CN108587983B

CN108587983B - Lactobacillus plantarum and application thereof in preparation of Sichuan sausage through fermentation

Info

Publication number: CN108587983B
Application number: CN201810580215.3A
Authority: CN
Inventors: 孙群; 田圆圆; 刘绒梅; 张国建; 刘维; 刘保
Original assignee: Sichuan University
Current assignee: Sichuan Shengmeisida Biotechnology Co ltd; Sichuan University
Priority date: 2018-06-07
Filing date: 2018-06-07
Publication date: 2020-07-31
Anticipated expiration: 2038-06-07
Also published as: CN108587983A

Abstract

The invention relates to microorganism and meat food fermentation, and particularly discloses lactobacillus plantarum and application thereof in preparation of Sichuan sausages through fermentation. The lactobacillus plantarum 1-1 provided by the invention is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms, and the preservation number is as follows: CGMCC NO.15467, preservation date: year 2018, month 3 and day 20. The strain is separated from the traditional sausage in Qingcheng mountain of Wei city of Yangjiang weir, Sichuan province, grows well on MRS agar culture medium, has main fermentation characteristics meeting the requirements of meat leavening agents, and is suitable for meat fermentation. Has the capability of degrading sarcoplasmic proteins and biogenic amines in an in vitro simulation system. The method is applied to producing Sichuan sausages, can promote the degradation of sarcoplasmic proteins of the Sichuan sausages and obviously reduce the content of biogenic amine in the Sichuan sausages, and has the potential of being applied to the actual production of the Sichuan sausages, thereby ensuring and improving the flavor, taste and safety of the Sichuan sausages.

Description

Lactobacillus plantarum and application thereof in preparation of Sichuan sausage through fermentation

Technical Field

The invention relates to the field of microorganism and meat food fermentation, in particular to lactobacillus plantarum (A)Lactobacillus plantarum) 1-1 and application thereof in Sichuan sausages.

Background

The Sichuan sausage is spicy and tasty, has unique flavor and moderate acidity, and is popular with a plurality of consumers. Sichuan sausages on the market are divided into Sichuan traditional sausages and Sichuan sausages produced in a modern large scale. The traditional Sichuan sausage belongs to naturally fermented meat products, and the fermented microorganisms of the traditional Sichuan sausage are mainly derived from raw meat and a processing environment, and contain rich strain resources with regional characteristics. The Sichuan traditional sausage has the advantages of unique flavor, texture, color, taste and low acidity, but has the defects of long production period and difficult guarantee of safety and sanitation. The Sichuan sausage produced in a modern large scale is safe, sanitary and stable in quality, but the taste and flavor of the Sichuan sausage are inferior to those of the Sichuan sausage. Although there are many research reports on the Sichuan sausage leavening agent, there is a certain distance to apply the selected leavening agent to the actual production, and there are a large number of commercial meat leavening agents abroad. If the existing foreign commercial leavening agent is used for producing the Sichuan sausage, the Sichuan sausage can lose local unique sensory characteristics, particularly the acidity is increased, and unpleasant sensory experience is caused.

The special flavor of the fermented sausage is attributed to the balance between volatile substances and nonvolatile substances, the lactobacillus can promote protein degradation, the protein degradation can generate nonvolatile substances such as polypeptide and free amino acid, some free amino acids and polypeptide are flavor substances, and some free amino acids generate aldehydes, alcohols, esters and other volatile substances through transamination, deamination and decarboxylation, so that the content of the volatile substances and the nonvolatile substances is adjusted. However, under the action of microbial amino decarboxylase, free amino acids generated by protein degradation can also form biogenic amine through decarboxylation, and the low pH environment caused by organic acids generated by lactic acid bacteria metabolism is also favorable for biogenic amine formation. Excessive biogenic amine intake can cause palpitations, headache, respiratory distress, high or low blood pressure and even death. Therefore, it is necessary to screen for lactic acid bacteria capable of degrading muscle proteins and biogenic amines simultaneously.

The research on protein and biogenic amine leavening agents is reported as follows, 1) separation and identification of fungal strains producing protease and lipase in Sichuan sausages and preliminary application thereof [ D ], the Sichuan agriculture university ] is primarily applied to Sichuan sausages in 2012, the Sichuan naturally fermented sausage is screened to produce penicillium chrysogenum with strong activity, and is subjected to a complex leavening agent with Lactobacillus pentosus L76 and Staphylococcus saprophyticus S25 stored in a laboratory, the inoculated leavening agent can promote protein degradation of Sichuan sausages and increase relative contents of alcohols, ketones, esters and flavor substances of the Sichuan sausages through protein hydrolysis index and volatile flavor substance analysis, but the problem that protein hydrolysis may cause increase of biogenic amine content is not concerned in the text, 2) the sausage is developed through protein hydrolysis index and volatile flavor substance analysis, the inoculated fermented sausage is inoculated to influence on the quality of Sichuan sausages and the leavening agent thereof, the concept of the fermented sausage is improved, the concept of the fermented sausage is improved through the biological leavening agent, the concept of the antibiotic.

Disclosure of Invention

Aiming at the problem that a meat leaven for ensuring the unique flavor and safety of the sausage is lacked in the production of the Sichuan sausage, the invention screens a lactobacillus plantarum (A) from the traditional sausage collected from Qingcheng mountain of Wei city of Yangjiang city, Sichuan provinceLactobacillus plantarum) 1-1, the main fermentation characteristics of the lactobacillus plantarum meet the requirements of meat leavening agents, and the lactobacillus plantarum can promote the meat leavening agents in an in vitro simulation system and Sichuan sausagesThe degradation of sarcoplasmic protein and biogenic amine is facilitated, the flavor, the taste and the safety of Sichuan sausage are improved, and the Sichuan sausage has the potential of being applied to the actual production of Sichuan sausage.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

lactobacillus plantarum (A)Lactobacillus plantarum) 1-1, deposited in China general microbiological culture Collection center (CGMCC for short, address: west road No.1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101), and the preservation number is CGMCC NO. 15467.

The lactobacillus plantarum 1-1 is obtained by separating traditional sausages collected from Qingcheng mountains of Wei city of Yangjiang province in Sichuan province.

The lactobacillus plantarum 1-1 grows well on a De Man Rogosa Sharpe (MRS) agar culture medium, and the bacterial colony is milky white, convex, round, smooth and neat in edge. The shape of the strain is observed by an optical microscope, and the strain cell is in a short rod shape and has no spores and flagella.

The lactobacillus plantarum 1-1 is obtained by amplifying the 16S rDNA by using the bacterial universal primers 27F and 1492R, and a target gene SEQUENCE consisting of 1403 base pairs (bp) is obtained, as shown in SEQUENCE L ISTING, and the gene SEQUENCE obtained by sequencing is input into an NCBI database for comparison, and is compared with the gene SEQUENCE in GenebankLactobacillus plantarumThe similarity rate of the standard strain MG983980.1 reaches 99%, and the strain can be preliminarily identified to be lactobacillus plantarum (A), (B), (C)Lactobacillus plantarum）。

The main fermentation characteristics of the lactobacillus plantarum 1-1 meet the requirements of meat leavening agents, namely, the lactobacillus plantarum has protease activity and does not produce NH₃Biogenic amines, H₂S and CO₂Can tolerate 6 percent of NaCl and 150 mg/kg of NaNO₂And can grow at pH4.2-6.0 and 10-30 deg.C.

Further, in order to investigate the capacity of lactobacillus plantarum 1-1 to degrade sarcoplasmic proteins and biogenic amines, lactobacillus plantarum 1-1 is inoculated to two in vitro simulation systems, namely pork sarcoplasmic protein extract and phosphate buffer containing tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine, respectively, and the lactobacillus plantarum 1-1 is proved to be capable of promoting the degradation of the pork sarcoplasmic proteins and biogenic amines in the in vitro simulation system.

Still further, in order to explore the influence of the lactobacillus plantarum 1-1 on the degradation condition of the muscle plasma protein and the content of the biogenic amine of the Sichuan sausage, the lactobacillus plantarum 1-1 is applied to the Sichuan sausage, and the lactobacillus plantarum 1-1 is proved to be capable of promoting the degradation of the muscle plasma protein of the Sichuan sausage and reducing the content of tryptamine, phenethylamine, putrescine, cadaverine, histamine and tyramine of the Sichuan sausage.

The research results show that the main fermentation characteristics of the lactobacillus plantarum 1-1 provided by the invention meet the requirements of meat leavening agents, have the capacity of degrading sarcoplasmic proteins and biogenic amines, and have the potential of being applied to the actual production of Sichuan sausages.

Compared with the prior art, the invention has the advantages and beneficial effects that:

1. the invention provides lactobacillus plantarum (A)Lactobacillus plantarum) 1-1, screening traditional sausage products separated from Qingcheng mountain of Wei city of Yangjiang river, Sichuan province, growing well on De Man Rogosa Sharpe (MRS) agar culture medium, and having main fermentation characteristics meeting the requirements of meat leavening agent, namely having protease activity and producing no NH₃Biogenic amines, H₂S and CO₂Can tolerate 6 percent of NaCl and 150 mg/kg of NaNO₂And can grow at pH4.2-6.0 and 10-30 deg.C, and the main fermentation characteristics of said strain are suitable for the condition of actual production.

2. The lactobacillus plantarum 1-1 provided by the invention has good capacity of degrading sarcoplasmic proteins and biogenic amines in an in vitro simulation system, and has strong application potential.

3. The lactobacillus plantarum 1-1 provided by the invention is applied to Sichuan sausage, not only can the sarcoplasmic proteins of the Sichuan sausage be obviously degraded, but also the contents of tryptamine, phenethylamine, putrescine, cadaverine, histamine and tyramine (P is less than 0.05) in the Sichuan sausage can be obviously reduced, so that the unique flavor, taste and safety of the Sichuan sausage are ensured and improved, and the organic combination of the traditional process and the modern industry of the Sichuan sausage is expected to be promoted.

Drawings

FIG. 1 is a colony morphology diagram of Lactobacillus plantarum 1-1 on MRS agar medium;

FIG. 2 is a cell morphology diagram of Lactobacillus plantarum 1-1 under an optical microscope;

FIG. 3 is SDS-PAGE of the band variation of the sarcoplasmic protein bar fermentation broth in different fermentation days, wherein M is protein Marker, and 0-4 are fermentation days (days);

FIG. 4 is SDS-PAGE (sodium dodecyl sulfate-PAGE) graph of sarcoplasmic protein band changes of Sichuan sausage at different processing stages, wherein M is a protein Marker, 1-5: respectively represent day 0, end of fermentation, day 2 of maturation, day 4 of maturation and end of drying;

fig. 5 is a schematic diagram of biogenic amine content in Sichuan sausage after drying is finished.

Detailed Description

The present invention is further illustrated with reference to the following examples, but the embodiments of the present invention are not limited thereto, and any technologies implemented based on the above contents of the present invention are within the scope of the present invention.

The following are the media used in the examples of the invention:

firstly, MRS agar culture medium: 10 g of peptone, 1 g of beef extract, 5g of yeast extract powder, 20g of glucose, 5g of sodium acetate, 2 g of monopotassium phosphate, 2 g of ammonium citrate and 0.5 g of MgSO 2₄•7H₂O、0.25 gMnSO₄•4H₂O, 1m L Tween 80, 18 g agar powder and 1L distilled water, and sterilizing at 121 ℃ for 15 min.

Secondly, detecting a culture medium for main fermentation characteristics:

1) nitrite resistance test medium: the same MRS liquid culture medium is added with NaNO₂So that NaNO is present₂To a final concentration of 150 mg/L.

2) Salt tolerance experiment culture medium: the same MRS liquid medium was used, but NaCl was added thereto so that the final concentration of NaCl was 6%.

3) The glucose gas production culture medium comprises 10 g of peptone, 5g of sodium chloride, 1L of distilled water, a bromocresol purple ethanol solution with the pH adjusted to 7.4 and the concentration of 1m L1.6.6 percent and 20g of glucose, and is subpackaged in test tubes, the Du's tubules are placed in the test tubes in an inverted mode, and the test tubes are sterilized for 30min at the temperature of 121 ℃.

4) NH produced by arginine₃Culture medium: 20g of arginine, 10 g of peptone, 5g of yeast extract powder, 5g of sodium acetate and 2 gK₂HPO4, 2 g sodium citrate, 0.5 g glucose, 0.5 g MgSO₄•7H₂O、0.25 g MnSO₄•4H₂O, 1m L Tween 80, 20g agar, 1L distilled water, and sterilizing at 121 deg.C for 15 min.

5) Produce H₂The S culture medium comprises 10 g of peptone, 10 g of beef extract, 5g of sodium chloride, 0.5 g of cysteine and 1L of distilled water, the pH value is adjusted to 7.0-7.4, and the sterilization is carried out for 20 min at 112 ℃.

6) The litmus milk medium comprises 10 g skimmed milk powder, 0.1L distilled water, 4 m of L25 g/L litmus, and is sterilized at 113 deg.C for 15 min.

7) Lipase activity detection medium: the same MRS solid culture medium is adopted, but 15 percent of lard oil and a neutral red indicator are added.

8) The amino acid decarboxylase test culture medium comprises 5g of peptone, 3 g of yeast extract, 1 g of glucose, 1m of L1.6.6% bromocresol purple ethanol solution, 5g of L-amino acid and 1L distilled water, the pH value is adjusted to 6.8, the control culture medium is not added with amino acid, the test tubes are separately filled with the amino acid decarboxylase test culture medium, a layer of liquid paraffin is added into the test tubes, and the test tubes are sterilized for 10min at the temperature of 115 ℃.

The percentages referred to in the above media are mass to volume ratios.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Example 1

Separation, culture and molecular identification of lactobacillus plantarum 1-1

Weighing 5g of a sheared sample under an aseptic condition, placing the sample in a triangular flask filled with 50m L sterile distilled water, shaking the flask in a shaking way at room temperature for 30min, standing for several minutes, sucking supernatant, sequentially carrying out 10-fold gradient dilution, selecting proper dilution, coating the diluted sample on an MRS solid culture medium, carrying out inversion culture at 37 ℃ for 48h, selecting different characteristic colonies according to the colony morphology on the culture medium and primary microscopic observation, carrying out multiple streaking separation and purification, carrying out enrichment culture on the purified strain by using a corresponding liquid culture medium, extracting strain genome DNA, amplifying 16S rDNA by using bacterial universal primers 27F and 1492R, wherein the amplification condition is pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30S, annealing at 58 ℃ for 45S, extension at 72 ℃ for 1 min for 30 cycles, final extension at 72 ℃ for 10min, and the amplification product is verified to be a single band by 2% agarose gel electrophoresis, and the band length is about 1500 bp, and carrying out sequencing.

The above-mentioned bacterial universal primers 27F and 1492R were synthesized by Invitrogen (Shanghai). The sequence of 27F is AGAGTTTGATCCTGGCTC, and the sequence of 1492R is CTACGGCTACCTTGTTACGA.

The sequencing length of the amplified fragment is 1403 bp, and the gene sequence obtained by sequencing is input into an NCBI database for comparison with that in GenebankLactobacillus plantarumThe similarity rate of the standard strain MG983980.1 reaches 99%, and the strain 1-1 is preliminarily identified as one strainLactobacillus plantarumI.e. lactobacillus plantarum.

And (3) streaking the lactobacillus plantarum 1-1 on an MRS agar culture medium, carrying out inverted culture at 37 ℃ for 48 hours, and observing the colony morphology of the strain. As shown in figure 1, the strain 1-1 grows well on the MRS culture medium, and the bacterial colony is milky white, convex, round, smooth and regular in edge.

As shown in FIG. 2, the shape of the Lactobacillus plantarum 1-1 cells was observed by an optical microscope, and the Lactobacillus plantarum 1-1 cells were short rods, and had no spores and flagella.

Example 2

Detection of major fermentation characteristics of Lactobacillus plantarum 1-1

1. Salt tolerance test: inoculating Lactobacillus plantarum 1-1 into a salt tolerance test culture medium, culturing at 37 deg.C for 24h, and measuring OD at 600 nm. NaCl tolerance (%) = (OD)_{600 (with addition of NaCl)}/OD_{600 (without NaCl)}）×100%

2. Nitrite resistance test: inoculating Lactobacillus plantarum 1-1 into nitrite-resistant test culture medium, culturing at 37 deg.C for 24 hr, and measuring OD value at 600 nm. NaNO₂Tolerance (%) = (OD)_{600 (with NaNO 2)}/OD_{600 (No addition NaNO 2)}）×100%

3. Growth conditions at different temperatures: inoculating Lactobacillus plantarum 1-1 into MRS liquid culture medium, culturing at different temperatures for 24h, and measuring OD at 600 nm.

4. Growth conditions at different pH: inoculating Lactobacillus plantarum 1-1 into MRS liquid culture medium with pH of 3.0, 4.0, 5.0, 6.0, and 7.0, respectively, culturing at 37 deg.C for 24 hr, and measuring OD at 600 nm.

5. Catalase production test: lactobacillus plantarum 1-1 was inoculated on MRS agar medium. Culturing at 37 deg.C for 24 h. 5% hydrogen peroxide solution is dripped on the lawn, and the generation of bubbles is positive, otherwise, the generation is negative.

6. Protein degradation test: inoculating Lactobacillus plantarum 1-1 into litmus milk culture medium, and culturing at 37 deg.C for 48 hr. Lactic acid bacteria produce acid by using lactose in the culture medium, the acidity is increased, and milk is coagulated. If the strain is protease-positive bacteria, the protein is decomposed, and the culture medium is clearer and slightly transparent.

7. Lipolysis test: 0.1 ml of the bacterial liquid of the lactobacillus plantarum 1-1 diluted to a proper concentration is uniformly coated on a fat culture medium, the culture is carried out for 24 hours at 37 ℃, whether red spots are generated on the culture medium or not is observed, the red spots appear as positive, and the red spots are negative on the contrary.

8. Production of NH₃And (3) testing: inoculating lactobacillus plantarum 1-1 into an ammonia-producing culture medium, making another blank control, culturing at 37 ℃ for 24h, sucking a small amount of culture solution and a Neisseria reagent, placing in a colorimetric disc, and uniformly mixing, wherein if yellow or brownish red precipitates are generated, the mixture is positive.

9. Amino acid decarboxylase test: the lactobacillus plantarum 1-1 is inoculated in an amino acid decarboxylase test culture medium and cultured for 24 hours at 37 ℃, as the amino decarboxylase positive strain can produce alkaline biogenic amine, the culture medium can change the color into purple, and the negative strain can metabolize glucose to produce organic acid, so that the culture medium can change the color into yellow. The control tube should be yellow.

10. Produce H₂S test: inoculating Lactobacillus plantarum 1-1 to produce H₂In S medium, a sterile strip of lead acetate was hung in an inoculation tube. The strain which turns black on the lead acetate paper strip is positive after being cultured for 24 hours at 37 ℃.

11. Glucose gassing test: inoculating the lactobacillus plantarum 1-1 into a glucose gas production culture medium for culture, and observing whether bubbles exist in the Duchenne tubule or not, wherein the bubbles are positive.

The main fermentation characteristics of Lactobacillus plantarum 1-1 detected by the above tests are shown in Table 1, and it can be seen from Table 1 that this strain has protease activity and does not produce NH₃Biogenic amines, H₂S and CO₂Can tolerate 6 percent of NaCl and 150 mg/kgNaNO₂And can grow at pH4.2-6.0 and 10-30 deg.C. The main fermentation characteristics of the lactobacillus plantarum 1-1 meet the requirements of meat leavening agents and are suitable for the conditions of actual production.

TABLE 1 major fermentation characteristics of Lactobacillus plantarum 1-1

Main fermentation characteristics	Results	Main fermentation characteristics	Results
				Decomposing protein	+	Decomposing lipid	-
Production of NH₃	-	Generation of biogenic amines	-
				Produce H₂S	-	Produce CO₂	-
Growth at pH4.2-6.0	+	Growth at 10-30 deg.C	+
				6% NaCl tolerance/%)	26.55 ±0.59	150mg/kg NaNO₂Tolerance/%	100.00±1.40

Note: + indicates a positive result, -indicates a negative result.

Example 3

In-vitro simulation system for exploring the capability of lactobacillus plantarum 1-1 in degrading sarcoplasmic proteins and biogenic amines

1. In vitro simulation system for exploring the ability of Lactobacillus plantarum 1-1 to degrade sarcoplasmic protein is prepared by weighing 6 g of minced pig longissimus dorsi, adding 10 times volume of iced deionized water, homogenizing for 1 min, standing the homogenate at 4 deg.C for 30min, centrifuging at 4 deg.C for 10min at 10000 × g, filtering the supernatant with Whatman No. 4 filter paper, supplementing 1% glucose into the filtrate, adjusting pH of the filtrate to 6.0, filtering the filtrate with 0.22 μm filter membrane for sterilization to obtain sarcoplasmic protein extract, and culturing 3 times of subcultured Lactobacillus plantarum 1-1 according to 10 times⁷Inoculating the final concentration of cell/m L into sarcoplasmic protein extract, mixing, culturing at 37 deg.C for 4 days, culturing under the same condition with sarcoplasmic protein extract without thallus for 4 days as control, sampling every day, analyzing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), sucking 120 μ L sarcoplasmic protein fermentation liquid and 30 μ L5 × sample buffer, mixing, boiling water bath for 5min, centrifuging at 8000 × g for 5min, collecting 15 μ L supernatant, performing electrophoresis, separating gel concentration of 12%, and concentrating gel concentration of 12%5 percent. The voltage of the sample in the concentrated gel is 80V, and the voltage of the sample after entering the separation gel is changed into 120V. After the electrophoresis, the gel was removed from the glass plate and stained in Coomassie brilliant blue R-250 staining solution for 30 min. And taking the gel out of the staining solution, placing the gel in a decoloring solution, and decoloring for multiple times until the protein bands become clear and the background is clean. The decolorized gel was photographed in a gel imaging system. As shown in FIG. 3, at day 4 of fermentation, Lactobacillus plantarum 1-1 was able to degrade and disappear the remaining bands except the band at 63.0 kDa, as compared to the control. The bacterial strain 1-1 has strong degradation effect on the sarcoplasmic protein and has the potential of improving the flavor and taste of the sausage.

2. The in vitro simulation system explores the capability of lactobacillus plantarum 1-1 in degrading biogenic amine: the thallus of Lactobacillus plantarum 1-1 after 3 subcultures is treated according to the ratio of 10⁷cell/m L was resuspended in 0.05 mol/L phosphate buffer (pH 7.0) containing 100 mg/L each of tryptamine, phenethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine, which had been sterilized by filtration through a 0.22 μm filter, and incubated at 37 ℃ for 48h, then incubated at 37 ℃ for 48h with biogenic amine-containing phosphate buffer without added cells, as a control, after 48h, centrifugation at 5000 × g for 10min, the supernatant was collected, pre-column derivatization was performed on the supernatant, 1m L supernatant was aspirated, 1.5 m L pH 11.0 buffer (50 m L0.5 mol/L NaHCO) was added to the supernatant₃Solution and 12 m L0.5 mol/L Na₂CO₃Mixing the solutions, adjusting pH to 9.2, and adding 16.65 g K₂CO₃) And 1m L10 mg/m L dansyl chloride acetone solution, uniformly mixing, carrying out dark reaction at 40 ℃ for 1 h, adding 0.2 m L0100 mg/m L prolinic acid solution, carrying out dark reaction at room temperature for 1 h, after the reaction is finished, adding 3 m L n-heptane, uniformly mixing, standing for a plurality of minutes, taking 1m L upper organic phase, carrying out nitrogen blow-drying at 40 ℃, adding 1m L chromatographic pure acetonitrile for dissolving, filtering a dissolved solution by using a 0.22 mu m filter membrane, measuring the content of the biogenic amine in a filtrate by using a high performance liquid chromatography (HP L C) method, wherein a chromatographic column is C18 (4.6 × 250 mm and 5 mu m), the column temperature is 40 ℃, the flow rate is 1m L/min, the detection wavelength is 254 mu m, and the sample injection amount is 20 mu L, and a gradient elution program is adopted, and the gradient elution program is shown in Table 2.

Biological amine degradation rate = (biological amine content in control group supernatant-biological amine content in experimental group supernatant)/biological amine content in control group supernatant × 100%

Table 2 gradient elution procedure for biogenic amine analysis

Time (min)	Water (%)	Acetonitrile (%)
			0	35	65
9	30	70
			28	0	100
32	0	100
			33	35	65

The degradation of biogenic amines by Lactobacillus plantarum 1-1 in an in vitro simulated system is shown in Table 3. As can be seen from Table 3, the Lactobacillus plantarum has a certain degradation effect on 8 biogenic amines, and the degradation rate is higher than 10%, wherein the degradation rate on putrescine is the highest and reaches 16.55%. The lactobacillus plantarum 1-1 is expected to be used for sausage production, so that the safety of the sausage is improved.

TABLE 3 degradation rate of biogenic amines by Lactobacillus plantarum 1-1

Biogenic amines	Degradation Rate (%)	Biogenic amines	Degradation Rate (%)
				Tryptamine	10.82 ± 109	Histamine	11.86 ± 1.06
Phenylethylamine	11.26 ± 0.24	Tyramine	14.41 ± 1.47
				Putrescine	16.55 ± 0.52	Spermidine	13.91 ± 1.52
Cadaverine	13.97 ± 0.74	Spermine	12.28 ± 0.82

Example 4

Application of lactobacillus plantarum 1-1 to Sichuan sausage

1. Preparing Sichuan sausage: mincing 10 kg raw meat (pork, weight ratio 1: 4), adding 250 g edible salt, stirring, and pickling at 4 deg.C for 4 hr. And adding 100 g of white granulated sugar, 100 g of white spirit, 100 g of pepper powder and 200 g of chili powder into the pickled sausage meat slices, and uniformly stirring. According to 10⁷Inoculating the bacterial suspension of the lactobacillus plantarum 1-1 into meat material according to the inoculation amount of cfu/g, uniformly mixing, and filling the meat material into a pig small intestine by using an enema machine, wherein the suspension liquid is prepared by suspending the bacterial body of the lactobacillus plantarum 1-1 in sterile normal saline (0.9% sodium chloride aqueous solution), the suspension method comprises the steps of centrifuging the bacterial liquid of the lactobacillus plantarum 1-1 subjected to twice subculture for 10min at 8000 × g at 4 ℃, collecting the bacterial body, uniformly mixing the sterile normal saline with the collected bacterial body, and finally regulating the concentration of the bacterial suspension to 10 by a blood counting plate cell counting method⁹cfu/m L, cleaning dirt on the surface of the sausage body with clear water, placing in a constant temperature incubator, fermenting at 20 ℃ for 12 h with relative humidity of 60-90%, after the fermentation is finished, maturing at 13 ℃ for 4 days with relative humidity of 50-70%, placing the mature sausage in an oven, drying at 55 ℃ for 24h, and using Sichuan sausage which is prepared by the same formula and processing technology and is not inoculated with lactobacillus plantarum 1-1 as a control.

2. SDS-PAGE analysis Sichuan sausage sarcoplasmic protein degradation, weighing 5g of the cut sample, adding 10 times the volume of ice deionized water, homogenizing for 1 min, homogenizing the homogenate, standing at 4 ℃ for 30min, and centrifuging at 4 ℃ for 10min at 10000 × g, filtering the supernatant with Whatman No. 4 filter paper to obtain sarcoplasmic protein extract, the SDS-PAGE analysis of the sarcoplasmic protein extract is the same as that of example 3. the result is shown in FIG. 4. the sarcoplasmic protein band of the control sausage is almost unchanged before drying and is degraded to some extent after drying is finished.

3. HP L C analyzes biogenic amine content of Sichuan sausage, namely, 5g of cut Sichuan sausage is added with 50m L7.5.5% of trichloroacetic acid solution, homogenate is carried out for 2 min, homogenate is carried out at 4 ℃ for 8000 × g and centrifuged for 10min, supernatant is collected to obtain biogenic amine extract, 1m L biogenic amine extract is absorbed, pre-column derivatization and HP L C measurement are carried out according to the method in implementation case 3, the result is shown in figure 5, the biogenic amine with the highest content in two types of sausages is tyramine, the biogenic amine, cadaverine and histamine, the content of the four biogenic amines in control sausages is 87.29 mg/kg, 40.90 mg/kg, 36.70 mg/kg and 21.88 mg/kg, the content of the four biogenic amines in inoculated sausages is 58.14 mg/kg, 40.87 mg/kg, 14.88 mg/kg and 9.68 mg/kg., the content of tryptamine, phenethylamine, histamine and the content of the biogenic amine in control sausages is lower than 6 biogenic amine in control sausagesP<0.05), which shows that the lactobacillus plantarum 1-1 inoculated to the Sichuan sausage can reduce the content of biogenic amine and improve the safety of the Sichuan sausage.

SEQUENCE LISTING

<110> Sichuan university

<120> Lactobacillus plantarum and application thereof in preparation of Sichuan sausage through fermentation

<160>1

<170>PatentIn version 3.3

<210>1

<211>1403

<212>DNA

<213> Lactobacillus plantarum (L Acobacillus plantarum)

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tcgccactgg tgttcttcca tatatctacg catttcaccg ctacacatgg agttccactg 780

tcctcttctg cactcaagtt tcccagtttc cgatgcactt cttcggttga gccgaaggct 840

ttcacatcag acttaaaaaa ccgcctgcgc tcgctttacg cccaataaat ccggacaacg 900

cttgccacct acgtattacc gcggctgctg gcacgtagtt agccgtggct ttctggttaa 960

ataccgtcaa tacctgaaca gttactctca gatatgttct tctttaacaa cagagtttta 1020

cgagccgaaa cccttcttca ctcacgcggc gttgctccat cagactttcg tccattgtgg 1080

aagattccct actgctgcct cccgtaggag tttgggccgt gtctcagtcc caatgtggcc 1140

gattaccctc tcaggtcggc tacgtatcat tgccatggtg agccgttacc ccaccatcta 1200

gctaatacgc cgcgggacca tccaaaagtg atagccgaag ccatctttca agctcggacc 1260

atgcggtcca agttgttatg cggtattagc atctgtttcc aggtgttatc ccccgcttct 1320

gggcaggttt cccacgtgtt actcaccagt tcgccactca ctcaaatgta aatcatgatg 1380

caagcaccaa tcaataccag agt 1403

Claims

1. Lactobacillus plantarum (Lactobacillus plantarum) for preparing Sichuan sausage by fermentationLactobacillus plantarum) 1-1, characterized in that: the lactobacillus plantarum is preserved in the general microbiological center of China Committee for culture Collection of microorganisms (CGMCC) in 2018, 3 and 20, and the preservation number is CGMCC NO. 15467.

2. Lactobacillus plantarum (L) according to claim 1Lactobacillus plantarum) 1-1 in the application of preparing Sichuan sausage by fermentation, which is characterized in that: mincing 10 kg pork raw material with weight-lean ratio of 1: 4, adding 250 g edible salt, stirring, and pickling at 4 deg.C for 4 hr; adding 100 g of white granulated sugar, 100 g of white spirit, 100 g of pepper powder and 200 g of chili powder into the pickled sausage meat slices, and uniformly stirring; according to 10⁷Inoculating the bacterial suspension of the lactobacillus plantarum 1-1 into the meat material according to the inoculation amount of cfu/g, and uniformly mixing; filling meat material into small intestine of pig with a sausage filler, cleaning dirt on the surface of the intestine with clear water, placing in a constant temperature incubator, fermenting at 20 deg.C for 12 hr, and controlling relative humidity at 60-90%; after fermentation, ripening for 4 days at 13 ℃ and the relative humidity is 50-70%; and finally, placing the mature sausage in an oven, and drying for 24 hours at 55 ℃ to obtain the Sichuan sausage finished product.

3. Lactobacillus plantarum (L) according to claim 2Lactobacillus plantarum) 1-1 in the application of preparing Sichuan sausage by fermentation, which is characterized in that: the bacterial suspension is prepared by suspending the thallus of lactobacillus plantarum 1-1 in sterile normal saline; the specific method comprises the following steps: the bacterial liquid of the lactobacillus plantarum 1-1 after twice subculture is addedCentrifuging at 8000 × g at 4 deg.C for 10min, collecting thallus, mixing with sterile physiological saline, and adjusting the concentration of the suspension to 10 by cytometry on a hemocytometer⁹cfu/m L.