Preparation method for reducing biogenic amine in red intestine by using lactobacillus plantarum diamine oxidase
By extracting and purifying diamine oxidase from Lactobacillus plantarum SH7 and applying it to the preparation process of red sausage, the problem of biogenic amine accumulation in red sausage was solved, thus improving safety and quality.
Patent Information
- Application Number
- CN202510462608.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-14
- Publication Date
- 2025-11-28
AI Technical Summary
Red sausages are prone to accumulating biogenic amines, such as histamine and tyramine, during fermentation, which can lead to health risks. Traditional control methods affect the flavor and microbial community structure of the product, and the targeted application of biogenic amine oxidases has not yet been reported.
Diamine oxidase was extracted from Lactobacillus plantarum SH7, purified by ammonium sulfate fractionation precipitation and anion exchange chromatography, and then freeze-dried. The resulting powder was added to the raw meat of red sausage, fermented, and air-dried to control the accumulation of biogenic amines.
It effectively reduces the content of biogenic amines in red sausage, improves product safety and quality, while maintaining the flavor and physicochemical properties.
Smart Images
Figure CN121014820A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of food processing technology, specifically to a method for reducing biogenic amines in red sausage using Lactobacillus plantarum diamine oxidase. Background Technology
[0002] Red sausage, a traditional fermented meat product, is beloved for its unique flavor. However, it easily accumulates biogenic amines, such as histamine and tyramine, during fermentation, and excessive intake can lead to health risks such as allergies and diarrhea. Traditional control methods, such as adding chemical inhibitors or natural extracts, can easily affect the product's flavor and microbial community structure. Biogenic amine oxidases (such as diamine oxidase) can degrade biogenic amines through oxidation reactions, representing a safe and efficient biological control method. However, the technology of extracting diamine oxidase from *Lactobacillus plantarum* and applying it specifically to control biogenic amines in red sausage has not yet been reported. Summary of the Invention
[0003] In view of this, the purpose of this invention is to provide a method for reducing biogenic amines in red sausage using diamine oxidase derived from Lactobacillus plantarum SH7, thereby achieving effective control of biogenic amines in red sausage and improving product safety and quality.
[0004] A method for preparing red sausage by using Lactobacillus plantarum diamine oxidase to reduce biogenic amines includes the following steps:
[0005] Strain culture and enzyme extraction: Lactobacillus plantarum SH7, isolated from traditionally fermented and dried intestines, was inoculated into MRS liquid medium and cultured at 37 ℃ for 18 h. After two consecutive subcultures, the supernatant was collected by centrifugation at 4 ℃ and 8000 rpm for 15 min to obtain crude enzyme solution.
[0006] Ammonium sulfate fractionation precipitation: Add ammonium sulfate to the crude enzyme solution to a concentration of 50%-70%, let stand at 4 ℃ for 8 h, centrifuge at 8000 rpm for 10 min to collect the precipitate, resuspend in phosphate buffer at pH 7.0, dialyze for 24 h to remove salts, and obtain a preliminarily purified enzyme solution.
[0007] Anion exchange chromatography purification: The dialyzed enzyme solution was loaded onto a DEAE anion exchange chromatography column and eluted with a gradient of 0-1 mol / L NaCl. The fraction with diamine oxidase activity was collected and lyophilized to obtain diamine oxidase lyophilized powder.
[0008] Red sausage preparation and enzyme powder addition: Diamine oxidase freeze-dried powder is added to the raw meat of red sausage at a ratio of 0.1%-0.5% (w / w). The raw meat formula is: 70 parts lean meat, 30 parts fat meat, 2 parts salt, 1 part glucose, and 0.5 parts spices. After mixing and chopping, the mixture is stuffed into sausages and fermented for 12 days at 25 ℃ and 80% relative humidity. Then it is air-dried for 7 days at 15 ℃ and 70% relative humidity.
[0009] Biogenic amine detection and quality evaluation: The biogenic amine content in red sausage was determined according to GB5009.229-2016, and the physicochemical indicators such as moisture content, water activity, pH value and color difference were also detected. Attached Figure Description
[0010] Figure 1 This is a flowchart illustrating the preparation of a method using Lactobacillus plantarum diamine oxidase to reduce biogenic amines in red intestines. Detailed Implementation
[0011] Example 1: Extraction and purification of Lactobacillus plantarum SH7 diamine oxidase.
[0012] Strain culture: Lactobacillus plantarum SH7 was inoculated into 500 mL of MRS liquid medium and cultured at 37 °C with shaking for 18 h. After two repeated subcultures, the culture was centrifuged at 4 °C and 8000 rpm for 15 min and the supernatant was collected.
[0013] Ammonium sulfate precipitation: Slowly add ammonium sulfate to the supernatant until the saturation is 60%, stir to dissolve, let stand at 4 ℃ for 8 h, centrifuge at 8000 rpm for 10 min, resuspend the precipitate in 50 mL of pH 7.0 phosphate buffer (0.05 mol / L), put it into a dialysis bag (molecular weight cutoff 10 kDa), and dialyze at 4 ℃ with the same buffer for 24 h, changing the dialysate 4 times during the period.
[0014] Anion exchange chromatography: The dialyzed enzyme solution was loaded onto a DEAE-Sepharose Fast Flow column (1.6 cm × 20 cm), equilibrated with 5 column volumes of buffer, and then eluted with a 0-1 mol / L NaCl gradient at a flow rate of 1 mL / min, collecting one tube every 2 min. The enzyme activity of each fraction was measured (using putrescine as a substrate, measuring the absorbance change at 340 nm), and the active peak fractions were combined and lyophilized to obtain diamine oxidase lyophilized powder with a specific activity of 15 U / mg.
[0015] Example 2: Preparation of low-biogenic amine red sausage.
[0016] Raw material preparation: Take 700 g of fresh lean pork and 300 g of fatty pork, cut them into small pieces, add 20 g of salt, 10 g of glucose and 5 g of spices (black pepper, nutmeg, etc.), and mix well.
[0017] Enzyme addition: Dissolve 0.5 g of the diamine oxidase freeze-dried powder (containing 7.5 U of enzyme) prepared in Example 1 in 10 mL of sterile water, add it to the chopped minced meat, mix thoroughly, stuff into natural sausage casings, and ferment in an incubator at 25 ℃ and 80% relative humidity for 12 days. During this period, the pH value is measured daily. When the pH value stabilizes at around 5.5, transfer to a drying room at 15 ℃ and 70% relative humidity for 7 days to air dry.
[0018] Preparation of control sample: Except for the absence of enzyme powder, the other steps are the same as in Example 2.
[0019] Determination of biogenic amines: Red sausage samples were collected after fermentation for 0, 4, 8, and 12 days, and after air drying. The contents of histamine, tyramine, putrescine, cadaverine, and phenylethylamine were determined by high-performance liquid chromatography (HPLC) according to GB5009.229-2016. The results showed that the total biogenic amine content in the red sausage with added enzyme powder was reduced by 35% compared to the control sample. The degradation rates of histamine and tyramine were 40% and 30%, respectively, and the color of the red sausage (…) L *、 a *、 b The * value, moisture content, and pH value were not significantly different from the control sample (P>0.05), indicating that the method maintained the quality characteristics of the sausage while reducing biogenic amines.
Claims
1. A method for preparing red sausage by using Lactobacillus plantarum diamine oxidase to reduce biogenic amines, characterized in that, Includes the following steps: Step 1: Culture Lactobacillus plantarum SH7 (preservation number: CGMCC No. 25287) and extract crude extracellular diamine oxidase solution; Step 2: Purify diamine oxidase by ammonium sulfate fractionation precipitation and anion exchange chromatography to obtain highly active enzyme powder; Step 3: Add the diamine oxidase powder to the red sausage raw material, and prepare low-biogenic amine red sausage through fermentation and air drying.
2. The preparation method according to claim 1, characterized in that, The Lactobacillus plantarum SH7 strain was isolated from traditionally fermented and dried intestines and has the characteristic of efficiently secreting diamine oxidase.
3. The preparation method according to claim 1, characterized in that, The concentration of the ammonium sulfate fractionation precipitate was 50%-70%, and the anion exchange chromatography was performed using a DEAE-Sepharose Fast Flow column with a gradient elution of 0-1 mol / L NaCl.
4. The preparation method according to claim 1, characterized in that, The amount of diamine oxidase powder added is 0.1%-0.5% of the weight of the raw meat for red sausage.