CN108977380A - A kind of lactobacillus plantarum strain and its application - Google Patents

A kind of lactobacillus plantarum strain and its application Download PDF

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CN108977380A
CN108977380A CN201810888146.2A CN201810888146A CN108977380A CN 108977380 A CN108977380 A CN 108977380A CN 201810888146 A CN201810888146 A CN 201810888146A CN 108977380 A CN108977380 A CN 108977380A
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strain
fermentation
bacterium
lactobacillus plantarum
meat
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CN108977380B (en
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剧柠
赵梅梅
王雪蓉
张喻琳
胡婕
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Ningxia Yijiaren Food Co ltd
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Ningxia University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • A23L13/70—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor
    • A23L13/72—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor using additives, e.g. by injection of solutions
    • A23L13/74—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor using additives, e.g. by injection of solutions using microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation

Abstract

The present invention relates to microorganisms technical fields, it is proposed a kind of lactobacillus plantarum strain and its application, it is fermented using the lactobacillus plantarum (Lactobacillus plantarum) to cured beef, obtained fermented beef shape of product rule, surface muscle is in rose, fat white；Elastic preferable, section is solid neat；It is moderate with soft fragrant sweet and sour.

Description

A kind of lactobacillus plantarum strain and its application
Technical field
The present invention relates to microorganisms technical field more particularly to a kind of lactobacillus plantarum strain and its applications.
Background technique
Fermentation meat product refers under nature or manual control condition, special using having for microbial fermentation effect production Flavor, color and quality, and there is the meat products compared with long storage life.By microbial fermentation, change the quality of raw material Kind, flavor increased, and nutritive value is improved.In fermentation meat product whole process, meat endogenous enzymes itself and Microbial enzyme meeting decomposing protein generates a large amount of amino acid, to improve human body to the digestibility of fermentation meat product.And this A little free amino acids are all much the essential amino acids that human body needs, have apparent bioactivity, this makes the guarantor of meat products Strong property and trophism are greatly improved.Amino acid itself generates flavor substance with other substance reactions, promotes meat products Fragrance and flavour.Meanwhile some amino acid and other substances are compound can play antioxidation in meat products, and The sense organ for improving fermentation meat product as color auxiliary, has an impact the quality of meat products.
China's meat products is divided into Western-style Meat and Chinese style meat products two major classes.Western-style Meat is with bowel lavage, ham, training Based on root three classes product, Chinese style meat products includes salt down cured based article, meat dried product and jam product three classes product.Western-style fermentation The production of meat product industry metaplasia is more mature, but product does not meet compatriots' eating habit more.
Although Chinese style meat products has the long history of edible fermentation meat product in ethnic minority live inconcentrated communities, mostly Square product, is limited by local environment and weather.Traditional marinated meat product, it is sharp in its natural state generally using pork as raw material Anaerobic fermentation is carried out with microorganism and is made.Have the following problems: one side pork fat content is high, easily leads to the fat and heart Vascular diseases, the another aspect spontaneous fermentation period is long, controls bad easily pollution microbes.
Beef provides the protein of high quality rich in nutritional factors such as minerals, B family vitamin, potassium, iron, zinc.Compared with pig Meat ratio, the high 5-10% of protein content, the low 20-30% of fat content.Current fermented beef product in the market, with jerked beef Based on, belong to slight fermented product.Lack the beef product of depth fermentation.
Currently there are all kinds of commercial fermented meat strains, produce company and product type compared with horn of plenty, and mostly with It delivers directly based on examination fermentation, it is easy to operate.But these strains belong to the Western-style meat strain of fermentation, the meat bacterium of Chinese style fermentation mostly Kind is rarely found.
Butcher's meat category Chinese style fermentation meat product, generallys use spontaneous fermentation, and the production cycle is long, miscellaneous bacteria easy to pollute.
Existing commercialization leavening is chiefly used in Western-style fermentation meat product, and it is same that flavor easily occurs in the product of production, indistinctive Problem.In addition, the leavening of dedicated Chinese style fermentation meat product is rare in the market, to need to open the commercialization of Chinese style fermented meat Send out leavening corresponding.
Beef is fermented to cause protein denaturation and degradation in beef, is converted into the substances such as peptone, peptide, amino acid, both changed Kind product quality keeps nutrition more abundant, also improves the absorptivity of protein, be easier to the utilization that is absorbed by the body.But in the market Depth fermented product about beef is few.
Summary of the invention
It is necessary to propose a kind of lactobacillus plantarum (Lactobacillus plantarum) strain, the strain is in 2018 On June 11, in is preserved in the Chinese microorganism strain preservation pipe that address is city of BeiJing, China Chaoyang District North Star West Road 1 institute 3 Reason committee common micro-organisms center (CGMCC), deposit number are CGMCC No.15923.
It there is a need to propose that a kind of lactobacillus plantarum (Lactobacillus plantarum) strain salts down in fermentation Chinese style Application in beef processed.
It is fermented using strain centering type cured beef proposed by the present invention, obtained fermented beef shape of product rule Then, surface muscle is in rose, fat white；Elastic preferable, section is solid neat；It is suitable with soft fragrant sweet and sour In.
This is different with the fermenting microbe to ferment in the prior art to Western-style meat, is a kind of marinated ox of Chinese style Meat tailored version zymophyte.
Detailed description of the invention
Fig. 1 is individual morphology of the obtained fermentative lactobacillus P6 of screening under 100 times of oil mirrors.
Fig. 2 is individual morphology of the obtained fermentative lactobacillus K12 of screening under 100 times of oil mirrors.
Fig. 3-Fig. 6 is the sensory evaluation column of the influence of different strain, different fermentations time to fermentation beefbacon quality Figure.It is followed successively by L3, K12, P6, ZC4.
Fig. 7-Figure 10 is different strain sensory evaluation and pH situation of change histogram at different temperatures.
Figure 11-Figure 14 is different strain sensory evaluation and pH situation of change histogram under different humidity.
Figure 15-Figure 18 is different strain sensory evaluation and pH situation of change histogram under different vaccination amount.
Figure 19-Figure 24 is that lactic acid bacteria produces viscous experimental result display figure.
Figure 25-30 is that lactic acid bacteria produces gas experimental result display figure.
Figure 31,32 be lactic acid bacteria production H2S experimental result display figure.
Figure 33 is the growth curve chart of 4 plants of bacterium.
Specific embodiment
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below in conjunction with embodiment and experimental program into Row explanation.
One, strain source
P6: separation screening obtains in Yinchuan of Ningxia Province area city home spontaneous fermentation pickles.
K12: separation screening obtains in the commercially available Kefir grain strain naturally fermented milk for originating from Linzhi Area of Tibet.
Two, strain separating screening technique
1, pouring plate: by triangular flask sterilized MRS+0.5% calcium carbonate agar medium and MSA agar train Base is supported, heating fusing is cooled to 45 DEG C or so, pouring plate, each plate about 20mL.
2, sample dilutes: the spontaneous fermentation pickle juice for the commercially available Kefir grain strain naturally fermented milk and 1mL that 1mL is above-mentioned is taken, It is separately added into 9mL sterile water, it is 10 that dilution, which is made,-1Zymophyte suspension and pickle juice bacteria suspension, continue to take dilution respectively 1mL is put in sterile test tube, add 9mL sterile water, is mixed, and is taken turns doing 10 times and is serially diluted, takes the hair of a small amount of dilution respectively Yeast-like fungi liquid and pickle juice bacterium solution do the separation of fermentative lactobacillus.3, plate separates: the inoculation of A. plate streaking: using oese respectively Picking zymocyte liquid and each ring of pickle juice bacterium solution, sterile working carry out streak inoculation on plate；After scribing line, cover Lid, is upside down in 40 DEG C of constant incubators and cultivates 48h.B. plate coating inoculation: taking two aseptic flat boards, sterile flat at two Add 0.1mL zymocyte liquid and pickle juice bacterium solution on plate respectively, applies stick with sterile glass and be coated with bacterium solution uniformly on plate, put down It is put in 20min on experimental bench, is then inverted in 25 DEG C of constant incubator culture 48h.
4, observe bacterium colony: selection bacterium colony is distributed preferable plate, first observes its colonial morphology, tentatively finds out hair Ferment lactic acid bacteria bacterium colony.(MRS+0.5% calcium carbonate solid Screening of Media standard: it is good in media surface or growth inside, White is glossy, and neat in edge easily chooses adhesion, there is transparent circle.MSA solid medium screening criteria: raw in media surface Long good, white is glossy, and neat in edge easily chooses adhesion.) 5, microscopy: oese picking colony, bacterium processed are used from plate Smear, with Gram's staining, oil mirror is observed to obtain the strain P6 and K12 of two kinds of forms, the individual morphology picture of two kinds of strains. As shown in Figure 1, Figure 2.
Three, strain idenfication and conclusion
Label is sampled to the strain of two kinds of individual morphologies of acquisition, respectively K2, K3, K5, K10, K12, K13, K17、K18、K19、K20、K21、K22、L1、L2、L5、FX3、ZC4、ZC5、 ZC7、FXH11、FH2、FH6、P6、P7、Y4、Y6、 Y9、Y10、L3、L7。
Seven kinds of comparative experimentss in following (four, strain properties comparative experiments) are successively carried out to the above strain, with And 4 plants of strains after screening are carried out with the fermenting experiment of single bacterial strain (five) respectively, obtain every characteristic of strain P6 and K12 Being significantly better than other strains, P6 and K12 is optimal to the effect that cured beef ferments.
Molecular biology identification is carried out to strain P6 and K12, is sequenced later using 16S rDNA amplification, through sequencing result It is compared with the sequence in BLAST database, finally obtain the qualification result of two plants of bacterium: bacterial strain P6 is accredited as the bright beading of goldbeater's skin Bacterium (Leuconostoc mesenteroides) bacterial strain；K12 is accredited as lactobacillus plantarum (Lactobacillus plantarum).See the 16S rDNA sequence of bacterial strain P6 and the 16S rDNA sequence of bacterial strain K12.
Four, strain properties comparative experiments and conclusion
4.1, strain characteristic
(1) Leuconostoc mesenteroides P6 and Lactobacillus plantarum K12 is tolerable 6% sodium chloride solution, i.e., for 24 hours, OD is reachable respectively under 600nm for 37 DEG C of cultures in the MRS fluid nutrient medium containing 6%NaCl 2.360 with 2.351.
(2) the tolerable Asia Leuconostoc mesenteroides P6 and Lactobacillus plantarum K12 Sodium nitrate solution, i.e., 37 DEG C of cultures measure OD value difference for 24 hours, under 600nm in the MRS fluid nutrient medium containing 150mg/kg 2.517 with 2.611.
(3) Leuconostoc mesenteroides P6 and Lactobacillus plantarum K12 is in MRS liquid In body culture medium after 37 DEG C of culture 28h, pH value is down to 3.78 and 3.71 respectively.
(4) two plants of bacterium are on MRS solid plate culture medium, 37 DEG C of culture 48h, and bacterium colony occurs without viscous situation is produced.
(5) two plants of bacterium are seeded in the glucose equipped with Du Shi tubule and produce in gas culture medium, and 37 DEG C are cultivated for 24 hours, without production gas Phenomenon occurs.
(6) two plants of bacterium percutaneous puncture-inoculations are producing H2In S culture medium, 37 DEG C are cultivated 24 hours, and no black precipitate line occurs, no Generate hydrogen sulfide.
(7) Oxford cup middle punching on the MRS solid plate culture medium containing 10% skimmed milk is used, is added in hole respectively The above-mentioned two plants of strain lactobacillus suspensions of 100 μ L, the hydrolysis ability for observing casein afterwards for 24 hours are strong.
That is: the above conclusion (of pressure testing) is to verify Leuconostoc mesenteroides by following seven groups of comparative tests P6 and Lactobacillus plantarum K12 is high with salt tolerance, resistance to sodium nitrite is high, produce acid height, does not produce mucus, no It produces gas, produce hydrogen sulfide gas, the good characteristic that protein active has been degraded.
(1) the salt tolerant experimental program and test data of lactic acid bacteria, is shown in Table 1.
Lactobacillus inoculum in addition 2.0%, 4.0%, the MRS fluid nutrient medium of 6.0%NaCl, 37 DEG C of cultures For 24 hours, OD value is measured under 600nm, while making blank control not connect bacterium MRS fluid nutrient medium, compares its growing state.
OD value of the 1 30 plants of bacterium of table under 2.0%, 4.0%, 6.0% salt
Compared by the OD value measured to bacterial strain, discovery K2, K3, K5, K10, K12, K13, K18, K20, K21, P6, P7, The resistance to NaCl higher of 20 plants of bacterium of Y4, Y10, ZC4, ZC5, ZC7, FX3, FXH11, FH2, L3, after selecting this 20 plants of bacterium to carry out Continuous test.
(2) experiment of resistance to sodium nitrite and test data of lactic acid bacteria, is shown in Table 2.
Lactobacillus inoculum in addition 50mg/kg, 100mg/kg, 150mg/kg NaN02MRS fluid nutrient medium in, OD value is measured under 600nm, while making blank control not connect bacterium MRS fluid nutrient medium, compares its growing state.
2 20 plants of bacterium of table are in 50mg/kg, 100mg/kg, 150mg/kg NaNO2Under OD value
Compared by the OD value measured to bacterial strain, discovery K2, K3, K5, K10, K12, K13, K18, K20, L3, P6, Y4, The resistance to NaN0 of this 15 plants of bacterium of FXH11, ZC4, ZC5, ZC72Property it is high.
(3) experiment of production acid and test data of lactic acid bacteria, is shown in Table 3.
Lactobacillus inoculum in MRS fluid nutrient medium, 37 DEG C of cultures, for 24 hours after measure its pH value for the first time, while it is every The pH value that secondary survey does not connect bacterium MRS fluid nutrient medium makees blank control, sees the production acid situation of each lactic acid bacteria.
3 20 plants of bacterium pH value of table
By comparing the pH value that strain culturing measures for 24 hours, discovery K2, K3, K5, K10, K12, K13, K18, K20, L3, The pH value decline of this 15 plants of bacterium of P6, Y4, FXH11, ZC4, ZC5, ZC7 is very fast, illustrates to produce sour effect preferable.
(4) experiment of production mucus and test result of lactic acid bacteria, is shown in Figure 19-24.
Lactobacillus inoculum on MRS solid plate culture medium, 37 DEG C of constant temperature incubation 48h are then placed, picking colony is straight Connect observation.
Bacterium colony is provoked using oese, observation bacterium colony whether there is wire drawing or adhesion, according to the observation in test panel Bacterium colony is vaccinated the state after ring is provoked, and can determine the bacterium colony not wire drawing of this 7 plants of bacterium of ZC4, K3, K12, L3, P6, Y4, K18, Adhesion does not produce mucus.
(5) experiment of production gas and test result of lactic acid bacteria, is shown in Figure 25-30.
Lactobacillus inoculum is produced in gas culture medium in the glucose equipped with Du Shi tubule, 37 DEG C of cultures are for 24 hours.Observe Du Shi Whether there is bubble formation in tubule.
By observe Du Shi tubule, determine K2, K3, K5, K10, K12, K13, K18, K20, L3, P6, Y4, FXH11, ZC4, It is generated inside the tubule of this 15 plants of bacterium of ZC5, ZC7 without bubble, does not produce gas.
(6) the production H of lactic acid bacteria2S experiment and test result, are shown in Figure 31,32.
Lactic acid bacteria percutaneous puncture-inoculation is being produced H2In S culture medium, 37 DEG C cultivate 24 hours, just if there is black precipitate line It is to have H2S is generated, and is otherwise exactly not produce H2S.Protein degrading activity experiment
(7) test method: with Oxford cup middle punching on the MRS solid plate culture medium containing 10% skimmed milk, in hole 100 μ l lactobacillus suspensions of interior addition observe casein hydrolysis circle afterwards for 24 hours.Having that it's too late according to hydrolysis circle, size judges bacterial strain to albumen The hydrolysis ability of enzyme.
Protein degrading activity experiment and result such as the following table 4:
The protein degradation test result of 4 different strains of table
Strain name Hydrolysis ability Strain name Hydrolysis ability Strain name Hydrolysis ability
K2 -- K13 -- Y4 +
K3 -- K18 + FXH11 ++
K5 -- K20 -- ZC4 ++
K10 -- L3 + ZC5 +
K12 ++ P6 ++ ZC7 --
The size of more transparent circle determines the hydrolysis ability of this 8 plants of bacterium of ZC4, ZC5, K12, K18, Y4, FXH11, P6, L3 By force.
4.2, growth curve is drawn
According to above-mentioned seven groups of tolerance tests as a result, aggregate analysis, filters out salt tolerance height, resistance to sodium nitrite height, produces acid It is high, do not produce mucus, do not produce gas, do not produce hydrogen sulfide gas, ZC4, K12, P6, L3, Y4, K18 that protein active has been degraded this 6 Strain bacterium draws growth curve.Such as Figure 33.
As can be seen, K12, P6, this 4 plants of bacterium of L3, ZC4 can reach stationary phase for 20 hours or so, and growth is vigorous, and K12, This 2 plants of bacterium speeds of growth of P6 are fast, can reach stationary phase at 9 hours, 20 hours or so respectively, and growth is vigorous, therefore after conduct Bacterial strain is applied in continuous production.
Five, the fermenting experiment of single bacterial strain
4 plants of strain inoculateds for choosing above-mentioned function admirable do fermentation test into cured beef, to prove that K12, P6 are being sent out Application and effect in ferment cured beef.
5.1, experimental strain be by above-mentioned " four, strain properties comparative experiments and conclusion " screening obtain it is functional 4 plants of bacterial strains.
Lactobacillus plantarum K12, Leuconostoc mesenteroides P6,
Lactobacillus alimentarius L3, Leuconostoc mesenteroides ZC4.
5.2, test method
The investigation of (1) four plant of different strain inoculum concentration: inoculum concentration presses 0.2%, 1.0%, 2.0% 3 group of different vaccination amount Production fermentation butcher's meat, the pH of survey in every 5 hours stop fermentation when pH reaches 5.2 or less.
(2) four plants of different strain fermentation temperatures: by lactic acid bacteria in the fermentation of 28 DEG C, 37 DEG C, 41 DEG C progress butcher's meats, every 4 is small When survey a pH, when pH reaches 5.2 or less stop fermentation.
(3) four plants of different strains fermentation humidity: the fermentation that lactic acid bacteria is carried out butcher's meat 85%, 90%, 95%, every 5 is small When survey a pH, until pH reach 5.2 or less stopping fermentation.
(4) four plants of different strain fermentation times: by lactic acid bacteria 6h, 12h, 18h, for 24 hours, 30h carry out butcher's meat hair Ferment, each time point carry out the sensory evaluation of marinated meat product.
5.3 subjective appreciation
Using balanced non-fully block design BIB, subjective appreciation is carried out to the butcher's meat for reaching fermentation termination, specific as follows: The professional for learning food processing forms 5 people and evaluates group, it is desirable that 12h non-smoking, no drinking before evaluating member, do not eat it is pungent Peppery equal irritable foods.Evaluating member keeps certain distance, and the inaccurate mutually talk of evaluation process is to fermentation beefbacon aesthetic quality Evaluation, mainly for its appearance, structural state and flavor this three aspects attribute are evaluated, and evaluation criteria is commented referring to (table 4) A fixed product is complete, is gargled with clear water, and interval 10min evaluates next sample again, finally fills in grade form and signs, will be each The evaluation result of evaluating member collects, and is analyzed.5 panelists carry out integrated sensory's scoring to every kind of product respectively, Range of giving a mark is 0~100 point.
The fermentation beefbacon subjective appreciation standard of table 4
5.4 experimental result
(1) influence of different strain, different fermentations time to fermentation beefbacon quality, such as Fig. 3-6.
By K12, P6, L3, ZC4, four plants of bacterium, 6h, 12h, 18h, for 24 hours, 30h, inoculum concentration 0.2%, temperature 28 DEG C, humidity is 85% fermentation for carrying out butcher's meat.The fermentation of different fermentations time different strain, the sensory evaluation of marinated meat product is as schemed Shown in 3-6.
The results showed that extension of four plants of bacterium with fermentation time, downward trend is totally presented in pH value；Institutional framework Aspect, flavour mark are higher and higher；Appearance color is dimmed, and fat slightly turns to be yellow, this may be with the fat and protein quilt in meat Lactic acid bacteria is related using aoxidizing.It is most fast that digestion lactobacillus K12 reaches fermentation termination, and it is one plant that pH, which can reach 5.1, within 24 hours With the bacterial strain compared with high acid potentiality.Although the more other three plants of bacterium of fermentability are weak, the sensory evaluation of Leuconostoc mesenteroides P6 Highest, it is 81 points that integrated sensory, which evaluates score value, relative to 2.1 points of L3 high, 0.2 point of K12 high, 0.5 point of ZC4 high, product, and appearance Shape is slightly irregular, and coffee-like, fat jaundice is presented in muscle surface part；In terms of structural state, elasticity preferably, tie by section It is real neat；The soft fragranced of flavor, tart flavour are moderate.
(2) different strain sensory evaluation and pH situation of change at different temperatures, such as Fig. 7-10.
It by four plants of bacterium, ferments at 28 DEG C, 37 DEG C, 41 DEG C respectively, keeps inoculum concentration 0.2%, humidity is 85% constant, Result is observed after fermentation 15h.Different strain ferments at a temperature of different fermentations, the sensory evaluation of marinated meat product and pH such as Fig. 7-10 It is shown.
Downward trend is integrally presented as temperature increases pH in the 15h the experimental results showed that this four plants of bacterium are fermented simultaneously, raw The fermented meat overall appearance regular shape of production, surface muscle is dimmed, fat jaundice, and preferably, section is neat for structural state elasticity, tool There is the fragrance of fermentation but lactic acid flavor is insufficient.Aggregate analysis, more other two fermentations of the sensory evaluation valence of four plants of bacterium at 41 DEG C Temperature score is high, is equally divided into 81.9 points, is higher by 7.6 points compared with 28 DEG C of fermentations, is higher by 3.5 points compared with 37 DEG C of fermentations.P6 feels at 41 DEG C It is 83.0 points that official, which evaluates highest scoring,.In conjunction with pH curve, it is known that it is still best that 41 DEG C of fermenting plant lactobacillus K12, which produce sour effect,.
(3) different strain sensory evaluation and pH situation of change, such as Figure 11-14 under different humidity.
It by four plants of bacterium, ferments under 85%, 90%, 95% humidity respectively, keeping inoculum concentration is 0.2%, temperature 28 DEG C, ferment 15h when after observe result.Figure 11-14 is shown in the fermentation of different fermentations humidity different strain, the sensory evaluation of marinated meat product It is shown.
The experimental results showed that the trend that four plants of bacterium rise after falling before as the different pH of humidity are integrally presented.Fermented meat It is dimmed that overall organoleptic shows as appearance surfaces muscle, and fat jaundice, structural state is flexible, and section is neat, there is crack but unknown It is aobvious, fermenting aroma foot.Aggregate analysis, when humidity 90% is fermented, the more other two fermentations humidity of the sensory evaluation valence of four plants of bacterium Score is high, is equally divided into 83.8 points, is higher by 8.0 points compared with 85% fermentation, is higher by 3.1 points compared with 95% fermentation.P6 is commented in 90% sense organ Valence highest scoring is 85.6 points.In conjunction with pH curve, it is known that 41 DEG C of fermenting plant lactobacillus L3 product acid activities are best.
(4) different strain sensory evaluation and pH situation of change, such as Figure 15-18 under different vaccination amount.
It by four plants of bacterium, decomposes and is inoculated with 0.2%, 1.0%, 2.0% inoculum concentration, kept for 41 DEG C of temperature, humidity is 85%, ferment 15h hours observation fermentation results.The fermentation of different fermentations temperature different strain, the sensory evaluation of marinated meat product and pH As shown in Figure 15-18.
The experimental results showed that downward trend, appearance surfaces muscle is integrally presented with the raising pH of additive amount in four plants of bacterium Dimmed, fat jaundice, structural state shape comparison rule, fermenting aroma is insufficient, there is lactic acid fermented fragrance.Aggregate analysis body Analysis, at 2.0%, the more other two fermentation humidity scores of the sensory evaluation valence of four plants of bacterium are high, are equally divided into 76.2 points, compared with 0.2% fermentation is higher by 8 points, is higher by 2.5 points compared with 1.0% fermentation.P6 is 77.8 points in 2.0% sensory evaluation highest scoring.In conjunction with PH curve, it is known that it is still best that 2.0% fermenting plant lactobacillus K12, which produces sour effect,.
In conclusion 28 DEG C of best fermentation times are 24 hours according to sensory evaluation and pH curve graph, temperature 41 DEG C, humidity 90%, additive amount 2.0%.Although obtaining two plants of optimal bacterium by hygrogram is P6 and L3 but time diagram, Hygrogram, addition spirogram obtain optimal bacterial strain be P6, K12, in conjunction with sensory evaluation, the optimal bacterial strain of final choice be P6 and K12。
Six, the preparation of fermentation cured beef
6.1, actication of culture
Lactobacillus plantarum K12 of the invention is activated, it is necessary to be carried out in aseptic operating platform.The single bacterial strain that will be inoculated with It is inoculated in the place close to flame, oese is burnt, can be inoculated with when oese reddens, but put It first to be cooled down on test tube wall when entering test tube and carry out picking, the strain access that picking goes out is prepared into culture before In base test tube, whole process must all be carried out in flame periphery.The culture in later period；It is cultivated in the incubator that temperature is 37 DEG C 24 hours.Secondary culture 3 times according to the above method make strain maintain vigour vigorous.The strain that culture obtains is placed in centrifuge Centrifugation, is centrifuged 5 minutes by 3000 turns of revolving speed, obtains wet bacterium mud precipitating.
6.2, beef process early period
1. stripping and slicing: fresh beef is cut into the cuboid that a length of 2cm, width 1cm, thickness are 1cm,
2. mixed spices: the Chinese prickly ash of 3% salt and 1.25% uniformly being mixed, mixed spices are made.
The condiment 3. small fire stir-fries；Do not stop to stir-fry during frying, prevents local stir-bake to brown.Until color yellowing.
4. salt bath: the mixed spices that small fire stir-fries, which are uniformly stirred the meat piece prevented, does not have salt bath to arrive.
5. marinated sealing: vessel the being sealed property inspection before sealing to butcher's meat is had to, it can to prevent have gas leakage Energy.
6.3, strain access and fermentation
Lactobacillus plantarum K12 strain inoculum concentration is 2.0% inoculation fermentation of the cured beef after above-mentioned pickle.It ferments wet Degree control is being 90%~95%, and temperature is 41 DEG C, ferments 15~24 hours, and the pH value of beef is not more than 5.2 after detection fermentation , fermentation cured beef is made.
6.4, ferment cured beef product characteristic
Using the moisture content 72% for the cured beef product that ferments made from the process and strain of the present invention, pH 5.1, POV value 2.0mg/100g, TVB-N values are 6.1 × 10-6mg/100g.The shape of product rule fermented under the process conditions, Surface muscle is in rose, fat white；Elastic preferable, section is solid neat；It is moderate with soft fragrant sweet and sour.
This is different with the fermenting microbe to ferment in the prior art to Western-style meat, is a kind of marinated ox of Chinese style Meat tailored version zymophyte.
Seven, the 16S rDNA sequence of bacterial strain P6 are as follows:
CAGTCGAACGCACAGCGAAAGGTGCTTGCACCTTTCAAGTGAGTGGCGA ACGGGTGAGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTT GGAAACAGATGCTAATACCGAATAAAACTTAGTGTCGCATGACACAAAGTT AAAAGGCGCTTCGGCGTCACCTAGAGATGGATCCGCGGTGCATTAGTTAGT TGGTGGGGTAAAGGCCTACCAAGACAATGATGCATAGCCGAGTTGAGAGA CTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGC TGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCG CGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATGGGAAGAA CAGCTAGAATAGGAAATGATTTTAGTTTGACGGTACCATACCAGAAAGGGA CGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCCCGAGCGTTAT CCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTTATTAAGTCTGATGT GAAAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGA GTGCAGTAGAGGTAAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATA TATGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACTGCAACTGACGT TGAGGCTCGAAAGTGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCC ACACCGTAAACGATGAACACTAGGTGTTAGGAGGTTTCCGCCTCTTAGTGC CGAAGCTAACGCATTAAGTGTTCCGCCTGGGGAGTACGACCGCAAGGTTG AAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGT TTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGAAG CTTTTAGAGATAGAAGTGTTCTCTTCGGAGACAAAGTGACAGGTGGTGCAT GGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCTTATTGTTAGTTGCCAGCATTCAGATGGGCACTCTAGCGAGAC TGCCGGTGACAAACCGGAGGAAGGCGGGGACGACGTCAGATCATCATGCC CCTTATGACCTGGGCTACACACGTGCTACAATGGCGTATACAACGAGTTGC CAACCCGCGAGGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCGGATTGT AGTCTGCAACTCGACTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAG CACGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACC ATGGGAGTTTGTAATGCCCAAAGCCGGTGGCCTAACCTT。
Eight, the 16S rDNA sequence of bacterial strain K12 are as follows:
CGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTG AGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGG ATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGG TCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCG TATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCG ACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCC TACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGG AGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGT TAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAAC CAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG GCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTT AAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACT GGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTG AAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGT CTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGAT ACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTC CGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACG GCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT GGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGA CATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATAC AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACT CTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA ATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTAC AACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAG TTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAAT CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC CCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTT AGGAACCAGCC。
The above disclosure is only the preferred embodiments of the present invention, cannot limit the right of the present invention with this certainly Range, those skilled in the art can understand all or part of the processes for realizing the above embodiment, and according to right of the present invention Equivalent variations made by it is required that, still belongs to the scope covered by the invention.

Claims (2)

1. a kind of lactobacillus plantarum (Lactobacillus plantarum) strain, the strain was in preservation on June 11 in 2018 In the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) for being located at BeiJing, China, deposit number is Deposit number is CGMCC No.15923.
2. lactobacillus plantarum (Lactobacillus plantarum) as described in claim 1 is in fermentation Chinese style cured beef In application.
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