CN1271201C - Pediococcus pentosaceus strain, ferment produced thereby and the use of ferment in meat ware - Google Patents

Abstract

The present invention discloses a pediococcus pentosaceus bacterial strain and leaven manufactured by the bacterial strain. The pediococcus pentosaceus I9 bacterial strain has the preservation number in the common micro-organism center of the CCCCM, which is CGMCC No. 0918. The pediococcus pentosaceus I9 bacterial strain of the present invention can bear 6.0% of table salt solution and nitrite solution with the concentration of 80 to 100 mg/Kg. The leaven made by using the pediococcus pentosaceus I9 bacterial strain as a bacterial strain can be applied to the fermentation production of meat food. A manufactured fermented sausage has palateful fermentation flavor, generates no foreign odor, generates a small amount of biogenic amine, has good color development effect, and improves the flavor quality of a product. The optimum temperature is 28 DEG C, and inactivation is generated within the range of 57 to 60 DEG C.

Description

A kind of Pediococcus pentosaceus bacterial strain and the starter and the application of this starter in the meat product production process made from this bacterial strain

Technical field

The starter and the application of this starter in the meat product production process that the present invention relates to a kind of Pediococcus pentosaceus bacterial strain and make with this bacterial strain.

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.Therefore, by purebred separation and Culture technology and the Microbial Breeding technology to organism of fermentation in the traditional zymotic meat product, the microbial starter culture bacterial classification that screening and structure have good character is the focus target of research.

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 starter that the purpose of this invention is to provide a kind of Pediococcus pentosaceus bacterial strain and make with this bacterial strain, this starter can be applied in the fermentation production process of meat product.

For achieving the above object, the technical solution used in the present invention is: a kind of Pediococcus pentosaceus I9 bacterial strain (Pediococcus Pentosaceus I9), its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is: CGMCC No.0918

Described Pediococcus pentosaceus I9 is placed on is cultured to logarithmic growth in the modified MRS culture medium and promptly obtains starter late period; The process for preparation of described modified MRS culture medium is as follows:

At first form the preparation nutrient solution according to following every liter of unit:

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 was cultivated in modified MRS culture medium, modified MRS culture medium was 28～32 ℃ of stationary states.

This starter can be applied in the meat product fermentative production, promptly can inoculate this starter in the fermented meat food production.

Described meat product is a fermented sausages, and inoculation quality ferments to pH5.0 in 20 ℃ of temperature, relative humidity 95% than the starter that is 0.5～1% before bowel lavage; Continue to ferment in 15 ℃ of temperature, relative humidity 90% to pH4.6.

After adopting technique scheme,, avoided the complicacy of compound ferment preparation because starter is a single strain; 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 6.0% salt solution through the advance copy starter culture.

(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 culture 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.Optimum temperuture is 28 ℃, deactivation in 57～60 ℃ of scopes.

Description of drawings

Fig. 1 is the separation process figure of bacterium;

Fig. 2 is that bacterium is at MRS-nutrient agar (+5%CaCO ₃) go up and produce sour situation observation figure;

Fig. 3 observes figure for morphological features;

Fig. 4 is a histamine growing amount comparison diagram in the different strains fermented sausages;

Fig. 5 is putrescine and a cadaverine growing amount comparison diagram in the different strains fermented sausages.

Pediococcus pentosaceus I 9 bacterial strains of the present invention (Pediococcus Pentosaceus I 9) are submitted China Committee for Culture Collection of Microorganisms common micro-organisms center on April 8th, 2003, and (be called for short: CGMCC) give preservation, deposit number is: CGMCC No.0918.

Embodiment

One, fermented sausages bacteria culture screening criteria

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.

2, 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, as Italian Salami, in addition can be higher, so the screening bacterial strain must have good salt tolerance, requirement can be under at least 3.0% common salt concn 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.

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, the fermenting-ripening phase of non-dry fermented sausages (final aw is between 0.94～0.96) and half-dried fermented sausages (final aw is between 0.90～0.95) is short, main nitrite and the color auxiliary sodium ascorbate of adding, 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.In the nitrite solution of advance copy invention starter bacterial strain under 80～100mg/Kg concentration conditions, still can grow.

(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.

4, has 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.

Two, strains separation

This strains separation substratum separates solid mediums such as adopting 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 ₂O0.05g, 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, NaCI 75g, 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 1.

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 is by 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 test of salt tolerance is the MRS liquid nutrient medium that the test bacterium is inoculated in pH4.5, observes its upgrowth situation; The mensuration of salt tolerance is with the mensuration of lactic acid tolerance.Whether gas and the research of mucus generation situation have gas to produce is adopted 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 μ l, includes 2.5mM Mg ²⁺, 20mM Tris-HCl (pH 9.0), 0.1%Triton-100 (v/v), the dNTP of 100 μ M, the 2.5UTaqDNA polysaccharase, the template DNA of 300ng, the primer of 40pmol covers and adds a cover with 25 μ 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 AmplitronI (Bamstead Thermolyne Corporation) and carries out.

Three, bacterial strain 16SrRNA order-checking and be the starter PCR product electrophoresis purifying made of bacterial classification with this bacterial strain after, 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.

1, 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.

2, 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 ' smanual of 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 HA, et al.Bergey ' s manual of determinative bacteriology (9 ^ThEdition) .Batimore:Williams﹠amp; Wilkins, 1993 carry out.

3, result and analysis

3.1, 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.

3.2, 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, separates 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 typeII 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.

3.3, identification of strains

3.3.1, the evaluation of I9

(1) morphological feature

The solid culture feature is after cultivating 2d under 30 ℃ of conditions, and I9 is at MRS+5%CaCO ₃Form the obvious transparent circle on the solid medium, smooth surface is moistening, as shown in Figure 2.

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 of cell dia, as shown in Figure 3.

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 table 1 isolated strains I9

Certified variety	The result	Certified variety	The result
				The catalase litmus milk produces the acids type from glucose and produces under acid, the aerogenesis pH9.6 condition under the pH4.5 condition 5%NaCl anaerobism from glucose and cultivate under the lower 10 ℃ of conditions of 45 ℃ of conditions of aerobic cultivation	Negative produce that acid is produced in yogurt acid but not aerogenesis-+-++-+	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 (table 1),, 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 institute

Have known array to carry out homology relatively, I9 of display separation strain as a result and Pediococcus pentosaceus (P.pentosaceus) have the homology (99%) of height.This confirms that from the molecular systematics angle strain isolated I9 is Pediococcus pentosaceus (Pediococcus pentosaceus), and the result is as follows:

CATGCAAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACT

GATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGT

GGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAA

TACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCT

GCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTA

AAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGG

CCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGT

AGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTG

AGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGGTTAAGAAGAACG

TGGGTAAGAGAACTGGTTACCCAGTGACGGTATTTAACCAGAAAGCCA

CGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGT

TATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCT

AATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGA

GACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAA

TGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTC

TGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGA

TACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGT

TTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGA

GTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCAC

AAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTAC

CAGGTCTTGACATCTTCTGACAGTCTAAGAGATTAGAGGTTCCCTTCGG

GGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAG

ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCA

GCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGA

AGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACAC

ACGTGCTACAATGGATGGTACAACGAGTCGCGAGACCGCGAGGTTAAG

CTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCC

TACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTG

AATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTT

GTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCTAA

GGTGGGACAGATGATTAGGGTGAAGTC

Above-mentioned Pediococcus pentosaceus I9 is placed on is cultured to logarithmic growth in the modified MRS culture medium and promptly obtains starter late period.

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 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 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 cultivated in modified MRS culture medium, modified MRS culture medium was 32 ℃ of stationary states.

Four, this bacterial strain application of starter in meat product of making

In the fermented meat food production, can inoculate starter of the present invention.As in the production process of fermented sausages, inoculation quality ferments to pH5.0 in 20 ℃ of temperature, relative humidity 95% than the starter that is 0.5% before bowel lavage; 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 slaking 70%～80% time.Ferment-fermented 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%.

Starter uses the single bacteria leaven of Pediococcus pentosaceus I9 (Pediococcus pentosaceus I9).Fermented sausages technical process: the first step: meat cutting, pre-freeze (18 ℃, 24h); Second step: cut and mix mixing; The 3rd step: inoculating starter (10 ⁷Individual/g); The 4th step: bowel lavage; The 5th step: (20 ℃ of temperature, relative humidity 95% are fermented to pH5.0 in fermentation; 15 ℃ of temperature, relative humidity 90% continue to ferment to pH4.6); The 6th step: ripe (14 ℃ of temperature, relative humidity are 70%-80%).Do not add spice.

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.Produce specificity analysis by biogenic amine, to examine or check its utility value on producing to preferred strain of the present invention.

1, 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.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. 4 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. 5 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 (table 2), 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 table 2 different strains fermented sausages

Table1 Other Biogenic amines in fermented sausage with different strains

Bacterial strain	Biogenic amine content (mg/Kg)
					Tyrasamine	Spermidine	Spermine	Tryptamines
	P.pentosaceus I9 L.sake 54 L.pentosus R1 P.parvus M7 Bactoferm TM F-1	8.41 9.28 5.92 6.18 5.24	2.60 2.51 2.29 2.02 3.27	4.03 3.89 3.29 3.88 3.50					Do not detect

2.3 the composite influence of bacterial strain to the biogenic amine growing amount

Normally used starter mostly is 2 kinds or the two or more composite starters that forms of bacterial strain greatly in the fermented sausages production.In fact can be regarded as a kind of special alternate in the starter between each single bacterial strain

Relation.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.Table 3 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.

Table 3 bacterial strain is composite to influence Table Effecf of coculture Starter on Biogenic amine content in fermented sausage to biogenic amine content in the fermented sausages

Bacterial strain	Biogenic amine content (mg/Kg)							Biogenic amine total amount (mg/Kg)
									Histamine	Putrescine	Cadaverine	Tyrasamine	Spermidine	Spermine	Tryptamines
	P.pentosaceus I9 P.pentosaceus I9+S.xylosus I2 Bactoferm TM F-1	1.42 do not detect 0.89	3.85 1.53 2.72	7.90 7.20 8.25	8.41 3.14 5.24	2.60 2.61 3.27	4.03 3.45 3.50									Do not detect	28.21 17.93 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 (5)

1, a kind of Pediococcus pentosaceus I 9 bacterial strains (Pediococcus Pentosaceus I9), its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is: CGMCCNo.0918.

2, a kind of starter made from the described Pediococcus pentosaceus bacterial strain of claim 1 is characterized in that: described Pediococcus pentosaceus I9 is placed on is cultured to logarithmic growth in the modified MRS culture medium and promptly obtains starter late period; The process for preparation of described modified MRS culture medium is as follows:

At first form the preparation nutrient solution according to following every liter of unit:

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 acid sodium 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.

3, starter as claimed in claim 2 is characterized in that: when Pediococcus pentosaceus I 9 was cultivated in modified MRS culture medium, modified MRS culture medium was 28～32 ℃ of stationary states.

4, claim 2 or 3 application of described starter in the meat product production process is characterized in that: this starter of inoculation in the fermented meat food production.

5, the application of starter as claimed in claim 4 in the meat product production process is characterized in that: described meat product is a fermented sausages, and inoculation quality ferments to pH5.0 in 20 ℃ of temperature, relative humidity 95% than the starter that is 0.5～1% before bowel lavage; Continue to ferment in 15 ℃ of temperature, relative humidity 90% to pH4.6.

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