CN104164384B - Lactobacillus curvatus and reduce the application of content of nitrosamines in air-dry sausage - Google Patents
Lactobacillus curvatus and reduce the application of content of nitrosamines in air-dry sausage Download PDFInfo
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- CN104164384B CN104164384B CN201410283158.4A CN201410283158A CN104164384B CN 104164384 B CN104164384 B CN 104164384B CN 201410283158 A CN201410283158 A CN 201410283158A CN 104164384 B CN104164384 B CN 104164384B
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Abstract
Lactobacillus curvatus and reduce the application of content of nitrosamines in air-dry sausage, belongs to microbial technology field.It is an object of the invention to the lactobacillus curvatus 2 having selected a strain to have fall nitrosamine effect, apply it in air-dry sausage, investigate the dynamic change of nitrosamine during the fermentation.Lactobacillus curvatus 2 of the present invention is to extract from fermentation yogurt and the strain of isolation identification, it is 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 strain is lactobacillus curvatus (Lactobacillus curvatus).The lactobacillus curvatus 2 that the present invention provides can effectively reduce the content of tetra-kinds of nitrosamine of NDEA, NDPA, NDphA and NPIP in air-dry sausage.
Description
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 reducing content of nitrosamines.
Background technology
Nitrosamine is the chemical substance that a class is nitrogenous, it is possible to teratogenesis, mutagenesis also induce cancer.
Containing nitrosamine in a lot of foods that the mankind take in, also having in some food processings can indirect shape
Becoming, the nitrite as added in meat products can form nitrosamine in sour environment, and nitric acid
What salt used in meat products has good colour generation effect, antioxidation and flavour enhancing effect,
Clostridium botulinum and the growth of many other types putrefaction bacteria can also be suppressed simultaneously, because of
And it is still the adding ingredient being difficult to substitute in meat products processing at present.The most both at home and abroad to nitrosamine
Formation mechanism conduct in-depth research, by control nitrosamine constructive ways reduce right
The harm that human body is likely to result in.
In meat products, the formation of N-nitrosamine is a complicated process, and has many kinds of substance
Nitrosation reaction can be affected.The initial feed that N-nitrosamine is formed in meat products have nitrate,
Nitrite, primary amine (primary amine), secondary amine (secondary amine), tertiary amine (tertiary amine), acyl
Amine, protein, polypeptide and aminoacid, or the precursor of these materials, made by microorganism
With being converted into N-nitrosamine precursor.
Air-dry sausage is China's traditional fermented meat products, and it is with a long history, because it has moisture
Low, length storage period, possess special quality and the feature of local flavor and northeastward area have wide
Consumption market.Nitrite is one of main component in air-dry sausage curing process, additionally in meat
Rich in protein, peptides, aminoacid, amine and amide substance etc., these are all to be formed
The precursor substance that nitrosamine is main.In addition, air-dry sausage is in its sweat, and microbes drops
Solve protein, the formation of reduction pH, beneficially nitrosamine, add the potential safety hazard of air-dry sausage.
European countries just introduce lactic acid bacteria, the forties as far back as the twenties in 20th century in Fermented Sausages
Being applied to by leaven on a large scale in the pickling of meat, shorten the production cycle, product quality obtains
Arrive raising.
Control method currently for N-nitrosamine compound mainly has three classes: one is to find
A kind of material can partly or completely substitute the precursor that can generate N-nitrosamine compound
Matter nitrite；Two is to find a kind of material or method to block the shape of N-nitrosamine in product
Become；Three is to find a kind of method to decompose nitrosamine established in product.For blocking
The research that in the meat products course of processing, N-nitrosamine is formed focus mostly on add ascorbic acid and α-
On tocopherol, but limit their application owing to dissolubility has limitation.About nitrous
The research of hydrochlorate substitute focuses mostly on chemosynthesis material, and this mode can not meet people
Requirement to food safety, microbe fermentation method obtains as a kind of safe alternative method
The concern of people.Myoglobin (Mb) is transformed into the derivant of redness by microbe, its
Middle most study is genus lactubacillus and staphylococcus.In meat products, add lactic acid bacteria, make
It produces lactic acid, reduces pH value, promotes the reduction of nitrite, and makes nitrite
Decompose, then can be substantially reduced nitrate residue, thus reduce the nitrous acid of residual further
Salt and amine react generation carcinogen nitrosamine.
Summary of the invention
It is an object of the invention to the lactobacillus curvatus 2 having selected a strain to have fall nitrosamine effect,
Apply it in air-dry sausage, investigate the dynamic change of nitrosamine during the fermentation.
The lactobacillus curvatus 2 (Lactobacillus curvatus 2) of the present invention is from fermentation yogurt
Middle extraction the strain of isolation identification, be deposited in China Committee for Culture Collection of Microorganisms general
Logical microorganism center (CGMCC), address: section in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Institute of microbiology of institute, its culture presevation is numbered: CGMCC No.9172, preservation date
Being on 05 16th, 2014, the Classification And Nomenclature of strain is lactobacillus curvatus (Lactobacillus
curvatus)。
Lactobacillus curvatus 2 in the present invention has a following biological characteristics:
(1) morphological characteristic: lactobacillus curvatus 2 Gram’s staining result is positive, bent stick
Shape, becomes short catenation, without brood cell, without pod membrane, atrichia.
(2) colony characteristics: lactobacillus curvatus 2 is good at MRS cultured on solid medium,
Colonial morphology is white, protruding, circular, and the smooth of the edge is fine and closely woven, diameter about 2～3mm.?
The most muddy growth in fluid medium.
Find that lactobacillus curvatus 2 liquid medium within has reduction by this research tested
The ability of nitrosamine, therefore the present invention adds lactobacillus curvatus in air-dry sausage and carries out nature and send out
Ferment, observes the dynamic change of nitrosamine in its sweat.Result shows, the present invention provides
Lactobacillus curvatus 2 can effectively reduce NDEA, NDPA, NDphA and NPIP in air-dry sausage
The content of four kinds of nitrosamine.
Preservation information:
Depositary institution's full name: in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart；
Depositary institution is called for short: CGMCC；
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Chinese Academy of Sciences microbe research
Institute；
Preservation date: CGMCC No.9172；
Preservation date: on 05 16th, 2014.
Accompanying drawing explanation
Fig. 1 is nitrosamine hybrid standard sample detection figure (20 μ g/mL)；
Fig. 2 be inoculation lactobacillus curvatus 2 culture medium in nitrosamine dynamic change；
Fig. 3 is the dimethylated dynamic change of nitrosamine in the air-dry sausage inoculating lactobacillus curvatus 2；
Fig. 4 is the dynamic change of nitroso-piperidine in the air-dry sausage inoculating lactobacillus curvatus 2；
Fig. 5 is the dynamic change of nitroso-group di-n-propylamine in the air-dry sausage inoculating lactobacillus curvatus 2；
Fig. 6 is the dynamic change of nitrosodiphenylamine in the air-dry sausage inoculating lactobacillus curvatus 2；
In figure, 1-nitrosodimethylamine, 2-nitrosomorpholine, 3-nitrosopyrolidine, 4-is sub-
Nitro diethylamine, 5-nitroso-piperidine, 6-nitroso-group di-n-propylamine, 7-nitrosoamide, 8-is sub-
Nitrodiphenylamine, a-f refers to that same nitrosamine, letter difference then illustrate significant difference (P ＜
0.05)。
Detailed description of the invention
Below technical scheme is further described, but limitation is not so, all
It is technical solution of the present invention to be modified or equivalent, without deviating from the technology of the present invention side
The spirit and scope of case, all should contain in protection scope of the present invention.
1. material and method
1.1 material
Pig buttocks meat, is purchased from big hair care supermarket；Containing the standard specimen of 8 kinds of nitrosamine compounds, including
Nitrosodimethylamine (NDMA), nitrosopyrolidine (NPYR), nitrosodiethylamine
(NDEA), nitrosomorpholine (NMor), nitrosoamide (NDBA), nitroso-piperidine
(NPIP), nitroso-group di-n-propylamine (NDPA), nitrosodiphenylamine (NDphA), be purchased from U.S.
Sigma company of state；Acetonitrile, Merck company of Germany；Dichloromethane, sodium chloride, sulphuric acid,
Sodium nitrite, glacial acetic acid, ammonium acetate, be purchased from Road Outside Area, Harbin contain reach assay apparatus sell public affairs
Department.Above reagent is in addition to acetonitrile is chromatographically pure, and remaining is analytical pure, and water is ultra-pure water.
1.2 instrument and equipment
Agilent1100 high performance liquid chromatograph is equipped with quaternary gradient pump and UV-detector, beautiful
Agilent company of state；K-D concentrating instrument, vapor distillation device, it is limited that vast science and technology is contained in Tianjin
Company；HWS-series constant temperature and humidity incubator, Tianjin Tai Zang Si Te Instrument Ltd..
1.3 experimental technique
1.3.1, culture medium has the screening of fall nitrosamine ability bacterial strain
(I) MRS (Man Rogosa and Sharp) culture medium
Peptone 10.0g, Carnis Bovis seu Bubali cream 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, seven
Magnesium sulfate heptahydrate 0.2g, manganese sulfate 0.05g, distilled water 1000mL, pH6.5～6.8.For
The activation of lactic acid bacteria, pass on.
(II) bacterial strain screening
Lactobacillus curvatus 2 bacterium solution 0.5mL activated is added in MRS fluid medium,
Add the mixed mark of nitrosamine that concentration is 20 μ g/mL so that it is final concentration reaches 1 μ g/mL.37℃
Under aerobic cultivation 12h, measure content of nitrosamines in 0h and 12h culture medium respectively.Due to Asia
Nitramine has volatility, bacterium solution changes isopyknic distilled water, under equal conditions in matched group into
Cultivate.Matched group 0h and 12h content of nitrosamines change, the most in the medium nitrosamine from
So volatile quantity.
1.3.2 the manufacture method of Harbin air-dry sausage
(I) supplementary material:
Lean meat (pig buttocks meat, pig tenterloin) 4.5kg, fat meat (back pork) 0.5kg, wine is (beautiful
Spring is big) 0.15kg, sodium nitrite 0.0045kg, glucose 0.25kg, gourmet powder 0.075kg,
Salt 0.125kg, mixed spices 0.025kg (mixed spices includes: Pericarpium Zanthoxyli, Fructus Anisi Stellati, Cortex cinnamomi japonici (Ramulus Cinnamomi),
Flos Caryophylli etc.).
(II) prepared by leaven
The lactobacillus curvatus 2 activated is inoculated in MRS culture medium and carries out fermentation culture,
Cultivate 12h for 37 DEG C.Fermentation liquid is under the conditions of 4 DEG C, after 10000g is centrifuged 5min, standby.
When adding leaven, lactic acid bacteria final concentration of 107CFU/g meat stuffing.
(III) air-dry sausage fabrication processing
(rejecting the connective tissues such as lymph, tendon, blood vessel) → dicing, (1cm is square for raw meat
Meat cubelets) → pickle (salt adding and flavouring agent etc.) → with leaven (matched group without)
→ 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 improve.Employing C18 post (Elite, 4.6mm × 250mm,
Filler is Hypersil ODS25 μm) chromatographic column, binary gradient eluting, aqueous phase mobile phase A
For 5mmol/L ammonium acetate one acetate buffer solution (pH 4.0), organic Mobile phase B is acetonitrile.Stream
Speed 1mL/min, sampling volume 20 μ L, column temperature is 35 DEG C, ultraviolet detection at 240nm
Device is measured.Concrete gradient elution program is as shown in table 1.
Table 1 elution program
Table1 elution program
1.3.3.2 sample treatment
Sample pre-treatments is according to the method for GB/T 5009.26-1996.After weighing 200g chopping
Sample, is placed in the alembic of vapor distillation device, adds 100mL ultra-pure water, 120g
Sodium chloride, shakes up, and adds 40mL dichloromethane and a small amount of ice cube, steam in conical flask
Steam distillation.Collect 400mL distillate, tapered receiver bottle adds 80g sodium chloride and 3mL
Sulphuric acid (concentrated sulphuric acid with water volume ratio be 1: 3).It is then transferred into the separatory leakage of 500mL
In bucket, vibrate 5min, stratification.Dichloromethane is divided to another conical flask, then uses
120mL dichloromethane divides 3 extraction water layers, merges 4 extracting solution, and cumulative volume is 160mL.
Transfer to organic layer K-D concentrator is concentrated into 1mL in 50 DEG C of water-baths, standby.
1.3.3.3 the preparation of standard curve
In 5 5mL volumetric flasks, add q. s. methylene chloride, then be separately added into 0.125,0.25,
0.375,0.5, the 200 μ g/mL hybrid standards of 0.625mL use liquid, fixed with dichloromethane
Holding, shake up, this solution every milliliter is respectively equivalent to 5,10,15,20,25 μ g nitrosamine,
Measure with high performance liquid chromatograph, make standard curve, such as Fig. 1.Chromatograph detection peak sequence
Be successively NDMA, NMor, NPYR, NDEA, NPIP, NDPA, NDBA and
NDphA。
2. result
From Fig. 2-6 it can be seen that in culture medium system, lactobacillus curvatus 2 is except right
NDMA, NMor have outside facilitation, and other six kinds of nitrosamine are all had fall in various degree
Low effect.Wherein, lactobacillus curvatus 2 is best to the inhibition of NPYR.At air-dry sausage
System has only detected tetra-kinds of nitrosamine of NDEA, NDPA, NDphA and NPIP.Curved
Bent lactobacillus 2 can significantly reduce during whole fermentation NDEA in air-dry sausage, NDPA,
The content (P ＜ 0.05) of NDphA and NPIP, when fermenting 9d, four kinds of nitrosamine
Content reduces 15.20%, 21.48%, 15.68% and 14.58% compared with matched group respectively,
And it is best to the inhibition of NDPA.
Claims (1)
1. a lactobacillus curvatus reduces by tetra-kinds of Asias of NDEA, NDPA, NDphA and NPIP in air-dry sausage
The application of nitramine content, it is the most micro-that described lactobacillus curvatus is preserved in China Committee for Culture Collection of Microorganisms
Bio-Centers, culture presevation is numbered: CGMCC No.9172.
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| CN110373353A (en) * | 2019-07-22 | 2019-10-25 | 东北农业大学 | A kind of mixed culture fermentation agent being able to ascend less salt air-dry sausage flavor |
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| CN104560808A (en) * | 2014-12-26 | 2015-04-29 | 中国肉类食品综合研究中心 | Lactobacillus plantarum strain and fermentation method of canned meat products |
| CN114451525A (en) * | 2022-02-16 | 2022-05-10 | 深圳市淳睿科技发展有限公司 | Preparation method of fermented meat product and fermented meat product |
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| CN101248887A (en) * | 2008-03-28 | 2008-08-27 | 东北农业大学 | Directly casting type composite leaven capable of quick fermenting air-dry sausage and preparation method |
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| CN101248887A (en) * | 2008-03-28 | 2008-08-27 | 东北农业大学 | Directly casting type composite leaven capable of quick fermenting air-dry sausage and preparation method |
Non-Patent Citations (3)
| Title |
|---|
| Influence of NaCl and NaNO2 on the Lactic Acid Fermentation of Lactobacillus curvatus Grown in MRS Medium;Maria Celia de Oliveira Hauly et al.;《Semina: Ci. Exatas Tecnol.》;20011231;第22卷(第1期);摘要 * |
| 乳酸菌发酵对风干香肠物理化学性质的影响;赵俊仁 等;《食品与发酵工业》;20101231;第36卷(第10期);全文 * |
| 自然发酵风干肠中乳酸菌的分离与鉴定;赵俊仁 等;《食品工业科技》;20101231;第31卷(第11期);全文 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373353A (en) * | 2019-07-22 | 2019-10-25 | 东北农业大学 | A kind of mixed culture fermentation agent being able to ascend less salt air-dry sausage flavor |
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