CN104164384A

CN104164384A - Lactobacillus curvatus and application thereof in reducing nitrosamine content in air-dried sausage

Info

Publication number: CN104164384A
Application number: CN201410283158.4A
Authority: CN
Inventors: 孔保华; 张宏伟; 李木子; 牛海力; 夏秀芳
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2014-06-23
Filing date: 2014-06-23
Publication date: 2014-11-26
Anticipated expiration: 2034-06-23
Also published as: CN104164384B

Abstract

The invention relates to lactobacillus curvatus and an application thereof in reducing nitrosamine content in an air-dried sausage, and belongs to the field of microbial technology. An object of the invention is to investigate dynamic changes of nitrosamine in a fermentation process by applying the lactobacillus curvatus 2 having the effect of reducing nitrosamine in the air-dried sausage. The lactobacillus curvatus 2 described by the invention is a strain extracted, separated and identified from fermented yogurt, is preserved in China General Microbiological Culture Collection Center (CGMCC) on May 16th, 2014 with a preservation number of CGMCC No.9172. The address of the CGMCC is the Institute of Microbiology Chinese Academy of Sciences, NO.1 West Beichen Road, Chaoyang District, Beijing, China. A classification name of the strain is lactobacillus curvatus. The lactobacillus curvatus 2 provided by the invention can reduce the content of four nitrosamines including NDEA, NDPA, NDphA and NPIP in the air-dried sausage effectively.

Description

Lactobacillus curvatus and reduce the application of content of nitrosamines in air-dry sausage

Technical field

The invention belongs to microbial technology field, relate to a strain lactobacillus curvatus (Lactobacillus curvatus), apply it in air-dry sausage, there is the effect that reduces content of nitrosamines.

Background technology

Nitrosamine is the nitrogenous chemical substance of a class, can teratogenesis, mutagenesis and bring out cancer.In a lot of foods that the mankind take in, contain nitrosamine, also have in some food-processings and can indirectly form, as the nitrite adding in meat product can form nitrosamine in sour environment, there is good colour generation effect, antioxygenation and increase taste effect and nitrate uses in meat product, simultaneously can also suppress the growth of clostridium botulinum and many other types spoilage organism, thereby be still at present in meat product processing and be difficult to alternative added ingredients.Both at home and abroad the formation mechanism of nitrosamine is conducted in-depth research at present, reduce the harm that may cause human body by the generation approach of controlling nitrosamine.

In meat product, the formation of N-nitrosamine is a complicated process, and has many kinds of substance can affect nitrosation reaction.The initial feed that N-nitrosamine forms in meat product has nitrate, nitrite, primary amine (primary amine), secondary amine (secondary amine), tertiary amine (tertiary amine), acid amides, protein, polypeptide and amino acid, or the precursor of these materials, be converted into N-nitrosamine precursor by microbial process.

Air-dry sausage is China's traditional zymotic meat product, and it is with a long history, has wide consumption market in area northeastward because it has the feature that moisture content is low, storage period long, possess special quality and local flavor.Nitrite is one of main component in air-dry sausage curing process, rich in protein, peptide class, amino acid, amine and amide substance etc. in meat in addition, and these are all to form the main precursor substance of nitrosamine.In addition, air-dry sausage is in its fermenting process, and microbes degrade proteins, reduction pH, be beneficial to the formation of nitrosamine, increased the potential safety hazard of air-dry sausage.European countries have just introduced milk-acid bacteria in Fermented Sausages as far back as the twenties in 20th century, be applied to starter on a large scale in pickling of meat the forties, shortened the production cycle, and quality product is improved.

At present, mainly contain three classes for the control method of N-nitrosamine compound: the one, find a kind of material and can partly or completely substitute the precursor substance nitrite that can generate N-nitrosamine compound; The 2nd, find a kind of material or method and block the formation of N-nitrosamine in product; The 3rd, find a kind of method established nitrosamine in product is decomposed.Focus mostly on and adding on xitix and alpha-tocopherol for the research that in the blocking-up meat product course of processing, N-nitrosamine forms, but limited their application because solvability has limitation.Focus mostly on chemosynthesis material about the research of nitrite surrogate, this mode can not meet the requirement of people to food safety, and microbe fermentation method has obtained people's concern as a kind of safe alternative method.Myohaemoglobin (Mb) is transformed into red derivative by using microbe, and wherein most study is genus lactubacillus and Staphylococcus.In meat product, add milk-acid bacteria, make it produce lactic acid, reduce pH value, promote the reductive action of nitrite, and nitrite is decomposed, and can greatly reduce nitrate residue, react generation carcinogenic substance nitrosamine with amine thereby further reduce residual nitrite.

Summary of the invention

The object of the invention is to select a strain to there is the lactobacillus curvatus 2 that falls nitrosamine effect, applied it in air-dry sausage, investigated the dynamic change of nitrosamine during the fermentation.

Lactobacillus curvatus 2 of the present invention (Lactobacillus curvatus 2) is from fermentation yogurt, to extract the also bacterial classification of isolation identification, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC No.9172, preservation date is on 05 16th, 2014, and the Classification And Nomenclature of bacterial classification is lactobacillus curvatus (Lactobacillus curvatus).

Lactobacillus curvatus 2 in the present invention has following biological characteristics:

(1) morphological specificity: lactobacillus curvatus 2 gramstaining results are positive, bending shaft-like, become short catenation, without brood cell, without pod membrane, atrichia.

(2) colony characteristics: lactobacillus curvatus 2 is well-grown on MRS solid medium, colonial morphology is white, projection, circular, the smooth of the edge is fine and closely woven, diameter 2～3mm.Evenly muddy growth in liquid medium within.

Research by this experiment finds in lactobacillus curvatus 2 liquid medium withins, to have the ability that reduces nitrosamine, and therefore the present invention adds lactobacillus curvatus and carries out spontaneous fermentation in air-dry sausage, observes the dynamic change of nitrosamine in its fermenting process.Result shows, lactobacillus curvatus 2 provided by the invention can effectively reduce the content of NDEA in air-dry sausage, NDPA, NDphA and tetra-kinds of nitrosamine of NPIP.

Preservation information:

Depositary institution's full name: China Committee for Culture Collection of Microorganisms's common micro-organisms center;

Depositary institution is called for short: CGMCC;

Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences;

Deposit number: CGMCC No.9172;

Preservation date: on 05 16th, 2014.

Brief description of the drawings

Fig. 1 is nitrosamine hybrid standard sample detection figure (20 μ g/mL);

Fig. 2 be inoculation lactobacillus curvatus 2 substratum in nitrosamine dynamic change;

Fig. 3 be inoculation lactobacillus curvatus 2 air-dry sausage in the dimethylated dynamic change of nitrosamine;

Fig. 4 is the dynamic change of nitroso-piperidine in the air-dry sausage of inoculation lactobacillus curvatus 2;

Fig. 5 is the dynamic change of nitroso-group dipropyl amine in the air-dry sausage of inoculation lactobacillus curvatus 2;

Fig. 6 is the dynamic change of nitrosodiphenylamine in the air-dry sausage of inoculation lactobacillus curvatus 2;

In figure, 1-nitrosodimethylamine, 2-nitrosomorpholine, 3-nitrosopyrolidine, 4-nitrosodiethylamine, 5-nitroso-piperidine, 6-nitroso-group dipropyl amine, 7-nitrosoamide, 8-nitrosodiphenylamine, a-f refers to same nitrosamine, and alphabetical difference illustrates significant difference (P < 0.05).

Embodiment

Below technical scheme of the present invention is further described, but does not limit to so, every technical solution of the present invention is modified or is equal to replacement, and do not depart from the spirit and scope of technical solution of the present invention, all should be encompassed in protection scope of the present invention.

1. materials and methods

1.1 material

Pig stern meat, is purchased from large profit and sends out supermarket; The standard specimen that contains 8 kinds of nitrosamine compounds, comprise nitrosodimethylamine (NDMA), nitrosopyrolidine (NPYR), nitrosodiethylamine (NDEA), nitrosomorpholine (NMor), nitrosoamide (NDBA), nitroso-piperidine (NPIP), nitroso-group dipropyl amine (NDPA), nitrosodiphenylamine (NDphA), be purchased from Sigma company of the U.S.; Acetonitrile, German Merck company; Methylene dichloride, sodium-chlor, sulfuric acid, Sodium Nitrite, glacial acetic acid, ammonium acetate, be purchased from Daowai District, Harbin Sheng Da assay apparatus sales company.Above reagent is except acetonitrile is chromatographically pure, and all the other are analytical pure, and water is ultrapure water.

1.2 instrument and equipment

Agilent1100 high performance liquid chromatograph is equipped with quaternary gradient pump and UV-detector, Agilent company of the U.S.; K-D concentrating instrument, wet distillation device, Tianjin Sheng Miao Science and Technology Ltd.; HWS-series fixed temperature and humidity incubator, Tianjin Tai Zang Si Te Instrument Ltd..

1.3 experimental technique

1.3.1 there is the screening of falling nitrosamine ability bacterial strain in substratum

(I) MRS (Man Rogosa and Sharp) substratum

Peptone 10.0g, extractum carnis 10.0g, yeast extract 5.0g, glucose 20.0g, sodium acetate 5.0g, ammonium citrate 2.0g, tween 80 1.0mL, dipotassium hydrogen phosphate 2.0g, bitter salt 0.2g, manganous sulfate 0.05g, distilled water 1000mL, pH6.5～6.8.For the activation of milk-acid bacteria, go down to posterity.

(II) bacterial strain screening

The lactobacillus curvatus having activated 2 bacterium liquid 0.5mL are added in MRS liquid nutrient medium, and adding concentration is the mixed mark of nitrosamine of 20 μ g/mL, makes its final concentration reach 1 μ g/mL.At 37 DEG C, aerobic cultivation 12h, measures respectively content of nitrosamines in 0h and 12h substratum.Because nitrosamine has volatility, in control group, change bacterium liquid into isopyknic distilled water, under equal conditions, cultivate.Control group 0h and 12h content of nitrosamines change, i.e. the natural volatile quantity of nitrosamine in substratum.

1.3.2 the making method of Harbin air-dry sausage

(I) supplementary material:

Lean meat (pig stern meat, pig tenterloin) 4.5kg, fat meat (pig back of the body fat) 0.5kg ， Qu wine (Yuquan great Qu) 0.15kg, Sodium Nitrite 0.0045kg, glucose 0.25kg, monosodium glutamate 0.075kg, salt 0.125kg, (mixed spices comprises mixed spices 0.025kg: Chinese prickly ash, aniseed, cassia bark, cloves etc.).

(II) starter preparation

The lactobacillus curvatus having activated 2 is inoculated in MRS substratum and carries out fermentation culture, cultivate 12h for 37 DEG C.Fermented liquid is under 4 DEG C of conditions, after the centrifugal 5min of 10000g, for subsequent use.While adding starter, milk-acid bacteria final concentration is 10 ⁷cFU/g meat stuffing.

(III) air-dry sausage fabrication processing

Raw meat (is rejected lymph, muscle tendon, reticular tissue such as blood vessel) → dice (meat cubelets that 1cm is square) → pickle (salt adding and food flavouring etc.) → with starter (control group does not add) → mix filling → record → fermented air-dried.

1.3.3 the mensuration of nitrosamine

1.3.3.1 high-efficient liquid phase chromatogram condition

With reference to the method for Luo Qian etc., slightly make improvements.Employing C18 post (filler is m) chromatographic column of Hypersil ODS25 μ for Elite, 4.6mm × 250mm, binary gradient elution, and water mobile phase A is 5mmol/L ammonium acetate one acetate buffer solution (pH 4.0), organic Mobile phase B is acetonitrile.Flow velocity 1mL/min, sampling volume 20 μ L, column temperature is 35 DEG C, in 240nm place, UV-detector is measured.Concrete gradient elution program is as shown in table 1.

Table 1 elution program

Table1?elution?program

1.3.3.2 sample preparation

Sample pre-treatments is according to the method for GB/T 5009.26-1996.Take the sample after 200g chopping, be placed in the matrass of wet distillation device, add 100mL ultrapure water, 120g sodium-chlor, shakes up, and adds 40mL methylene dichloride and a small amount of ice cube in Erlenmeyer flask, carries out vapor distillation.Collect 400mL distillate, in taper receiving bottle, add the sulfuric acid (vitriol oil and water volume ratio are 1: 3) of 80g sodium-chlor and 3mL.Then transfer in the separating funnel of 500mL vibration 5min, stratification.Methylene dichloride is divided to another Erlenmeyer flask, then divide and extract water layer 3 times with 120mL methylene dichloride, merge No. 4 times extracting solution, cumulative volume is 160mL.Organic layer is transferred in K-D thickener and be concentrated into 1mL in 50 DEG C of water-baths, for subsequent use.

1.3.3.3 the preparation of typical curve

In 5 5mL volumetric flasks, add appropriate methylene dichloride, add respectively again 0.125,0.25,0.375,0.5, the 200 μ g/mL hybrid standards of 0.625mL use liquid, with methylene dichloride constant volume, shake up, every milliliter of this solution is equivalent to respectively 5,10,15,20,25 μ g nitrosamine, measure with high performance liquid chromatograph, make typical curve, as Fig. 1.It is NDMA, NMor, NPYR, NDEA, NPIP, NDPA, NDBA and NDphA successively that chromatogram detects peak sequence.

2. result

From Fig. 2-6, can find out, in substratum system, lactobacillus curvatus 2, except NDMA, NMor are had promoter action, all has reducing effect in various degree to other six kinds of nitrosamine.Wherein, lactobacillus curvatus 2 is best to the inhibition of NPYR.NDEA, NDPA, NDphA and tetra-kinds of nitrosamine of NPIP in air-dry sausage system, are only detected.Lactobacillus curvatus 2 between whole yeast phase, can significantly reduce air-dry sausage in the content (P < 0.05) of NDEA, NDPA, NDphA and NPIP, in the time of fermentation 9d, the content of four kinds of nitrosamine has reduced respectively 15.20%, 21.48%, 15.68% and 14.58% compared with control group, and best to the inhibition of NDPA.

Claims

1. lactobacillus curvatus, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation is numbered: CGMCC No.9172.

2. lactobacillus curvatus as claimed in claim 1 reduces the application of content of nitrosamines in air-dry sausage.