CN113373097A

CN113373097A - Lactobacillus plantarum and application thereof

Info

Publication number: CN113373097A
Application number: CN202110849825.0A
Authority: CN
Inventors: 杨勇; 王艺伦; 裴慧洁; 邓霖; 兰沁洁; 刘雨萱; 黄晓红; 敖晓琳; 刘书亮
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2021-07-27
Filing date: 2021-07-27
Publication date: 2021-09-10
Anticipated expiration: 2041-07-27
Also published as: CN113373097B

Abstract

The invention provides a lactobacillus plantarum and application thereof, wherein the lactobacillus plantarum is named as: LPZN19, preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO: 22770. the lactobacillus plantarum is used as a food starter to prepare fermented food, such as Sichuan-style sausage, and can degrade biogenic amine in the Sichuan-style sausage, wherein the degradation rate of the biogenic amine in the total biogenic amine is 28.79% when fermentation is completed (30d), and the degradation rates of histamine, tyramine, cadaverine, putrescine, tryptamine, phenethylamine and spermidine reach 41.95%, 24.07%, 54.19%, 10.76%, 24.90%, 8.07% and 5.3%, respectively. The lactobacillus plantarum is obtained by screening fermented sausages and has excellent probiotic characteristics, so the lactobacillus plantarum is particularly suitable for production of fermented meat products such as preserved meat, ham and the like, and has important practical significance for research and development of functional lactobacillus leavening agents, establishment of lactobacillus strain resource libraries and solving of food safety problems to a certain extent.

Description

Lactobacillus plantarum and application thereof

Technical Field

The invention belongs to the technical field of microbial fermented foods, and particularly relates to lactobacillus plantarum and application thereof.

Background

The Sichuan flavor sausage is one of the traditional special fermented meat products in Sichuan, usually takes hot pepper, pepper and pepper (three peppers) as auxiliary materials, generally needs to be fermented and smoked, has the characteristic of heavy spicy flavor, is named as spicy sausage, and is deeply loved by consumers due to strong aroma and delicious taste.

The fermentation process of Sichuan flavor sausage mainly depends on the action of microorganisms in the environment and raw meat, and the microorganisms can produce Biogenic Amines (BAs). BAs are biologically active, small molecular weight nitrogen-containing organic compounds formed by decarboxylation of amino acids or amination of aldehydes and ketones. In the fermented sausage, common BAs mainly comprise histamine, tyramine, cadaverine, putrescine, tryptamine, phenylethylamine and spermidine, wherein the histamine has the strongest toxicity and the highest content and has the greatest harm to human bodies. BAs are precursors of hormones, alkaloids, nucleic acids and protein synthesis, and are essential for maintaining normal visceral function and metabolic activity of the immune system. Biogenic amines play an important role in bioactive cells, can promote growth, enhance metabolic activity, enhance intestinal system immunocompetence, control blood pressure, eliminate free radicals and other physiological functions, and play an active role in a nervous system. However, excessive biogenic amines not only cause harm to human health, resulting in enlargement of blood vessels, arteries and capillaries, such as hypertension, headache, abdominal cramps, diarrhea and vomiting, but also cause damage to human heart and central nervous system, etc., and also affect flavor of the product. Therefore, it is necessary to control the biogenic amine content in the fermented sausages.

Disclosure of Invention

Aiming at the problems of fermentation of the existing Sichuan flavor sausage, the invention provides a Lactobacillus plantarum capable of efficiently degrading biogenic amine and application thereof, wherein the Lactobacillus plantarum (Lactobacillus plantarum) is named as: LPZN19, preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO: 22770. and (4) storage address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North. The preservation date is as follows: the lactobacillus plantarum is applied to fermentation of Sichuan flavor sausages in 2021, 6 months and 24 days, and the degradation rate of total biogenic amine can reach more than 28%. The technical scheme of the invention is as follows:

in a first aspect, the present invention provides a strain of lactobacillus plantarum, designated: LPZN19, preserved in China general microbiological culture Collection center, with the preservation number of CGMCC NO: 22770.

further, the lactobacillus plantarum has a biogenic amine degrading property.

In a second aspect, the present invention provides a food leavening agent comprising the above lactobacillus plantarum, pediococcus pentosaceus and staphylococcus.

Further, the preparation method of the food leavening agent comprises the following steps:

(1) strains of lactobacillus plantarum, pediococcus pentosaceus and staphylococcus were treated in a cell number of 1: 1: 1, uniformly mixing, and activating in a liquid culture medium at 37 ℃ for 3-5 times, wherein each time lasts for 8-12 hours;

(2) and (4) centrifuging the activated bacterial solution for 10-15min at the temperature of 4 ℃ and under the condition of 4000-.

In a third aspect, the invention provides the use of the above mentioned leavening agent in the preparation of a fermented food product.

Preferably, the fermented food is a fermented meat food.

Further, the fermented meat food comprises Sichuan-style sausage, preserved meat and ham.

Further, the application of the fermented meat food comprises the following steps: and adding the leaven into the pickled raw materials for fermentation.

Preferably, the control parameters of the Sichuan flavor sausage fermentation are as follows: the fermentation temperature is 20-22 ℃, the fermentation relative humidity is 74-78%, and the fermentation time is 2-3 d; the ripening temperature is 12-14 ℃, the relative humidity of ripening is 60-70%, and the ripening time is 28-30 d.

In a fourth aspect, the invention provides a Sichuan-style sausage, which is obtained by adopting the application method.

Compared with the prior art, the beneficial effects of the invention are summarized as follows, and further proved in the specific examples:

the experimental results show that: the lactobacillus plantarum can degrade biogenic amine in the Sichuan flavor sausage, the degradation rate of the biogenic amine in the fermentation process (30d) is 28.79%, and the degradation rates of histamine, tyramine, cadaverine, putrescine, tryptamine, phenethylamine and spermidine reach 41.95%, 24.07%, 54.19%, 10.76%, 24.90%, 8.07% and 5.3% respectively. The lactobacillus plantarum is obtained by screening fermented sausages and has excellent probiotic characteristics, so the lactobacillus plantarum is particularly suitable for production of fermented meat products such as preserved meat, ham and the like, and has important practical significance for research and development of functional lactobacillus leavening agents, establishment of lactobacillus strain resource libraries and solving of food safety problems to a certain extent.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is the color development result of the culture medium after separation and purification of the amine degrading lactic acid bacteria in example 1 of the present invention, wherein FIG. 1-1 is the color development result of the non-amine degrading lactic acid bacteria, and FIG. 1-2 is the color development result of the amine degrading lactic acid bacteria.

FIG. 2 is an HPLC chromatogram of a mixed standard of biogenic amines in example 1 of the present invention.

FIG. 3 is an electrophoretogram of a PCR product of 16S rDNA of the strain isolated in example 1 of the present invention.

FIG. 4 is the growth ability of Lactobacillus plantarum obtained in example 1 of the present invention.

FIG. 5 shows acid productivity of Lactobacillus plantarum obtained in example 1 of the present invention.

FIG. 6 shows the salt tolerance of Lactobacillus plantarum obtained in example 1 of the present invention.

FIG. 7 shows the nitrite resistance of Lactobacillus plantarum obtained in example 1 according to the invention.

FIG. 8 is the growth capacity at different pH values of Lactobacillus plantarum obtained in example 1 according to the invention.

FIG. 9 is the growth capacity of Lactobacillus plantarum obtained in example 1 according to the invention at different temperatures.

FIG. 10 shows the change of histamine content of Chuanwei sausage processed by Lactobacillus plantarum in example 2 of the present invention.

FIG. 11 shows the change of tyramine content in the processing of Sichuan style sausage by Lactobacillus plantarum in example 2 of the present invention.

FIG. 12 shows the change of cadaverine content in the processing of Sichuan flavor sausage by Lactobacillus plantarum in example 2 of the present invention.

Fig. 13 shows the change of the putrescine content in the processing process of the Sichuan flavor sausage by lactobacillus plantarum in example 2 of the present invention.

FIG. 14 shows the change of tryptamine content in the processing of Sichuan flavor sausage by Lactobacillus plantarum in example 2 of the present invention.

Fig. 15 shows the change of the phenylethylamine content in the processing process of the Sichuan flavor sausage by lactobacillus plantarum in example 2 of the invention.

FIG. 16 shows the change of spermidine content in the processing of Sichuan flavor sausage by Lactobacillus plantarum in example 2 of the present invention.

Detailed Description

The initial meat sample employed in the present example was purchased from the Yaan farm trade market, Sichuan.

In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.

Example 1:

screening and identification of biogenic amine degrading bacteria

1.1 separation and purification of lactic acid bacteria for reducing biogenic amine

Homogenizing 25g meat sample in 225mL sterile normal saline, mixing, adding 1mL into MRS broth culture medium for enrichment culture, and performing gradient dilution (from 10%) with 0.8% normal saline^-1To 10^-7) 200 mu L of the dilution solution is taken and spread on an MRS solid culture medium containing 0.8 percent of calcium carbonate to be cultured for 24h, and strains with calcium-dissolving rings are selected to be developed on a chromogenic medium producing biogenic amine, and each appropriate dilution is carried out 3 times in parallel. As shown in FIG. 1, purple bacteria are amine-producing bacteria and yellow bacteria are non-amine-producing bacteria within 5min, and the color is developed twice in parallel. Separating and purifying the strain which shows yellow twice on an MRS culture medium for 3 times, and storing the strain on a slope at 4 ℃ for later use.

1.2 preliminary screening of the Strain

Lactic acid bacteria capable of being used as leaven for fermenting meat products should tolerate 6% NaCl and 150mg/kg NaNO₂Strong acid production capacity, no gas production of fermented glucose and no H production₂O₂、H₂S, ammonia and mucus. Because the protein is a precursor substance of the biogenic amine, the strain also has no obvious decomposition effect on the protein, and in addition, the strain with oxidase activity can degrade the biogenic amine. Accordingly, the following screening experiments were performed: salt resistance test, nitrite resistance test, viscosity production test, glucose gas production test and H production test₂S test, H₂O₂The test method comprises the following steps of testing, catalase testing, pigment production testing, arginine ammonia production testing, V-P testing, litmus milk testing, nitrate reduction testing, protein degradation activity testing and oxidase activity testing, wherein the tests are conventional tests in the field, and various culture medium and reagent formulas of a primary screening test are only given in the invention as follows:

MRS liquid medium: 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate tetrahydrate, 2g of dipotassium hydrogen phosphate, 2g of ammonium citrate, 5g of sodium acetate, 801 mL of tween-801, pH 6.2-6.4, and high-pressure steam sterilization at 121 ℃ for 20 min.

(1) Salt-resistant experimental culture medium

MRS liquid medium was supplemented with 20, 40 and 60g/L NaCl, respectively.

(2) Nitrite-resistant experimental culture medium

Adding 50, 100 and 150mg/kg NaNO into MRS liquid culture medium respectively₂。

(3) Mucus production culture medium

MRS solid medium, replacing glucose with 5% sucrose.

(4) Glucose gas production culture medium

MRS liquid medium, ammonium citrate with ammonium sulfate replacement, add 1.4mL of 1.6% bromocresol purple and inverted Du's tubule.

(5) Produce H₂S medium

10g of peptone, 5g of sodium chloride, 10g of beef extract, 5mL of 10% ferrous chloride, 1000mL of distilled water and pH 7.0-7.4.

(6)H₂O₂Production of assay Medium

Basic culture medium: beef extract 0.5g, yeast extract 0.5g, Tween-800.05 mL, MnSO₄0.01g, agar 1.5g, pH 6.5, 121 ℃ 15 min. Sterile sugar 1% separately sterilized and pour plate. Pouring a thin layer (1-2 ml) of the same culture medium on the upper layer, adding 4% MnO₂. Fresh culture in H₂O₂Test medium was streaked and cultured for 5 days. The colony periphery was observed and clarified to be H₂O₂And (4) positive.

(7) Catalase assay

And picking colonies on the solid culture medium by using an inoculating loop, placing the colonies in a clean test tube, dropwise adding 2mL of 5% hydrogen peroxide solution, and observing the result. The cell was positive in the case of generating air bubbles within half a minute, and negative in the case of not generating air bubbles.

(8) Detection of pigmentation

Sterilized whole milk (30%) was added to the medium, and a fresh culture strain was inoculated, cultured at 37 ℃ for 24 hours, and then cultured at room temperature for 3 days to observe the presence or absence of pigment production.

(9) Arginine ammonia-producing culture medium

MRS medium (without beef extract, 0.2% sodium citrate instead of ammonium citrate, glucose concentration 0.05%), 3% arginine, 1mL of 0.006% bromocresol purple, 1000mL of distilled water, pH 5.3. Neel's reagent A solution: 20g of potassium hydroxide and 50mL of distilled water; and B, liquid B: 5g of potassium iodide, 10g of mercury iodide and 50mL of distilled water. Mixing the solution A and the solution B, filtering, and reserving in a brown bottle for later use.

(10) V-P reaction medium

Solution A: 5g of alpha-naphthol and 100mL of 95% ethanol; and B, liquid B: 80g of potassium hydroxide, 0.6g of creatine and 200mL of distilled water. After 0.6mL of solution A and 0.2mL of solution B were added to 1mL of the culture medium, the mixture was shaken to show a positive red color.

(11) Litmus milk culture medium

4mL of 2.5% litmus alcohol solution and 100mL of skim milk. Mixing to obtain lilac purple, subpackaging in test tubes, sterilizing at 121 ℃ for 20-30 min, wherein the milk height is 4 cm.

(12) Nitrate reduction medium

10g of beef extract, 5g of peptone and KNO₃1g, pH 7.0-7.6, and sterilizing at 121 ℃ for 15-20 min. Grignard reagent: solution A: 0.5g of sulfanilic acid and 150mL of dilute acetic acid (10%); and B, liquid B: 0.1g of alpha-naphthol, 150mL of dilute acetic acid (10%) and 20mL of distilled water. Diphenylamine reagent: 0.5g of diphenylamine, 100mL of concentrated sulfuric acid and 20mL of distilled water.

(13) Protein degradation activity test medium

Adding 15% of skim milk or skim milk powder on the basis of MRS solid culture medium.

(14) Oxidase test

The negative bacteria are subjected to oxidase activity test, and the experimental method comprises the following steps: taking white clean filter paper to dip a colony, adding a drop of 1% dimethyl p-phenylenediamine hydrochloride solution to obtain a pink positive bacteria which is gradually deepened, adding a drop of 1% alpha-naphthol ethanol solution to obtain a blue positive bacteria within half a minute and a non-discoloring negative bacteria within 2min, and obtaining a primary screening strain, wherein the primary screening result is shown in table 1:

TABLE 1 Primary fermentation characteristics of the strains

Note: +: the reaction is positive; -: the reaction was negative.

Due to the loss of water, the concentrations of salt and nitrite can reach about 60g/L and 0.015 percent when the sausage is mature. Therefore, strains that can be used as leavening agents should tolerate 60g/L of common salt and 0.015% of nitrite. Mucus can affect the tissue state of sausage, gas production can affect the compactness of sausage structure, H₂S、 H₂O₂And NH₃And the flavor of the sausage can be influenced by the bad gases, and the sense of the sausage can be influenced by the pigment. Therefore, the starter should not produce slime, H₂S、H₂O₂、NH₃And pigments and the like. In addition, nitrite not only has a chromogenic effect, but also inhibits Clostridium botulinum, and thus the strain should also have the ability to reduce nitrate. Since the protein is a precursor of biogenic amine, it is preferable that the fermentation agent does not have a protein degradation ability, and the strain having an oxidase activity can degrade biogenic amine.

Of the 256 isolated strains, total 17 strains with oxidase activity meeting the relevant standards for leavening agents for fermented meat products, numbered 2, 4, 19, 25, 33, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, and the results of the preliminary screening are shown in Table 1. The other strains do not meet the requirements of the fermentation agent to a certain extent, have no oxidase activity and are not suitable for being used as the fermentation agent, so the 17 strains are used for subsequent experiments to measure the degradation capability of the histamine.

1.3 Re-screening of the strains

(1) Standard solution preparation and pre-column derivatization

Accurately weighing biogenic amine (histamine, tyramine, cadaverine, putrescine, tryptamine, phenethylamine and spermidine) standard substances 0.0100g respectively, diluting to 10mL with 0.1mol/L hydrochloric acid, sucking 5, 10, 20, 50, 100, 200, 500 and 1000 μ L standard solutions respectively to constant volume to 10mL, sequentially adding 200 μ L2 mol/L NaOH and 300 μ L saturated NaHCO 1 to 1mL respectively₃Mixing with 2mL of 10mg/mL dansyl chloride acetone solution, dark reacting at 40 deg.C for 45min, and adding 100 μ L of NH₄OH remove residual dansyl chloride, add acetonitrile to 5mL and filter through 0.45 μm filter for labeling, as shown in FIG. 2.

(2) The preparation method of the 17 samples to be tested comprises the following steps: separating and purifying the primary screened strain for 3 times, activating the primary screened strain in an MRS liquid culture medium for 3 times, washing the primary screened strain for 2 times by using 0.05mol/L phosphate with the pH value of 7.0, collecting thalli, inoculating the thalli into PBS buffer solution (0.02M, Ph7.0) containing 300 mu g/mL biogenic amine (histamine, tyramine, cadaverine, putrescine, tryptamine, phenethylamine and spermidine), culturing the thalli at 37 ℃ for 48 hours, taking 1mL sample, adding 1mL of 0.1mol/L hydrochloric acid, and then testing the sample in a refrigerator at-20 ℃. A blank was prepared from 1mL of PBS buffer containing 300. mu.g/mL of biogenic amine. Samples were run in triplicate and averaged. The pre-column derivatization step is the same as (1).

(3) Chromatographic conditions

The chromatographic column is C₁₈(4.6 mm. times.250 mm,5 μm), a flow rate of 1mL/min, an ultraviolet detection wavelength of 254nm, a sample introduction amount of 10 μ L, a column temperature of 30 ℃, a mobile phase A of ultrapure water, a mobile phase B of acetonitrile, and a gradient elution program shown in Table 2.

TABLE 2 gradient elution procedure

Tables 3 and 4 also provide seven biogenic amine regression equations and coefficients of determination as well as recovery test results.

TABLE 3 regression equation and coefficient of determination for seven biogenic amines

TABLE 4 recovery test results

Table 5 provides the results of the degradation of biogenic amines by 17 strains.

TABLE 5 degradation rate of biogenic amines by the strains

After the 17 strains are screened again, strains (19, 41, 46, 47 and 48) with the highest total biogenic amine degradation rate of 5 are selected for subsequent strain identification.

1.4 identification of the Strain

(1) Morphological characterization

Colony morphology: the morphology (shape, color, etc.) of the colonies isolated was observed and the results were recorded.

Strain morphology: the isolated strain was gram-stained, and the morphology (size, shape, etc.) of the cells was observed under a 100-fold oil-scope, and the observation results were recorded. The results of morphological identification of the strains and colonies are shown in Table 6.

TABLE 6 identification of strain morphology and colony morphology

(2) Physiological and biochemical characterization

And (3) performing physiological and biochemical identification on the 5 screened strains by using a micro-fermentation tube, and analyzing physiological and biochemical characteristics of the strains by referring to identification standards of lactic acid bacteria. The results are shown in Table 7.

TABLE 7 physiological and biochemical identification results of the strains

Note: +: the reaction is positive; -: the reaction is negative; /: not detected.

(3)16S rDNA genetic identification

Culturing the separated strain in MRS liquid culture medium at 37 ℃ for 24h, extracting DNA according to the instruction of a bacterial genome DNA extraction kit of Tiangen company, detecting by 1% agarose gel electrophoresis, and carrying out PCR amplification. The selected primers are universal primers: the forward primer was 27f (5'-AGA GTT TGA TCC TGG CTC AG-3') and the reverse primer was 1492r (5'-TAC GGC TAC CTT GTT ACG ACT T-3'). The PCR reaction was a 25. mu.L system: includes 1 μ L of template DNA, 1 μ L of each of the upstream primer and the downstream primer, 12.5 μ L of 2 XTaq Master MIX, and 9.5 μ L of sterile double distilled water. PCR reaction procedure: pre-denaturation at 95 ℃ for 5min, 30 cycles (denaturation at 95 ℃ for 60 s; annealing at 55 ℃ for 90 s; extension at 72 ℃ for 2 min), and final extension at 72 ℃ for 10 min. The PCR product was detected by 1% agarose gel electrophoresis, sequenced by Duckoku, and compared with known sequences in NCBI databases.

DNA of 5 strains was extracted using the kit, 16S rDNA of 5 strains was amplified with universal primers, and 5 amplified products of about 1500bp length were obtained after electrophoresis in 1% agarose gel, as shown in FIG. 3. Lane 1 is Marker DL 2000, and

lanes

2, 3, 4, 5, and 6 are PCR products of

strains

19, 41, 46, 47, and 48, respectively. The results of the PCR sequencing were compared with known sequences in the NCBI database and are shown in Table 8. Finally, it was judged that 19 th was Lactobacillus plantarum (Lactobacillus plantarum), 41 th was Pediococcus pentosaceus (Pediococcus pentosaceus), 46 th was Weissella confusa (Weissella convusa), and 47 th and 48 th were Enterococcus faecium (Enterococcus faecalis), and the results are shown in Table 8.

TABLE 8 identification results of the strains in molecular biology

As the enterococcus faecium may have drug resistance, the three safe and food-borne lactic acid bacteria of No. 19, No. 41 and No. 46 are selected for subsequent research according to the strain identification result.

1.5 biological Properties of the Strain

(1) Determination of growth ability and acid production ability of strain

The strains No. 19, 41 and 46 were inoculated into MRS liquid medium, cultured at 37 ℃ for 24 hours, and absorbance and pH at 600nm were measured every 2 hours using a spectrophotometer and a pH meter, and the corresponding blank medium was used as a control, and 3 times in parallel were averaged. The bacterial strain which can be used as the sausage starter needs to have stronger growth capability and acid production capability. As can be seen from FIGS. 4 and 5, the growth capacity and acid production capacity of the three strains are 19 th strains higher than 41 th strains and 46 th strains, and the pH value of the culture medium after 24h culture is lower than 5.0, which indicates that the three strains have good acid production capacity and meet the basic requirements for producing fermented sausages.

(2) Determination of salt resistance and nitrite resistance of strain

Strains No. 19, 41 and 46 were inoculated into MRS liquid media containing 0, 20, 40, 60, 80g/L NaCl and 0, 50, 100, 150mg/kg NaNO2, respectively, and cultured at 37 ℃ for 24 hours, and the absorbance at 600nm was measured with a spectrophotometer, and blank media was used as a control, and 3-fold averaging was performed. The strain which can be used in the sausage starter needs to be able to tolerate salt at a concentration of 60g/L and nitrite at a concentration of 150 mg/kg. As can be seen from FIGS. 6 and 7, when the salt concentration is lower than 60g/L, the three strains can grow, and when the concentration is increased to 80g/L, the OD values of the three strains after 24h culture are all lower than 0.2, because the higher salt concentration increases osmotic pressure and inhibits the growth activity of the strains; the nitrite concentration is within the range of 0-200 mg/kg, and the three strains can grow well. The results show that the three strains can tolerate salt and nitrite with certain concentration, and meet the basic requirements of the leaven for producing the fermented sausage.

(3) Determination of growth ability of strains at different temperatures and acidity

The strains No. 19, 41 and 46 are inoculated into MRS liquid culture medium, cultured for 24h under the conditions of 10 ℃, 20, 30, 40 and 50 ℃ respectively, the absorbance value at 600nm is measured by a spectrophotometer, and a corresponding blank culture medium is used as a control, and the average value is taken for 3 times in parallel. Inoculating the strain into MRS liquid culture medium with pH of 3.5, 4.5, 5.5, 6.5, 7.5, culturing at 37 deg.C for 24 hr, measuring absorbance at 600nm with spectrophotometer, comparing with corresponding blank culture medium, and averaging 3 times in parallel. The fermentation and maturation temperature of the sausage is usually between 10 and 20 ℃, and the pH value of the sausage is usually between 5.2 and 5.6 when the sausage is mature. As can be seen from FIGS. 8 and 9, all three strains can grow at a certain low temperature and low pH value, and can meet the requirements of sausage fermentation.

In conclusion, the 19 bacteria not only have stronger biogenic amine degradation capability, but also have stronger growth capability, acid production capability and salt and sodium nitrite tolerance than other two bacteria, so the 19 bacteria are selected to be prepared into microbial inoculum for preparing the Sichuan flavor sausage. The strain is named as: LPZN19, preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO: 22770 Lactobacillus plantarum.

Example 2:

the application of the lactobacillus plantarum 19 of example 1 in the preparation of Sichuan-style sausage comprises the following specific steps:

2.1 processing recipe

8kg of beef, 2kg of pig fat, 0.25kg of salt,0.1kg of white granulated sugar, 0.1kg of chilli powder, 0.04kg of pepper powder, 0.005kg of thirteen-spices, 0.015kg of monosodium glutamate, 0.01kg of glucose, 0.005kg of garlic powder, 0.002kg of black pepper and NaNO₂0.00075kg and 0.1kg of white spirit.

2.2 processing operating points

Raw meat: removing skin, bone and connective tissue of beef, and cleaning with clear water;

stranding: precooling raw meat at 4 ℃, putting the beef and the pig fat together into a sausage slicer for slicing;

pickling: adding a salt curing agent, and curing for 4h at 4 ℃;

stirring: adding spices and auxiliary materials into the pickled meat;

inoculating a leavening agent: inoculation concentration of 10⁷CFU/g, the leaven is prepared by mixing lactobacillus plantarum, pediococcus pentosaceus and staphylococcus;

performing clysis: filling the minced meat into a small intestine casing of a pig with the diameter of about 28mm, wherein the small intestine casing is compact and full in a section of 10-12 cm, and exhausting air by an exhaust needle;

fermentation: wiping off oil stains on the surface of the sausage casing by using a clean rag, transferring the sausage casing into a fermentation box, uniformly airing and hanging the sausage casing, and fermenting under certain temperature and humidity conditions;

and (3) maturing: ripening under certain temperature and humidity conditions;

and (3) drying: and (4) transferring the sausage into an oven to be dried at a certain temperature, and taking out the finished product of the fermented sausage.

2.3 preparation of the leaven

Strains of lactobacillus plantarum, pediococcus pentosaceus and staphylococcus were treated in a cell number of 1: 1: 1, uniformly mixing, and activating for 5 times in a liquid culture medium corresponding to 37 ℃, wherein each time is 12 hours; centrifuging the activated bacteria liquid at 4000rpm at 4 deg.C for 15min, washing with physiological saline for 1 time, centrifuging, collecting thallus, diluting to obtain leaven, counting by hemacytometer method, and inoculating 10% leaven⁷CFU/g。

2.4 Collection of samples

Grouping experiments: group A is a control group (without starter) and group B is a inoculation group (with starter). Sampling 10 process points in the sausage fermentation process, namely 0 (raw meat), 2, 5, 8, 12, 15, 18, 21, 24, 27 and 30 d.

2.5. Biogenic amine content determination

5g of meat was homogenized by adding 20mL of 0.1mol/L hydrochloric acid, centrifuged at 4 ℃ at 4000g for 10min, and the precipitate was extracted once more as before. Taking the supernatant liquid for 2 times, using 0.1mol/L hydrochloric acid to fix the volume to 50mL, taking 1mL of sample liquid to carry out pre-column derivatization and computer measurement, wherein the derivatization step is the same as that of 1.3(1), and the chromatographic condition is the same as that of 1.3 (3).

2.6. Statistical analysis

Experimental data were processed and plotted by Excel software. The results are shown in FIGS. 10-16.

Biogenic amine degradation rate (%) - (W)₀-W₁)*100/W₀

(W₀The content of biogenic amine in the control is mu g/mL; w₁For inoculation of biogenic amine content, μ g/mL)

2.7. Results and analysis

2.7.1 degradation ability of Lactobacillus plantarum on biogenic amine in Sichuan-style sausage

2.7.1.1 Effect of Lactobacillus plantarum on Histamine in Sichuan flavor sausage processing

As can be seen from fig. 10, the histamine content of the group a and the group B tended to increase as the ripening proceeded during the processing of the sikawa sausage. Histamine is a biogenic amine with the highest toxicity, and excessive intake of histamine causes hypertension, diarrhea, and the like. The U.S. FDA limits the biogenic amines in fish to 50 mg/kg; the limit of histamine in the temporarily fermented meat products of food and drug administration in Shanghai is lower than 100 mg/kg. The content of histamine in the raw meat is very low; the histamine content of the sausages in the two groups of fermented 2d, A, B is obviously increased, which is probably due to the fact that histidine in raw meat forms histamine under the action of histidine decarboxylase; the histamine content in the sausages of group a was significantly lower than in group B, probably due to the growth inhibition of histamine-producing microorganisms by the starter inoculum of group a, thereby inhibiting histamine formation. During the maturation process, the increase of histamine in Sichuan sausage in group A is significantly lower than that in group B, which shows that the inoculated leaven has stronger histamine metabolic enzyme activity, thereby reducing the accumulation of histamine; the histamine content of the sausage in the group A is 93.50mg/kg after the sausage is matured for 30 days, the histamine content of the sausage in the group B is 161.08mg/kg, the histamine content of the sausage in the group A is lower than 100mg/kg, the sausage in the group A is matured for 30 days in a safer range, the group A is obviously lower than the sausage in the group B, the degradation rate reaches 41.95 percent, and the degradation effect of the leavening agent on the histamine is obvious.

2.7.1.2 influence of Lactobacillus plantarum on tyramine in Sichuan flavor sausage processing process

As can be seen from FIG. 11, the tyramine content tends to increase throughout the processing of the Sichuan style sausage. As with histamine, only a small amount of tyramine was detected in the raw meat; during fermentation 2d, the tyramine content in the sausages in both groups increased significantly, probably due to the tyrosine decarboxylase itself present in the raw meat to form tyramine. In the early maturation stage (5-24 d), the difference of the tyramine content of the group A and the group B is not obvious, which is probably caused by the weak degradation capability of the starter inoculated in the group A to the tyramine; at the late maturation stage (27-30 d), the tyramine content in group B is significantly increased, probably because more tyrosine is generated by protein degradation, resulting in tyramine accumulation. When the sausage is matured for 30 days, the tyramine content in the sausage group A is 95.18mg/kg, the tyramine content in the sausage group B is 125.35mg/kg, and the degradation rate is 24.07 percent, which indicates that the leaven has a certain degradation effect on the tyramine.

2.7.1.3 Effect of Lactobacillus plantarum on cadaverine in Sichuan flavor sausage processing

As can be seen in fig. 12, there was a tendency for the cadaverine content of group a and group B to increase during the processing of the sichuan sausages. The cadaverine can be used as an evaluation index of the freshness of the meat, and the cadaverine content in the raw meat is very low, which indicates that the raw meat is relatively fresh. Fermentation 2d, the cadaverine content increased significantly in both groups of sausages, probably due to decarboxylation of the amino acids produced by the degradation of the proteins at higher fermentation temperatures to form cadaverine. During the maturation process, the cadaverine content of the sausages in group B showed a significant trend of rising, which is probably due to the fact that during the maturation process, proteins are continuously degraded to generate lysine, and the lysine forms cadaverine under the action of lysine decarboxylase, so that the cadaverine is greatly accumulated; the group A has a slow increasing trend, because the activity of lysine decarboxylase is inhibited by the inoculated leaven, so that the accumulation of cadaverine is inhibited, and the degradation effect of the leaven on the cadaverine is obvious. After the fermentation liquid is matured for 30 days, the cadaverine content (36.57mg/kg) of the group A is obviously lower than that of the group B (79.83mg/kg), the degradation rate reaches 54.19%, and the degradation effect of the fermentation agent on the cadaverine is obvious.

2.7.1.4 Effect of Lactobacillus plantarum on putrescine in Sichuan flavor sausage processing

As can be seen from fig. 13, the content of putrescine in group a and group B gradually increased during the processing of the chuanwei sausage. After 2d of fermentation, the putrescine content in the two groups of sausages is not increased remarkably, which is probably because the raw material meat contains less precursor substances of putrescine and less amino acid is generated by protein degradation in a short time; in the early stage of maturation (2-12 d), the content of putrescine in the group A and the group B is slowly increased, which is probably because the degradation reaction of protein in the early stage is not obvious; in the late maturation stage (12-30 d), the putrescine content in groups A and B increased more and more, probably due to the increase of ornithine and arginine generated by protein degradation. When the fermented liquid is matured for 30 days, the putrescine content (57.98mg/kg) of the group A is lower than that of the group B (64.97mg/kg), and the degradation rate is 10.76%, which indicates that the leavening agent has a certain degradation effect on putrescine.

2.7.1.5 influence of Lactobacillus plantarum on tryptamine in Sichuan flavor sausage processing process

As can be seen from fig. 14, the tryptamine contents of group a and group B gradually increased throughout the processing of the chuanwei sausage. In fermentation 2d, the tryptamine content in the Sichuan sausages in group A and group B was significantly increased, but the difference between the two groups was not significant, probably due to the presence of a large amount of tryptophan in the raw meat itself, and the higher fermentation temperature favoured the decarboxylation of tryptophan by tryptophan decarboxylase to form a large amount of tryptamine. In the early maturation stage (2-18 d), the tryptamine content in the Sichuan sausages in the group A and the group B is in a slow increasing trend, which is probably because less tryptophan is generated by the degradation of proteins in the early maturation stage; in the late maturation period (18-30 d), the tryptamine content in the Sichuan sausages in group A and group B is obviously increased, which is probably because the tryptophan content is continuously increased and the microbial growth is inhibited by low moisture, and the content of group A is lower than that of group B, which is probably because the inoculated leavening agent has tryptamine oxidase so as to degrade tryptamine. After the fermentation liquid is matured for 30 days, the tryptamine content of the group A is 27.57mg/kg and is obviously lower than that of the group B (36.71mg/kg), and the degradation rate is 24.90%, which indicates that the leavening agent has a certain degradation effect on tryptamine.

2.7.1.6 influence of Lactobacillus plantarum on phenylethylamine in processing of Sichuan flavor sausage

As can be seen from fig. 15, the content of phenethylamine in group a and group B was gradually increased during the whole processing of the sichuan sausages. In fermentation 2d, the phenylethylamine content was significantly higher than that of the feed meat, probably due to the substantial accumulation of phenylethylamine due to decarboxylation of phenylalanine decarboxylase in the feed meat at higher fermentation temperatures. In the early stage of maturation (2-12 d), the content difference of phenylethylamine in the group A and the group B is not obvious, which probably is because the degradation effect of inoculated leaven on phenylethylamine is not obvious; at mature 15d, the content of phenylethylamine in group A (13.52mg/kg) is significantly lower than that in group B (18.90mg/kg), which is probably because the phenylalanine generated by the early-stage degradation of group B generates phenylethylamine under the action of microorganisms, and the result is consistent with that the content of phenylalanine in group A is higher than that in group B; in the late maturation stage (15-30 d), the content of phenylethylamine in group A and group B is in a slow increasing trend, which is probably because the high-salinity environment in the late maturation stage is not beneficial to the production of phenylalanine decarboxylase by microorganisms. After the fermentation liquid is matured for 30 days, the content of phenylethylamine in the group A is reduced by 8.07 percent compared with that in the group B, which shows that the fermentation liquid has a certain degradation effect on the phenylethylamine.

2.7.1.7 influence of Lactobacillus plantarum on spermidine in Sichuan flavor sausage processing process

As can be seen from FIG. 16, the spermidine content in the Sichuan flavor sausage tends to increase as a whole in the whole processing process, the spermidine content is 2.0-6.0 mg/kg, and the spermidine content is always at a low level. The spermidine content of mature 12d, group a and group B was significantly increased, probably due to more arginine produced by protein degradation, which further decarboxylates to spermidine; the difference in spermidine content between sausages in group a and group B was not significant during maturation, probably because spermidine was mainly present in the raw meat and could be utilized during sausage maturation. After the fermentation liquid is matured for 30 days, the content of phenylethylamine in the group A is reduced by 5.3 percent compared with that in the group B, which shows that the leavening agent has a certain degradation effect on spermidine.

2.8 influence of Lactobacillus plantarum on Total Amines during Sichuan style sausage processing, as shown in Table 9.

TABLE 9 Total biogenic amine amount variation during Sichuan sausage processing

Note: it shows that the difference between the data of the same line is significant; different lower case letters indicate significant differences between the data in the same column (P < 0.05).

The total amount of biogenic amine changes throughout the processing in the Sichuan style sausages are shown in Table 9. As can be seen from Table 9, the total amount of biogenic amine in sausage in both group A and group B increased during processing, and the rate of increase in group B was higher than that in group A. For fermentation 2d, the total amount of biogenic amine in group a was significantly higher than that of the raw meat (P < 0.05), probably due to the presence of more biogenic amine precursor amino acids in the raw meat; the total amount of biogenic amines in group a was significantly lower than in group B, probably due to the degradation of biogenic amines by group a starter cultures. During the maturation process, the total content of the sausage biogenic amine in the A group and the B group is in a remarkable increasing trend (P < 0.05). At the mature 30d, the total biogenic amine content of the sausages in the group A is 340.60mg/kg which is obviously lower than that in the group B (422.13mg/kg), which indicates that the inoculated leavening agent has obvious degradation effect on the total biogenic amine content (P is less than 0.05).

In conclusion, the lactobacillus plantarum provided by the invention can degrade biogenic amine in Sichuan flavor sausages, the degradation rate of total biogenic amine is 28.79% after fermentation is completed (30d), and the degradation rates of histamine, tyramine, cadaverine, putrescine, tryptamine, phenethylamine and spermidine reach 41.95%, 24.07%, 54.19%, 10.76%, 24.90%, 8.07% and 5.3% respectively. The lactobacillus plantarum is obtained by screening fermented sausages and has excellent probiotic characteristics, so the lactobacillus plantarum is particularly suitable for production of fermented meat products such as preserved meat, ham and the like, and has important practical significance for research and development of functional lactobacillus leavening agents, establishment of lactobacillus strain resource libraries and solving of food safety problems to a certain extent.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A lactobacillus plantarum strain is characterized in that: is named as: LPZN19, preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO: 22770.

2. the lactobacillus plantarum of claim 1, wherein: the lactobacillus plantarum has the property of degrading biogenic amines.

3. A food leaven, which is characterized in that: comprising the Lactobacillus plantarum, Pediococcus pentosaceus and Staphylococcus according to claim 1 or 2.

4. The food leavening agent of claim 3, wherein: the preparation method of the food leavening comprises the following steps:

5. Use of the food ferment of claim 3 or 4 for the preparation of fermented food products.

6. Use according to claim 5, characterized in that: the fermented food is fermented meat food.

7. Use according to claim 6, characterized in that: the fermented meat food comprises Sichuan sausage, preserved meat and ham.

8. Use according to claim 7, characterized in that: the application of the fermented meat food comprises the following steps: and adding the leaven into the pickled raw materials for fermentation.

9. Use according to claim 8, characterized in that: the control parameters of the fermentation of the Sichuan flavor sausage are as follows: the fermentation temperature is 20-22 ℃, the fermentation relative humidity is 74-78%, and the fermentation time is 2-3 d; the ripening temperature is 12-14 ℃, the relative humidity of ripening is 60-70%, and the ripening time is 28-30 d.

10. A Sichuan flavor sausage is characterized in that: is obtained by the application method of any one of claims 5 to 9.