strains maintained stable growth performance with an endpoint OD600 between 1.2 and 3.0 even under special process conditions. This characteristic significantly distinguishes it from conventional lactic acid bacteria starter cultures and solves the problems of slow growth and low survival rate of exogenous strains in the smoked horse sausage environment.

[0016] This invention features an optimized freeze-drying protection system, combining whey powder, skim milk powder, trehalose, and maltodextrin in a specific ratio. This effectively maintains the integrity of the microbial cells during low-temperature freezing and vacuum drying, resulting in a freeze-dried starter culture survival rate of 85%–95%. Furthermore, after six months of storage at room temperature, it retains over 80% viable cell count. This high level of stability ensures the starter culture maintains its activity under actual industrial production and logistics conditions, overcoming the bottleneck of unstable application effects caused by activity decay in existing starter cultures.

[0017] The fermentation agent of this invention not only achieves a high degree of matching with the process conditions of smoked horse sausage, but also significantly improves the controllability and stability of the preparation and application of the agent, providing a practical new solution for the industrial production of smoked horse sausage. Attached Figure Description

[0018] The invention will now be further described with reference to the accompanying drawings.

[0019] Figure 1 Characteristics of lactic acid bacteria colonies and cell morphology; Figure 2 For the determination of salt tolerance of lactic acid bacteria; Figure 3 To determine the nitrite tolerance of lactic acid bacteria; Figure 4 For the determination of acid resistance of lactic acid bacteria; Figure 5 This is a graph showing the growth curve of lactic acid bacteria. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments. However, the present invention is not limited to the following embodiments. Equivalent adjustments made without departing from the spirit and essence of the present invention should also be considered to fall within the protection scope of the present invention.

[0021] Example 1: Verify the activity retention of the smoked horse sausage fermentation agent prepared under low ratio conditions.

[0022] S1, weigh out 15 parts of Lactobacillus weisseri MR1, 5 parts of Lactobacillus plantarum MR2, 5 parts of Lactobacillus plantarum MR3, 3 parts of Lactobacillus plantarum MC1, 3 parts of Lactobacillus plantarum MC2, and mix them together; add 5 parts of whey powder, 5 parts of skim milk powder, 3 parts of trehalose, and 5 parts of maltodextrin to make a mixture; S2, the above-mentioned lactic acid bacteria were cultured in MR liquid medium at 37℃ for 18 hours. The endpoint OD600 was 1.6, and the bacterial cells were collected. S3, mix the collected bacterial cells with the protectant and pre-freeze at -50℃ for 4 hours; S4 is vacuum freeze-dried, passed through a 60-mesh sieve, and then sealed in nitrogen-filled packaging.

[0023] Example 2: The stability and antibacterial effect of the smoked horse sausage fermentation agent prepared under intermediate ratio conditions were verified.

[0024] S1. Weigh out 110 parts of Lactobacillus weisseri MR1, 10 parts of Lactobacillus plantarum MR2 and MR3, and 16 parts of Lactobacillus plantarum MC1 and MC26 and mix them together; add 10 parts of whey powder, 8 parts of skim milk powder, 6 parts of trehalose, and 10 parts of maltodextrin to make a mixture. S2 was cultured in MR liquid medium at 25°C for 20 hours, with an endpoint OD600 of 2.0, and the bacterial cells were collected. S3: Mix the bacterial cells and the protectant thoroughly, and pre-freeze at -55°C for 3 hours; S4 is subjected to vacuum freeze drying, passed through an 80-mesh sieve, and then sealed in nitrogen-filled packaging.

[0025] Example 3: Verify the upper limit activity and long-term storage performance of the smoked horse sausage fermentation agent prepared under higher formulation conditions.

[0026] S1. Weigh out 115 parts of Lactobacillus weisseri MR1, 15 parts of Lactobacillus plantarum MR2 and MR3, and 10 parts of Lactobacillus plantarum MC1 and MC2 and mix them together; add 15 parts of whey powder, 12 parts of skim milk powder, 10 parts of trehalose, and 15 parts of maltodextrin to make a mixture. S2 was cultured in MR medium at 20°C for 24 hours until the endpoint OD600 was 2.3, at which point the bacterial cells were collected. S3: Mix the bacterial cells with the protectant and pre-freeze at -60°C for 6 hours; S4 is subjected to vacuum freeze drying, passed through a 100-mesh sieve, and then sealed in nitrogen-filled packaging.

[0027] Comparative Example 1 To verify the stability and antibacterial effect of the horse sausage fermentation agent when Lactobacillus plantarum MC1 and MC2 are missing.

[0028] S1, weigh out 10 parts of Lactobacillus weisseri MR1, 10 parts of Lactobacillus plantarum MR2 and 10 parts of MR3 and mix them together; add 10 parts of whey powder, 8 parts of skim milk powder, 6 parts of trehalose and 10 parts of maltodextrin to make a mixture; S2 was cultured in MR liquid medium at 25°C for 20 hours, with an endpoint OD600 of 2.0, and the bacterial cells were collected. S3: Mix the bacterial cells and the protectant thoroughly, and pre-freeze at -55°C for 3 hours; S4 is subjected to vacuum freeze drying, passed through an 80-mesh sieve, and then sealed in nitrogen-filled packaging.

[0029] Comparative Example 2 The stability and antibacterial effect of the smoked horse sausage starter were verified when only maltodextrin was used as a protectant.

[0030] S1, weigh out 10 parts of Lactobacillus weisseri MR1, 10 parts of Lactobacillus plantarum MR2 and MR3, and 16 parts of Lactobacillus plantarum MC1 and MC26 and mix them together; add 34 parts of maltodextrin to make a mixture; S2 was cultured in MR liquid medium at 25°C for 20 hours, with an endpoint OD600 of 2.0, and the bacterial cells were collected. S3, mix the bacterial cells with a single protectant and pre-freeze at -55°C for 3 hours; S4 is subjected to vacuum freeze drying, passed through an 80-mesh sieve, and then sealed in nitrogen-filled packaging.

[0031] Performance testing 1. Viable bacteria count detection Take the prepared fermentation sample and serially dilute it 10-fold with sterile physiological saline. Spread an appropriate amount of the diluted solution onto MR solid medium plates. After incubation at 37°C for 48 hours, count the number of typical colonies, calculate and convert the viable cell count of the sample, and express the result as CFU / g.

[0032] 2. Salt and acid resistance tests The revival broth of the fermentation agent was inoculated into MR liquid medium containing different sodium chloride concentrations (9%, 10%, 12%) and MR medium with different pH values ​​(4.5, 5.0, 6.5), with an inoculation amount of 2%. After incubation at 30°C for 24 hours, the OD600 value was measured using a spectrophotometer to compare the cell growth under different conditions. If the OD600 value is between 1.2 and 3.0, it is considered to have good salt and acid tolerance.

[0033] 3. Antibacterial performance test The antibacterial activity of the starter culture against common pathogenic bacteria was evaluated using the agar diffusion method. Logarithmic phase cultures of Staphylococcus aureus, Escherichia coli, and Salmonella were spread onto nutrient agar plates. 6 mm diameter wells were punched in the plates, and the supernatant of the starter culture (after centrifugation to remove bacterial cells) was added. After incubation at 30°C for 24 hours, the diameter of the inhibition zone was measured and recorded. A diameter of 10–20 mm was considered the effective inhibition range.

[0034] 4. Detection of freeze-dried survival rate and moisture content After freeze-drying, the starter culture sample was diluted in equal volumes, and the viable cell count was measured. The viable cell count was compared with that before freeze-drying, and the survival rate was calculated. The results were expressed as a percentage. Moisture content was determined using a 105℃ constant-temperature drying method. A certain amount of sample was weighed, dried to constant weight, and the moisture content was calculated.

[0035] 5. Storage stability test The prepared starter culture samples were sealed in aluminum foil bags and stored at room temperature for 0, 3, and 6 months. The viable cell count was then measured, and the viable cell retention rate was calculated. A viable cell retention rate of ≥80% was used as the evaluation index.

[0036] Table 1 Performance test results of smoked horse sausage fermentation agent ; Table 2. Antibacterial activity of lactic acid bacteria ; Table 1 shows that comparing the performance test results of Examples 1-3 with Comparative Examples 1 and 2 clearly reveals the differences in activity, stability, and antibacterial effect among the different schemes. Example 2 showed the most outstanding performance, with a total viable count of 1.0 × 10^12 CFU / g, significantly higher than the 6.0 × 10^11 CFU / g of Example 1 and the 5.5 × 10^11 CFU / g of Example 3. Comparative Examples 1 and 2 only showed 3.0 × 10^11 CFU / g and 2.0 × 10^11 CFU / g, respectively, showing a more significant difference. This indicates that a reasonable combination of five strains can significantly improve the viable count of the starter culture. Regarding the freeze-drying survival rate, Example 2 achieved 95%, higher than the 88% of Example 1 and 87% of Example 3, while Comparative Examples 1 and 2 decreased to 75% and 65%, respectively, indicating that the composite protectant plays a crucial role in maintaining cell integrity during freeze-drying. The moisture content test further confirmed this. Example 2 had the lowest moisture content at 3.5%, which was better than 4.2% in Example 1 and 4.3% in Example 3. In contrast, Comparative Examples 1 and 2 had moisture contents of 4.8% and 5.0%, respectively, indicating poor powder stability. The results of the antibacterial performance were particularly evident. Example 2 showed inhibition zone diameters of 14–20 mm against Staphylococcus aureus, Escherichia coli, and Salmonella, significantly larger than 12–16 mm in Example 1 and 11–15 mm in Example 3. In contrast, Comparative Examples 1 and 2 only showed 8–11 mm and 7–10 mm, respectively, indicating that the introduction of Lactobacillus plantarum played a key role in enhancing antibacterial activity. Regarding salt and acid tolerance, Example 2 maintained an OD600 of 1.8–2.8 under conditions of 9%–12% sodium chloride and pH 4.5–6.5, demonstrating strong adaptability. Examples 1 and 3 maintained growth between 1.4–2.2 and 1.3–2.0, respectively, but not as well as Example 2. Comparative Example 1 saw its OD600 decrease to 0.9–1.2 under 12% NaCl conditions, and Comparative Example 2's OD600 was less than 1.0 under pH 4.5 conditions, indicating a significant decrease in cell activity. Long-term storage tests further highlighted the differences. Example 2 maintained 90% viable cell count after 6 months of storage at room temperature, while Examples 1 and 3 maintained 82% and 81%, respectively. In contrast, Comparative Examples 1 and 2 only maintained 70% and 58%, respectively, showing significant deficiencies. Comprehensive analysis results show that Example 2 is superior to Example 1 and Example 3 in terms of fermentation activity, freeze-drying survival rate, antibacterial performance, salt and acid resistance, and long-term stability. Meanwhile, the overall performance of Example 1 and Example 3 is far superior to that of Comparative Example 1 and Comparative Example 2, which fully demonstrates the significant technical effects and innovative value brought about by the synergistic optimization of specific strain combinations and composite protection systems.

[0037] Figure 1The colony morphology and microstructure of five isolated strains, MR1, MR2, MR3, MC1 and MC2, were shown. It can be seen that the colonies all exhibit typical characteristics of lactic acid bacteria. Under the microscope, the bacterial cells are intact and evenly arranged, indicating that each strain is a pure isolate and has the basis for application as a core strain of fermentation agent. Figure 2 The results showed that under 9% and 12% sodium chloride conditions, MR1 and MC2 maintained high OD600 values, demonstrating outstanding salt tolerance. MR2 and MC1 showed moderate performance, while MR3 was relatively weak. However, all of them still had the ability to survive in high-salt environments, which meets the requirements of the high-salt process for smoked horse sausage. Figure 3 The study showed the tolerance of the five strains to sodium nitrite. MC2 had the highest OD600 value, indicating that it had the best activity under this condition. MR1 was next, and MR2 was at a moderate level. MR3 and MC1 were significantly inhibited under high concentrations of sodium nitrite, which indicates that MC2 is the key strain in the complex system to resist sodium nitrite stress. Figure 4 The results further revealed the differences in growth of the strains under acidic conditions. MR1 and MR2 maintained a high OD600 under pH 4.5 to 5.5 conditions, showing good acid resistance. MC1 was at a moderate level, while MR3 and MC2 had insufficient activity under acidic conditions. This indicates that MR1 and MR2 are the main strains that improve the acid resistance of the composite system. Figure 5 The growth curves of the five strains are shown. MR1 grew the fastest, reaching its peak and remaining stable around 12 hours. MC2 reached a high level between 16 and 20 hours, exhibiting strong mid-to-late-stage activity. MR2, MR3, and MC1 grew more slowly, with lower peak values. In summary... Figures 1 to 5 The results show that the five strains each have their own advantages and disadvantages in terms of salt tolerance, acid tolerance, sodium nitrite tolerance, and growth kinetics. A single strain cannot simultaneously meet the requirements of the complex fermentation environment of smoked horse sausage, but the combined application can achieve a complementary effect: MR1 and MR2 enhance acid tolerance, MC2 improves salt and sodium nitrite tolerance, MR1 provides the advantage of rapid fermentation start-up, and MC2 maintains the activity in the later stage. Thus, the combined fermentation agent exhibits excellent adaptability and stability in the special environment of smoked horse sausage, which is far superior to the control group of single strains or unreasonable combinations.

[0038] In summary, this invention has developed a highly adaptable and stable composite fermentation agent specifically for smoked horse sausage by isolating and screening Lactobacillus weisseri MR1, Lactobacillus plantarum MR2, MR3 and Lactobacillus plantarum MC1, MC2 from the natural fermentation environment of smoked horse sausage, and combining them with a composite protectant of whey powder, skim milk powder, trehalose and maltodextrin. Figures 1 to 5The experimental results clearly demonstrate that different strains possess unique advantages in salt tolerance, acid tolerance, sodium nitrite tolerance, and growth kinetics. Through rational combination, these advantages can be complementary, significantly improving overall performance. Compared to the comparative example, the starter culture in the examples showed superior performance in terms of total viable cell count, freeze-drying survival rate, inhibition zone diameter, and long-term stability. Example 2, in particular, achieved optimal levels in multiple indicators. Therefore, this invention not only effectively solves the problems of unstable flavor, insufficient salt and acid tolerance, and short shelf life in the traditional natural fermentation process of smoked horse sausage, but also provides a feasible technical path for achieving low-salt, standardized, and industrialized production of smoked horse sausage, demonstrating significant innovation and application value.

Claims

1. A fermentation agent for smoked horse sausage, characterized in that, The raw materials include the following parts by weight: Lactobacillus weisseri MR15-15 parts; Lactobacillus plantarum MR25-15 parts; Lactobacillus plantarum MR35-15 parts; Lactobacillus plantarum MC13-10 parts; Lactobacillus plantarum MC23-10 parts; whey powder 5-15 parts; skim milk powder 5-12 parts; trehalose 3-10 parts; maltodextrin 5-15 parts; wherein the total viable count of the starter culture is 1.0×10^10-1.0×10^12 CFU / g, and the endpoint OD600 of the culture for 24 hours under the culture conditions of pH 4.5-6.5 and 9%-12% sodium chloride is 1.2-3.

0.

2. The fermentation agent for smoked horse sausage according to claim 1, characterized in that, The *Lactobacillus weisseri* MR1, *Lactobacillus plantarum* MR2, MR3, and *Lactobacillus plantarum* MC1, MC2 exhibited inhibition zones of 10–20 mm in diameter against *Staphylococcus aureus*, *Escherichia coli*, and *Salmonella* in vitro.

3. A fermentation agent for smoked horse sausage according to any one of claims 1 or 2, characterized in that, The viable cell retention rate of the freeze-dried starter culture is 85%–95%, and the moisture content is 2%–5%.

4. A fermentation agent for smoked horse sausage according to any one of claims 1 to 3, characterized in that, The total amount of whey powder, skim milk powder, trehalose, and maltodextrin accounts for 20% to 40% of the total weight of the starter culture.

5. A method for preparing a smoked horse sausage fermenting agent, wherein the smoked horse sausage fermenting agent is as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1, Lactic acid bacteria were isolated from traditionally naturally fermented smoked horse sausage samples to obtain Lactobacillus weisseri MR1, Lactobacillus plantarum MR2, MR3 and Lactobacillus plantarum MC1, MC2; S2 was identified by 16S rRNA sequencing, and strains that met the requirements for salt tolerance, acid tolerance and antibacterial properties were screened. S3 was cultured in MR medium until the logarithmic growth phase was reached, and the bacterial cells were collected when the OD600 at the end of the culture was between 1.5 and 2.

5. S4, mix the bacterial cells with whey powder, skim milk powder, trehalose and maltodextrin, and pre-freeze at -50℃ to -60℃; S5 is subjected to vacuum freeze-drying, sieved, and sealed with nitrogen to obtain the smoked horse sausage fermentation agent.

6. The method for preparing a smoked horse sausage fermentation agent according to claim 5, characterized in that, The culture temperature in step S3 is 20-30℃, and the culture time is 18-24 hours.

7. The method for preparing a smoked horse sausage fermentation agent according to claim 5, characterized in that, The pre-freezing time in step S4 is 2 to 6 hours.

8. The method for preparing a smoked horse sausage fermentation agent according to claim 5, characterized in that, The finished powder obtained in step S5 has a moisture content of 3% to 5%, and the viable bacteria count is maintained at 80% to 90% after 6 months of storage.

Images