CN1566323A - Staphylococcus xylosus I2 strain, composite ferment produced thereby and the use of ferment in meat ware - Google Patents
Staphylococcus xylosus I2 strain, composite ferment produced thereby and the use of ferment in meat ware Download PDFInfo
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Abstract
The invention relates to an xylose staphylococcus I2 strain, whose docket number in the Chinese Microbiological Culture Preservation Administration Commission Common Microbiological Center is CGMCC No.0919, the leaven can be applied into the production process for meat product e.g. fermentation sausage.
Description
Technical field
The present invention relates to a kind of staphylococcus xylosus and the application in meat product with its starter of making and this starter.
Technical background
Fermented sausages is by a kind of fermented meat prods of roman's invention at first, has formed broad variety and multiple style at present.The fermented sausages effect that undergoes microbial fermentation in the course of processing is converted into various acid and alcohol with sugar, and the pH value of sausage is reduced; Fat is become aldehyde and ketone with proteolytic degradation, make sausage have good fermented flavour; And the drying dehydration descends Aw.Therefore, fermented sausages has unique flavor quality, and has long shelf-lives.The fermentation mode of fermented meat prods mainly contains two kinds of non-inoculation fermentation method and inoculation fermentation methods.Wherein non-inoculation fermentation is subject to microbial contamination, and fermentation starting is slow, and fermentation period is long; And the inoculation fermentation method is exactly to carry out suitability for industrialized production by inoculating good meat product fermented bacterium.Inoculation fermentation has shortened fermentation period, has improved the stability of quality product, helps technology controlling and process.Though fermented sausages is various in style, technology has nothing in common with each other, but inoculation fermentation has become the main method that present production is used, therefore, the basis that to screen good meat product organism of fermentation be inoculation fermentation, and milk-acid bacteria, micrococci and non-virulent staphylococcus are the inoculation fermentation bacterial classifications of normal use.
At present, developed country's meat product deep processing ratio is up to more than 30%, and wherein fermented meat prods occupies critical role.Current American-European countries, by the characteristic of microbial fermentation meat product has been carried out comparatively deep research, finished from the transformation of traditional spontaneous fermentation to the suitability for industrialized production of microorganism directional inoculation fermentation, the production of fermented meat prods has possessed quite sophisticated production technique.At present, one of external main research contents of fermented meat prods is the seed selection work of having strengthened good microbial fermentation bacterial classification.By purebred separation and Culture technology and the Microbial Breeding technology to organism of fermentation in the traditional zymotic meat product, screening and structure have the microbial starter culture bacterial classification of good character.
1940, American L.B.Jenson described the application of milk-acid bacteria in fermented sausages for the first time in patent, thereby had begun the first step of the microbial starter culture production fermented sausages of use pure culture; Discovery sheet coccuses such as Deibel can be used as the bacterial classification of making the semi dry sausages starter in 1961; Everson in 1974 etc. have applied for the patent of plant lactobacillus, meanwhile, and the research of other bacterial strains such as micrococci, suis and milk-acid bacteria and other fermented by mixed bacterium agent and application also development to some extent.The starter that is applied at present on the meat product mainly comprises bacterium, yeast and mould etc.
China region is wide, the ecological condition complexity, production practice show, extensively exist good natural bacterial strain in China tradition meat product main producing region, but since separation screening comparatively difficulty and to excellent species the awareness of importance is not enough aborning, make China's fermented meat prods milk-acid bacteria The Study on Resources with utilize still very weak so far.For this reason, we systematically examine or check the fermented meat prods Microbial resources of China, wish therefrom to optimize the bacterial strain that is fit to production requirement.
Summary of the invention
The purpose of this invention is to provide a kind of staphylococcus xylosus I2 bacterial strain.
The present invention also aims to provide a kind of compound ferment and the application of this starter in meat product made from staphylococcus xylosus I2 and Pediococcus pentosaceus I9.
For achieving the above object, the technical solution used in the present invention is: staphylococcus xylosus I2 (Staphylococcus xylosus) bacterial strain, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.0919.
A kind of compound ferment, it is that Pediococcus pentosaceus I9 (Pediococcus pentosaceus) and staphylococcus xylosus I2 (Staphylococcus xylosus) are cultured to logarithmic growth respectively after late period in modified MRS culture medium, again according to
Volume ratio mixed starter, wherein the deposit number of Pediococcus pentosaceus I9 at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCCNo.0918, and the deposit number of staphylococcus xylosus I2 at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.0919.
The process for preparation of described modified MRS culture medium is as follows:
At first by following formulated nutrient solution: (unit/L)
Peptone 10g yeast powder 5g extractum carnis 10g
MgSO
4·7H
2O?0.58g???????MnSO
4·4H
2O?0.25g???????K
2HPO
4?2g
Dibasic ammonium citrate 2g sodium acetate 5g glucose 20g
Tween80 1mL cysteine hydrochloride 0.5g tomato juice 10～20% (v/v)
The nutrient solution that will prepare is according to the above ratio then transferred pH to 6.2-6.4,115 ℃ of moist heat sterilization 30min.
When Pediococcus pentosaceus I9 and staphylococcus xylosus I2 were cultivated in modified MRS culture medium, modified MRS culture medium was 28～32 ℃ of stationary states.
The above-mentioned starter of inoculation in the fermented meat food production.
The inoculation weight ratio is 0.5～1% starter before the bowel lavage in the fermented sausages production process, ferments to pH5.0 in 20 ℃ of temperature, relative humidity 95%; Continue to ferment in 15 ℃ of temperature, relative humidity 90% to pH4.6.
After adopting technique scheme, this starter has good characteristic aspect the fermentation adaptability:
(1) salt tolerance
The addition of sodium-chlor in fermented sausages is generally 2.5～3.0%, and at some type fermented sausages, can be higher as the addition among the Italian Salami, so the screening bacterial strain must have good salt tolerance, requirement can be under at least 3.0% common salt concn can normal growth.Can tolerate 5.0% salt solution through the advance copy starter.
(2) anti-NaNO
2Or NaNO
3
Fermented sausages (except the dried fermented sausages) generally adds nitrite, adds concentration and can reach 150mg/Kg; And dried fermented sausages adds nitrate usually, addition 200～600mg/Kg, in addition higher.But in the course of processing, what play color development and bacteriostatic action mainly is Sodium Nitrite, the growth that the general requirement bacterial classification at least can be good under the nitrite concentration of 50mg/Kg.In the nitrite solution of advance copy starter under 80～100mg/Kg concentration conditions, still can grow.
Simultaneously, this is ferment-fermented with rich flavor, does not produce peculiar smell, and the amount that produces biogenic amine is very little, and color development is respond well, has improved the flavor quality of product.This starter can be grown in 27～43 ℃ of scopes, and optimum temperuture is 32 ℃, deactivation in 57～60 ℃ of scopes.Fermenting power was moderate when starter of the present invention was used in the fermented sausages, and course of fermentation is mild, and tart flavour is soft, and the ripe secondary fermentation of product gives off a strong fragrance, and color development is good, and product section property is good.
Description of drawings
Fig. 1 is that Pediococcus pentosaceus I9 is at MRS-nutrient agar (+5%CaCO
3) go up and produce sour situation observation figure;
Fig. 2 is that Pediococcus pentosaceus I9 morphological features is observed figure;
Fig. 3 is that staphylococcus xylosus I2 is at MRS-nutrient agar (+5%CaCO
3) go up and produce sour situation observation figure;
Fig. 4 is that staphylococcus xylosus I2 morphological features is observed figure;
Fig. 5 is the separation process figure of Pediococcus pentosaceus I9 and staphylococcus xylosus I2 bacterial strain;
Fig. 6 is the comparison diagram of histamine growing amount in the different strains fermented sausages;
Fig. 7 is the comparison diagram of putrescine and cadaverine growing amount in the different strains fermented sausages.
Staphylococcus xylosus I2 bacterial strain provided by the invention, on April 8th, 2003 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.0919.
Embodiment
Staphylococcus xylosus I2 bacterial strain provided by the invention, on April 8th, 2003 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.0919.
Pediococcus pentosaceus I9 and staphylococcus xylosus I2 are cultured to logarithmic growth respectively after late period in modified MRS culture medium, again according to
Volume ratio mix and can make compound ferment of the present invention, wherein the deposit number of Pediococcus pentosaceus I9 at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.0918, and the deposit number of staphylococcus xylosus I2 at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.0919.
Described modified MRS culture medium can be prepared in following ratio:
At first by following formulated nutrient solution: (unit/L)
Peptone 10g yeast powder 5g extractum carnis 10g
MgSO
4·7H
2O?0.58g??????MnSO
4·4H
2O?0.25g?????K
2HPO
4?2g
Dibasic ammonium citrate 2g sodium acetate 5g glucose 20g
Tween80 1mL cysteine hydrochloride 0.5g tomato juice 10% (v/v)
The nutrient solution that will prepare is according to the above ratio then transferred pH to 6.4,115 ℃ of moist heat sterilization 30min.When Pediococcus pentosaceus I9 and staphylococcus xylosus I2 cultivated in modified MRS culture medium, modified MRS culture medium was 30 ℃ of stationary states.
When the above-mentioned modified MRS culture medium of preparation, tomato juice also can be prepared in the ratio of 15% (v/v), and will transfer PH to 6.3,115 ℃ of moist heat sterilization 30min in this ratio prepared culture.When Pediococcus pentosaceus I9 and staphylococcus xylosus I2 cultivated in modified MRS culture medium, modified MRS culture medium was 28 ℃ of stationary states.
When the above-mentioned modified MRS culture medium of preparation, tomato juice also can be prepared in the ratio of 20% (v/v), and will transfer PH to 6.2,115 ℃ of moist heat sterilization 30min in this ratio prepared culture.When Pediococcus pentosaceus I9 and staphylococcus xylosus I2 cultivated in modified MRS culture medium, modified MRS culture medium was 32 ℃ of stationary states.
1, fermented sausages bacteria culture screening criteria among the present invention
1.1 security
As the fermented sausages bacteria culture, must be harmless, particular content comprises: 1. must be gram-positive microorganism, no pathogenicity must not produce bacteriotoxin (especially in staphylococcic screening); 2. do not produce biogenic amine.Some bacterium has degrade proteins and amino acid whose ability, and some meta-bolites in the degradation process has certain harm to human body.Wherein, some bacterium has the amino acid decarboxylase ability, can metabolism amino acid generates biogenic amine, and biogenic amine (especially histamine) during too high levels, may cause anaphylaxis after the people is edible in leavened food; 3. do not produce urethanum (EC).EC is a kind of common carcinogenic substance in the spontaneous fermentation food, and it is mainly produced by some LAB metabolism arginine, and the screening by strain excellent can overcome this problem.
1.2 fermentation adaptability
(1) salt tolerance
The addition of sodium-chlor in fermented sausages is generally 2.5～3.0%
[1], and at some type fermented sausages, as Italian Salami, in addition can be higher, so the screening bacterial strain must have good salt tolerance, requirement can be at least 3.0%
[1]Common salt concn down can normal growth.
(2) anti-NaNO
2Or NaNO
3
Fermented sausages (except the dried fermented sausages) generally adds nitrite, adds concentration and can reach 150mg/Kg; And dried fermented sausages adds nitrate usually, addition 200～600mg/Kg, in addition higher.But in the course of processing, what play color development and bacteriostatic action mainly is Sodium Nitrite, the growth that the general requirement bacterial classification at least can be good under the nitrite concentration of 50mg/Kg.
(3) produce sour characteristic
Why fermented sausages has long shelf-lives at normal temperatures, is because it self inherent " two low height ", low pH value, low moisture and high saltiness.This just requires LAB energy quick fermentation glucose or sucrose to produce acid, and this is an important process condition of decision fermentation success or not and leavened food security.This standard is primarily aimed at bacillus and the part coccus among the LAB.For acid production speed bacterial classification more slowly, require in 3d, to make the pH value of fermented sausages reduce to 5.1 ± 0.1; And for the fast bacterial strain of acid production speed, can reduce to below 5.0 in the pH value fermented sausages about 40h.
1.3 fermentation character
Good fermented sausages square section weave construction is fine and close fine and smooth, but color and luster is people's rose, middle high-fat emulsion white or faint yellow, and these The apparent phenomenon are all relevant with the fermentation character of microorganism.
(1) NO is arranged
2 -And NO
3 -Reducing power
The chromophoric mechanism of meat product is that nitrate is under the effect of reducing bacteria, be reduced into nitrite, subsequently with meat in the lactic acid formation nitrous acid that reacts, the latter decomposes generation nitrogen oxide (NO), NO combines generation nitroso-group flesh (blood) red eggs with myohaemoglobin or oxyphorase white, makes meat have bright-coloured rose.Chromophoric mechanism thus, selected bacterial strain must have good nitrate reduction ability.But from modern fermented sausages complete processing, non-dry fermented sausages (final a
wBetween 0.94～0.96) and half-dried fermented sausages (final a
wBetween 0.90～0.95) the fermenting-ripening phase short, mainly add nitrite and color auxiliary sodium ascorbate, not too strict to the requirement of adding bacterial classification nitrate reduction ability; And dried fermented sausages is taken into account color development and local flavor two aspect factors, mainly adds nitrate, and this just requires selected bacterial strain to have good nitrate reduction ability.
(2) the catalase test positive
The generation of nitrosomyoglobin or nitrosohaemoglobin is the major cause of sausage color development, but the H that microorganism may form in the fermenting-ripening process
2O
2Can cause the sausage color to become the ash obfuscation.H
2O
2The enzyme positive bacterial strain can produce H
2O
2Reductase enzyme is the H that generates
2O
2Resolve into H
2O and O
2Therefore, if use single strain fermentation, this bacterium is necessary for the catalase positive strain; If composite strain fermentation so wherein must have the catalase positive strain to exist.
(3) must not there be a large amount of gases to produce in the fermenting process, must not have mucus to generate
During the fermentation, as milk-acid bacteria heterofermentation bacterial strain, produce a large amount of CO
2, can have influence on the compact structure of sausage; And mucus shape material is arranged in the meta-bolites of some mushroom, equally also can damage the interior tissue state of sausage.In the screening of bacterial strain, these standards must be considered.
(4) selected bacterial classification can make sausage produce good fermented flavour in its growth metabolism process
The local flavor of fermented sausages is formed very complicated, and it comprises volatile component and involatile constituent.From present research, the local flavor of fermented sausages forms and relates to fat, proteinic degraded and amino acid whose alienation etc.Micrococci and staphylococcus are the main fungus strains that flavour substances forms, but some LAB such as Pediococcus bacterium also have similar characteristics.Micrococci or staphylococcus secretion lipase are cut and are split the lipid acid of macromole fat for short chain or side chain; Extracellular proteinase, degrade proteins become various peptides and amino acid, and amino acid further is dissimilated as various aldehydes or ketones class flavour substancess by its corresponding enzyme again.In addition, in microbial metabolism, also may produce meta-bolites, as H with unpleasant odor
2S.In view of above analysis, the micrococci of screening or staphylococcicly should have following feature: the fat of 1. degrading; 2. can decomposing protein; 3. can alienation amino acid; 4. the undesirable substance of local flavor does not exert an influence.
1.4 have anti-lyophilize characteristic
Vacuum freeze-drying method is generally adopted in starter for fermentative meat production, therefore requires preferred strain that anti-strongly lyophilize ability must be arranged.Mainly show: 1. under optimal culture condition, have than mcroorganism amount (nectar degree＞10
9Cfu/ml); 2. has higher survival rate after the freeze-drying.Though influence the factor a lot (protective material, freeze-dry process etc.) of survival rate, still there is very big difference in anti-cryodesiccated ability between different strains; 3. the vigor of freeze-drying lactobacillus can keep the long period.General requirement freeze-dried vaccine powder viable count after depositing a year is not less than 10
9Cfu/g.
At present, developed country's meat product deep processing ratio is up to more than 30%, and wherein fermented meat prods occupies critical role.Current American-European countries, by the characteristic of microbial fermentation meat product has been carried out comparatively deep research, finished from the transformation of traditional spontaneous fermentation to the suitability for industrialized production of microorganism directional inoculation fermentation, the production of fermented meat prods has possessed quite sophisticated production technique.At present, one of external main research contents of fermented meat prods is the seed selection work of having strengthened good microbial fermentation bacterial classification.By purebred separation and Culture technology and the Microbial Breeding technology to organism of fermentation in the traditional zymotic meat product, screening and structure have the microbial starter culture bacterial classification of good character.
China region is wide, the ecological condition complexity, production practice show, extensively exist good natural bacterial strain in China tradition meat product main producing region, but since separation screening comparatively difficulty and to excellent species the awareness of importance is not enough aborning, make China's fermented meat prods milk-acid bacteria The Study on Resources with utilize still very weak so far.For this reason, we systematically examine or check the fermented meat prods Microbial resources of China, wish therefrom to optimize the bacterial strain that is fit to production requirement.This research obtains the milk-acid bacteria that a strain is fit to the single strain fermentation by to screening from the milk-acid bacteria in the fermented meat prods under China's different ecological condition.
2, the sepn process of Pediococcus pentosaceus I9 and staphylococcus xylosus I2 bacterial strain is:
This strains separation substratum adopts solid mediums such as MRS-agar, modified MRS-agar, MSA-agar; The MRS liquid nutrient medium is adopted in activation.Culture medium prescription following (1L):
(1) MRS substratum: yeast extract 4g, extractum carnis 8g, bacto peptone 10g, glucose 20g, Trisodium Citrate 2g, sodium-acetate 5g/L, KH
2PO
42g, MgSO
47H
2O 0.2g, MnSO
44H
2O 0.05g, Tween80 1mL uses the 400mL dissolved in distilled water.In addition,, after in boiling water bath, dissolving fully, add above-mentioned solution, be settled to 1000mL, with NaOH or the 1M HCl adjustment pH to 5.0 of 1M with 500mL dissolved in distilled water 20g agar.121 ℃ of sterilization 15min.
(2) modified MRS culture medium prescription: peptone 10g; Yeast powder 5g; MgSO
47H
2O 0.58g; MnSO
44H
2O 0.25g; Extractum carnis 10g; K
2HPO
42g; Dibasic ammonium citrate 2g; Sodium acetate 5g; Glucose 20g; Tween80 1mL; Tomato juice 10% (v/v); Cysteine hydrochloride 0.5g/L; Transfer pH to 6.2-6.4,115 ℃ of moist heat sterilization 30min.
(3) MSA substratum: extractum carnis 1g, bacto peptone 10g, D-N.F,USP MANNITOL 10g, NaCI75g, phenol red 0.025g with 400mL dissolved in distilled water (except phenol red), transfers pH to 7.4 ± 0.2 with 1M HCl or NaOH; Add 1% phenol red solution 2.5ml, mixing; In addition,, after in boiling water bath, dissolving fully, add above-mentioned solution, be settled to 1000mL with 500mL dissolved in distilled water 20g agar.121 ℃ of sterilization 15min.
Adopt plate streak that the lactic-acid-bacterium in the spontaneous fermentation meat product (ham and sausage) is separated.Get 25g sample and 225ml physiological saline in aseptic homogeneous bag, with bouncing formula homogenizer Bagmixer homogeneous 120sec (sausage) or 210sec (ham).Draw the wine sample of 0.1ml and rule on isolation medium, observation bacterium colony formation situation behind the cultivation 2d is separated through repeatedly ruling, up to the pure bacterium colony of acquisition under 30 ℃ of conditions.Separation process as shown in Figure 3.
The mensuration of nectar degree adopts colony counting method, and count results also is converted into colony-forming unit (cfu/mL).The equal triplicate of each culture experiment, results averaged.Lactic acid is measured and is adopted vapor-phase chromatography.The mensuration of pH value and total acid is undertaken by the GB method.Viability test adopts maximum bacterium densimetry.Having nitrate or nitrite reducing power is undertaken by " industrial microbiology experimental technique " (Du Lianxiang writes).H
2O
2Enzyme assay is operated by " microbiology experiment " (Fan Xiurong writes).The mensuration of salt tolerance:, observe its upgrowth situation testing the MRS liquid nutrient medium that bacterium is inoculated in pH4.5; The mensuration of salt tolerance is with the mensuration of lactic acid tolerance.Whether there is gas to produce and adopts semi-solid soft agar post method; Whether have mucus to produce can directly observe from the state of provoking bacterium colony.
Adopt PCR method that the 16SrRNA gene of strain isolated I9 is increased.PCR employing prokaryotic organism universal primer 27f (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1541R (5 '-AAGGAGGTGATCCAGCC-3 ').The PCR reaction conditions: the reaction mixture cumulative volume is 50 ì l, includes 2.5mM Mg
2+, 20mM Tris-HCl (pH9.0), 0.1%Triton-100 (v/v), the dNTP of 100 ì M, the 2.5UTaqDNA polysaccharase, the template DNA of 300ng, the primer of 40pmol covers and adds a cover with 25 ì l mineral oil.Before the PCR reaction was carried out, 94 ℃ of sex change 5min of dna profiling carried out 30 circulations subsequently, and each circulation comprises 94 ℃ of sex change 1min, 52 ℃ of renaturation 2min, and 72 ℃ are extended 3min, and last circulates in 72 ℃ and extends 2-3h.PCR is reflected among the Thermolyne Amplitron I (Barnstead Thermolyne Corporation) and carries out.
The 16SrRNA order-checking:
Behind the PCR product electrophoresis purifying, be bound up among the plasmid pSK-T, carry among the recombinant plasmid transformed E.coli DH5 α of 16SrRNA, on the flat board that contains acillin Ap/IPTG/X-gal, screen hickie then, extract plasmid with the alkaline hydrolysis method, after enzyme is cut, use the polyoxyethylene glycol purified product.The order-checking of 16SrRNA is finished by Institute of Microorganism, Academia Sinica, carries out homology analysis relatively with the 16SrRNA sequence that BLAST software will be measured among sequence and the GenBank.
Main laboratory apparatus and equipment:
Portable high-pressure sterilizing pot, 101C type electric drying oven with forced convection, JA3003 electronic balance, the DMB5-5 digit microscope, Ph/ORH acidity ionometer, anaerobic operation incubator, the YJ Bechtop, micropipet (Shanghai refinement), BS-2F shaking culture case, pHB5 type acidometer, Ultralow Temperature Freezer, Lyostar vacuum freeze drier, GM-21 low-temperature and high-speed whizzer, BS-2F constant incubator, proving room automatically.
Strain identification:
Individual morphology feature, colony morphology characteristic, Gram dyeing, catalase experiment, difference is coerced " lactic-acid-bacterium classification identify and experimental technique " and the Holt J G of the mensuration of the tolerance of environment and other physiological and biochemical properties by the Ling Daiwen chief editor, Krieg N R and Snath P H A, et al.Bergey ' s manualof determinative bacteriology (9
ThEdition) .Batimore:Williams ﹠amp; Wilkins, the method for 1,993 two reference is carried out; Strain identification is pressed Holt J G, Krieg N R and Snath P H A, et al.Bergey ' s manual of determinative bacteriology (9
ThEdition) .Batimore:Williams﹠amp; Wilkins, 1993 carry out.
Result and analysis:
(1), the separating resulting of organism of fermentation
Screen good meat product fermentation strain, should be according to the fermentation character of bacterial classification, the bonded products characteristics are screened bacterial strain.The main contents of preferred strain fermentation character research comprise: 1. salt tolerance (＞3% salt solution); 2. nitrite tolerance (under the 80-100mg/Kg concentration conditions, still can grow); 3. can grow in 27-43 ℃ of scope, optimum temperuture is 30～32 ℃; 4. fermentation byproduct does not produce peculiar smell; 5. no pathogenicity; 6. it is little to produce the histamine amount.
(2), strain separating
This experiment from JINHUA HUOTUI, abroad ferment ham, external starter for fermentative meat, from habitats such as DSM C type and Yogurt type II type milk ferments, be divided into strains of lactic acid bacteria surplus obtain 120, separating resulting is as follows:
1. from JINHUA HUOTUI, separate 29 strains
It is an amount of to get JINHUA HUOTUI with aseptic technique, adds sterile saline, and homogeneous is drawn supernatant liquor and made gradient dilution, and pour plate is cultivated.According to colony characteristics, picking list bacterium colony one by one, with twice purifying of plate streaking, the inclined-plane of transferring afterwards, 4 ℃ of preservations, standby.
2. from external fermentation ham, separate 29 strains
It is an amount of to get JINHUA HUOTUI with aseptic technique, adds sterile saline, and homogeneous is drawn supernatant liquor and made gradient dilution, and pour plate is cultivated.According to colony characteristics, picking list bacterium colony one by one, with twice purifying of plate streaking, the inclined-plane of transferring afterwards, 4 ℃ of preservations, standby.
3. from external starter for fermentative meat, separate 12 strains
It is an amount of to get starter with aseptic technique, adds the sterile saline gradient dilution, and pour plate is cultivated.According to colony characteristics, picking list bacterium colony one by one, with twice purifying of plate streaking, the inclined-plane of transferring afterwards, 4 ℃ of preservations, standby.
4. from DSM C type and Yogurt type II type milk ferments, separate totally 11 strains
It is an amount of to get starter with aseptic technique, adds the sterile saline gradient dilution, and pour plate is cultivated.According to colony characteristics, picking list bacterium colony is secondarily purified with plate streaking one by one, the inclined-plane of transferring afterwards, and 4 ℃ of preservations, standby.
In addition, separation obtains 42 strain isolates from other fermented meat prods samples.
(3) identification of strains:
The evaluation of Pediococcus pentosaceus I9:
1. morphological feature
The solid culture feature is after cultivating 2d under 30 ℃ of conditions, and I9 is at MRS+5%CaCO
3Form obvious transparent circle, smooth surface moistening (as shown in Figure 1) on the solid medium.
Cell morphological characteristic I9 bacterial strain Gram-positive is observed down with opticmicroscope (1600 *) after the methylenum coeruleum simple stain, cell spheroid, and tetrad or slabbing are arranged, the about 1 μ m (as shown in Figure 2) of cell dia.
The liquid culture feature is under aseptic condition, with the 30 ℃ of static cultivations in clarifying MRS liquid nutrient medium of 1 bacterium colony of inoculating needle picking, bacterium poor growth and evenly in substratum, reach maximum opacity after, thalline is deposited to the bottom of substratum gradually, and thalline is white in color.
2. physiological and biochemical property
The phenotypic characteristic of isolated strains I9
| The certified variety result | The certified variety result |
| The negative litmus milk of catalase produce acid from glucose produce acids type lactic acid from glucose do not produce that acid is produced in acid, aerogenesis but not under the aerogenesis pH9.6 condition-the pH4.5 condition under+5%NaCl-anaerobism cultivation+aerobic cultivation+45 ℃ condition under under-10 ℃ of conditions+ | Carbohydrate produces acid: lactose+rhamnose+ribose+maltose+sucrose+amarogentin+gluconate-sweet mellow wine-melibiose-mannose- |
According to I9 bacterium phenotypic characteristic and Physiology and biochemistry characteristics,, tentatively I9 can be classified as Pediococcus pentosaceus (Pediococcuspentosaceus) according to the standard of perfection of the relevant milk-acid bacteria of Bergey ' s Bacteria Identification handbook.
3. the 16SrRNA sequencing analysis of I9
By to the 16SrRNA sequence of isolated strains (length: 1477bp) with the GenBank gene pool in all known arrays carried out homology relatively, the I9 of display separation strain as a result and Pediococcus pentosaceus (P.pentosaceus) have homology (99%) highly.This confirms that from the molecular systematics angle strain isolated I9 is Pediococcus pentosaceus (Pediococcus pentosaceus), and the result is as follows:
CATGCAAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTT
GTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTG
AGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCT
GGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGT
TTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCG
CGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAG
TGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGAC
TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAA
TCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGA
GTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGGTTAAGAAG
AACGTGGGTAAGAGAACTGGTTACCCAGTGACGGTATTTAACC
AGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACG
TAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCG
CAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCG
AAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGG
ACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGG
AAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGA
CGCTGAGGCTC
GAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCAT
GCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTC
AGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACG
ACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCAC
AAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAAC
CTTACCAGGTCTTGACATCTTCTGACAGTCTAAGAGATTAGAGG
TTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTC
AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC
AACCCTTATTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTG
AGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCA
AATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAAT
GGATGGTACAACGAGTCGCGAGACCGCGAGGTTAAGCTAATCT
CTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTA
CACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCG
GTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT
GAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGA
GCTAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAAGTC
The evaluation of staphylococcus xylosus I2:
1. morphological feature
The solid culture feature forms circular, protruding, white colony after cultivating 48 under 30 ℃ of conditions, the about 1mm of colony diameter, and I2 is at MRS+5%CaCO
3Do not form the obvious transparent circle on the solid medium, smooth surface moistening (as shown in Figure 3).
Cell morphological characteristic I2 bacterial strain Gram-positive is observed down with opticmicroscope (1600 *) after the methylenum coeruleum simple stain, cell spheroid, and the about 1 μ m of cell dia, paired or tetrad is arranged (as shown in Figure 4).
The liquid culture feature is under aseptic condition, with the 30 ℃ of static cultivations in clarifying MRS liquid nutrient medium of 1 bacterium colony of inoculating needle picking, bacterium poor growth and evenly in substratum, reach maximum opacity after, thalline is deposited to the bottom of substratum gradually, and thalline is white in color.
2. I2 physiological and biochemical property
The phenotypic characteristic of isolated strains I2
| The certified variety result | The certified variety result |
| The positive oxidase negative of catalase from glucose produce acids type lactic acid from glucose produce acid, aerogenesis produces acid seldom, not under the aerogenesis pH9.6 condition+the pH4.5 condition under+NO3 -Under ℃ condition of-anaerobism cultivation+aerobic cultivation+45 under-10 ℃ of conditions+18%NaCl+ | Carbohydrate produces acid: cellobiose-fructose-galactolipin+w lactose-maltose+sweet mellow wine+w raffinose+w sucrose+wood sugar+mannose+melibiose+ |
According to I2 bacterium phenotypic characteristic and Physiology and biochemistry characteristics,, tentatively I2 can be classified as staphylococcus xylosus (Staphylococcusxylosus) according to the standard of perfection of the relevant milk-acid bacteria of Bergey ' s Bacteria Identification handbook.
3. the 16SrRNA sequencing analysis of I2
By to the 16SrRNA sequence of isolated strains (length: 1477bp) with the GenBank gene pool in all known arrays carried out homology relatively, the I2 of display separation strain as a result and staphylococcus xylosus (Staphylococcus xylosus) have homology (99%) highly.This confirms that from the molecular systematics angle strain isolated I2 is a staphylococcus xylosus, and the result is as follows:
GGCCTTCCGATACGGCTACCTTGTTACGACTTCACCCCAATCATTTGTCCC
ACCTTCGACGGCTAGCTCCATAAATGGTTACTCCACCGGCTTCGGGTGTT
ACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTA
TTCACCGTAGCATGCTGATCTACGATTACTAGCGATTCCAGCTTCATGTAG
TCGAGTTGCAGACTACAATCCGAACTGAGAACAACTTTATGGGATTTGCA
TGACCTCGCGGTTTAGCTGCCCTTTGTATTGTCCATTGTAGCACGTGTGTA
GCCCAAATCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCC
GGTTTGTCACCGGCAGTCAACCTAGAGTGCCCAACTTAATGATGGCAACT
AAGCTTAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGAC
ACGAGCTGACGACAACCATGCACCACCTGTCACTTTGTCCCCCGAAGGG
GGAAGGCTCTATCTCTAGGAGTTTTCAAAGGGATGTCAAGATTTGGTAAG
GGTCTTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGGCTTGGGCG
GGTCCCCGNCAATTCCTTTTGAGGTTTCAACTTTNNCGGGCCGTANCTCC
CCAAGGCCGGAGTGCTTAAATGCGTTTAGCTGCCACACTTAAGGGGGCG
GGAAAACCCCTTACACTTACACTTNAATCGTTA
NCGGGNGNGGACTCCCNAGGGNATCTAANTCCTTTNTTGGATCCCCACG
CTTTTCCACANTAANNGNTANTTACAGGACNANAAANGGNNCCTTCCCC
CCNGGGGGNTCCTCCANATTTTTTGNNNATTTNACCGCTCCCAATGGAA
NTCNCNTTTNNTTTTTTNCCTTAAANTTTTCNGGTTCNAANACCCTTTCC
CNGTTNACCNGNGNNTTTTNTTTNNNNTTNNNTNANCNNCCCCCCCCCC
CCCT
In the fermented meat food production, can inoculate compound ferment of the present invention.As in the production process of fermented sausages, the inoculation weight ratio is 0.5% starter before bowel lavage, ferments to pH5.0 in 20 ℃ of temperature, relative humidity 95%; Continue to ferment to pH4.6 in 15 ℃ of temperature, relative humidity 90%, the fermentation back is 14 ℃ of temperature, and relative humidity is carried out maturation 70%～80% time.Compound ferment fermenting power of the present invention is moderate, and course of fermentation is mild, and tart flavour is soft, and the ripe secondary fermentation of product gives off a strong fragrance, and color development is good, and product section property is good.
In the production process of fermented sausages, also can be by the arbitrary proportion inoculating starter between 0.8%, 1% or 0.5%～1%.
Below be elaborated to comparing compound ferment result of use of the present invention by the use of different fermentations agent in the fermented sausages production process:
In leavened food such as fermented sausages, sour milk, pickles and grape wine, mostly contain biogenic amine.Amine has the important physical function in life cell, but when absorption of human body is excessive, may cause anaphylaxiss such as headache, respiratory disorder, palpitaition, blood pressure.In addition, biogenic amine still is the precursor of nitrosamine, and the latter has carcinogenic effect.Vandekerckhove (1977) once was reported in the fermented sausages of aged and mould adult form, therefore the content of biogenic amine can reach 100mg/Kg, sometimes even up to 1000mg/Kg,, in recent years, the biogenic amine content problem in the leavened food has caused people's attention.
Biogenic amine is produced by amino acid decarboxylase in the fermented sausages.Its growing amount depends on the abundance of raw meat histaminase and microbial enzyme vigor and biogenic amine precursor (amino acid, oligopeptides).By adopting the fresh raw meat of fine, suitable starter and rational zymotechnique, can reduce the biogenic amine output in the product.Wherein, the related amino acid decarboxylase of starter culture directly affects the content of biogenic amine in the product, have related amino acid decarboxylation ability because produce most of bacterial strains of the employed milk-acid bacteria of fermented sausages such as Bacterium lacticum, sheet coccus and micrococci etc., thereby can produce biogenic amine during the fermentation.Mutant strain low by screening amino acid decarboxylase enzymic activity or that do not have a decarboxylase has great importance for the biogenic amine content that reduces in the fermented sausages.
1 materials and methods
1.1 bacterial classification
For examination bacterial classification: Pediococcus pentosaceus I9 (Pediococcus pentosaceus I9), rice wine milk bacillus 54 (Lactobacillus sake 54), Lactobacillus pentosus R1 (Lactobacillus pentosus R1), Pediococcus parvulus M7 (Pediococcus parvus M7) and staphylococcus xylosus I2 (Staphylococcus xylosus I2).Above test strain be in 2001-2002 separate in by spontaneous fermentation meat product (ham, sausage and bacon) fermented meat prods main producing regions such as Hunan, Zhejiang and Sichuan obtain 120 surplus screen in the strain microorganism and obtain, the evaluation of bacterial classification is finished by Institute of Microorganism, Academia Sinica.
Commercial starter: the German CHR.HANSEN Bactoferm of company
TMF-1 starter for fermentative meat (lyophilize bacterium powder).
1.2 technical process
The fermented sausages technical process: meat cutting, cut mix, mix the back inoculating starter (10
7Individual/g), (20 ℃ of temperature, relative humidity 95% are fermented to pH5.0 in bowel lavage and fermentation afterwards; 15 ℃ of temperature, relative humidity 90% continue to ferment to pH4.6; ), carry out maturation (4 ℃ of temperature, relative humidity are 70%-80%) at last.Do not add spice.
1.3 the mensuration of biogenic amine
Derivatization method behind the mensuration employing reversed phase ion coupled columns of biogenic amine (histamine, cadaverine, putrescine, tyrasamine, spermidine, spermine and tryptamines).
High performance liquid chromatograph: day island proper Tianjin LC10A type; Chromatographic column: C
18Reversed-phase column (DISCOVERY); Ion pair reagent: perfluorooctane sulfonate; Moving phase: 10mmol/L perfluorooctane sulfonate, 0.1mol/L sodium acetate solution and acetonitrile; The liquid of deriving: 0.1 o-phthalaldehyde(OPA) solution; Excitation wavelength: 340nm; Emission wavelength: 440nm; Flow rate of mobile phase: 1.0ml/min; Flow velocity: 0.5ml/min derives.
Get an amount of sample with going up the machine analysis behind the perchloric acid extraction of 0.6mol/L.
2. result and analysis
2.1 the histamine growing amount of different strains
Histamine is one of most important biogenic amine composition in the fermented sausages, is formed by the Histidine decarboxylation.In biogenic amine, the physiology toxicity of histamine is the strongest.Biogenic amine can be degraded by some enzymatic metabolic reactions in human body cell, but alcohol and some medicine can suppress the activity of these enzymes, thereby has reduced detoxification efficient
[4]For a long time, investigators think that having only Pediococcus to belong to bacterium can produce histamine, but studies show that, some bacterial strains of lactobacillus (Lactobacillus) and grape wine Pseudomonas (Staphylococcus) also have L-Histidine decarboxylase. (histidine decarboxylase, HDC) therefore activity also can produce histamine.
Can find out that from Fig. 6 all strains testeds all can produce histamine during the fermentation, but content is different along with the difference of bacterial strain, promptly exists the bacterial strain effect.The histamine generation of L.sake 54 is the highest, reach 1.98mg/Kg, and the growing amount of L.pentosus R1 is minimum, is 0.831.98mg/Kg, and the histamine output of P.pentosaceusI9 and P.parvus M7 also is higher than contrast.
2.2 the putrescine of different strains and cadaverine growing amount
The Methionin decarboxylation generates cadaverine, and the effect of spermine acid decarboxylation base generates putrescine.Similar to the physiological action of histamine, cadaverine and putrescine also have the effect that rises blood pressure, have stronger physiology toxicity simultaneously, and the two is fishery products and the putrid and deteriorated feature product of meat product especially.Fig. 7 shows, can both produce the cadaverine and the putrescine of inequality during the fermentation for 4 strain bacterial strains of examination, and the cadaverine amount that all bacterial strains produce is all than putrescine amount height, and exists the bacterial strain effect.With contrast (Bactoferm
TMF-1) compare, the putrescine that Bacterium lacticum (L.sake 54 and L.pentosus R1) produces is relative lower than sheet coccus (P.pentosaceus I9 and P.parvus M7).
This experiment is also measured the content of the tyrasamine in the fermented sausages, spermidine, spermine and tryptamines, studies show that (seeing the following form), and all bacterial strains can both produce tyrasamine, spermidine and spermine, but does not produce tryptamines.This shows that strains tested does not all have the tryptophan decarboxylase activity.
Other kind biogenic amine content in the different strains fermented sausages
Biogenic amine content (mg/Kg)
Bacterial strain
Tyrasamine spermidine spermine tryptamines
P.pentosaceus I9 8.41 2.60 4.03 does not detect
L.pentosus R1 5.92 2.29 3.29 does not detect
P.parvus M7 6.18 2.02 3.88 does not detect
Bactoferm
TMF-1 5.24 3.27 3.50 does not detect
2.3 the composite influence of bacterial strain to the biogenic amine growing amount
Normally used starter mostly is two or more the composite starter that forms of bacterial strain greatly in the fermented sausages production.In fact can be regarded as a kind of special intergrowth relation in the starter between each single bacterial strain.Generally speaking, the effect of milk-acid bacteria in the starter such as Bacterium lacticum (Lactobacillus), sheet coccus (Pediococcus) mainly is in the short period of time the pH of sausage to be reduced to below 5.0, to suppress other harmful microbes growths.And the staphylococcus of alternate (Staphylococcus), micrococci (Micrococcus) local flavor main and sausage is formed with substantial connection with it.In sausage fermentation matrix, each component bacterial strain can be lived separately, but when they are grown in same matrix, can influence the other side's metabolism behavior again by Metabolic activity separately.Following table shows, after Pediococcus pentosaceus I9 and staphylococcus xylosus I2 are composite, can significantly reduce biogenic amine content in the fermented sausages.Wherein, the content of histamine drops to and can not detect from 1.42mg/Kg, and the content of putrescine and tyrasamine also has tangible reduction.In addition, the biogenic amine total amount of I9 and the composite ferment-fermented generation of I2 also is lower than the biogenic amine total amount of single strain fermentation of I9 and commercial ferment-fermented generation.
The composite influence of bacterial strain to biogenic amine content in the fermented sausages
Biogenic amine content (mg/Kg) biogenic amine total amount
Bacterial strain
Histamine putrescine cadaverine tyrasamine spermidine spermine tryptamines (mg/Kg)
P.pentosaceus I9 1.42 3.85 7.90 8.41 2.60 4.03 does not detect 28.21
P.pentosaceus I9+S.xylosus I2 does not detect 1.53 7.20 3.14 2.61 3.45 and does not detect 17.93
Bactoferm
TMF-1 0.89 2.72 8.25 5.24 3.27 3.50 does not detect 23.87
Annotate: the compound proportion of P.pentosaceus I9 and S.xylosus I2 is 1: 1
3. conclusion and discussion
Because the effect of enzyme and bacterium, protein, fat and carbohydrate take place to decompose and change and putrid and deteriorated in the meat.In the meat decay process, protein decomposes generation ammonia (NH
3) and amine (R-NH
2) wait alkalescence nitrogenous toxic substance such as tyrasamine, histamine, cadaverine, putrescine and tryptamines etc., they have been referred to as toxic amine.There is toxic amine to have certain toxicity, can causes food poisoning.Can cause vasoconstriction as tyrasamine, histaminergic distends the blood vessels, and cadaverine, putrescine etc. also can cause tangible toxic reaction, therefore, will reduce the content of biogenic amine in the product by screening excellent fermentation agent and technological measure in fermented sausages produces.This experimental result shows that in the sausage fermentation process, all strains testeds and commercial starter can both produce histamine, putrescine, cadaverine, tyrasamine, spermidine and spermine, but do not produce tryptamines, and the growing amount of each strains tested biogenic amine exists the bacterial strain effect.With sheet coccus and the composite compound starter inoculation fermentation that makes of staphylococcus, can significantly reduce in the product the especially content of histamine, putrescine and tyrasamine of biogenic amine total amount.This result of study also provides reference frame for screening the low natural bacterial strain of good production performance and biogenic amine turnout.
Claims (6)
1, staphylococcus xylosus I2 bacterial strain, its deposit number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.0919.
2, a kind of compound ferment is characterized in that: it is that Pediococcus pentosaceus I9 and staphylococcus xylosus I2 are cultured to logarithmic growth respectively after late period in modified MRS culture medium, again according to
Volume ratio mixed starter, wherein the deposit number of Pediococcus pentosaceus I9 at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.0918, and the deposit number of staphylococcus xylosus I2 at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.0919.
3, starter as claimed in claim 2 is characterized in that: the process for preparation of described modified MRS culture medium is as follows:
At first by following formulated nutrient solution: (unit/L)
Peptone 10g yeast powder 5g extractum carnis 10g
MgSO
4·7H
2O?0.58g???MnSO
4·4H
2O?0.25g???K
2HPO
4?2g
Dibasic ammonium citrate 2g sodium acetate 5g glucose 20g
Tween80 1mL cysteine hydrochloride 0.5g tomato juice 10～20% (v/v)
The nutrient solution that will prepare is according to the above ratio then transferred pH to 6.2-6.4,115 ℃ of moist heat sterilization 30min.
4, as claim 2 or 3 described starters, it is characterized in that: when Pediococcus pentosaceus I9 and staphylococcus xylosus I2 were cultivated in modified MRS culture medium, modified MRS culture medium was 28～32 ℃ of stationary states.
5, the application of the described starter of claim 2 in meat product is characterized in that: this starter of inoculation in the fermented meat food production.
6, the application of starter as claimed in claim 5 in meat product is characterized in that: described meat product is a fermented sausages, and the inoculation weight ratio is 0.5～1% starter before bowel lavage, ferments to pH5.0 in 20 ℃ of temperature, relative humidity 95%; Continue to ferment in 15 ℃ of temperature, relative humidity 90% to pH4.6.
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Assignee: LUOHE SHUANGHUI HAIYING FLAVORING FOOD Co.,Ltd. Assignor: HENAN SHUANGHUI INVESTMENT & DEVELOPMENT Co.,Ltd. Contract record no.: 2011410000039 Denomination of invention: Staphylococcus xylosus I2 strain, composite ferment produced thereby and the use of ferment in meat ware Granted publication date: 20070131 License type: Exclusive License Open date: 20050119 Record date: 20110413 |
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