CN102199566B - Lactic acid bacteria strain for protecting meat product color - Google Patents
Lactic acid bacteria strain for protecting meat product color Download PDFInfo
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Abstract
The invention discloses a lactic acid bacteria strain for protecting meat product color. The strain belongs to Lactobacillussalivarius and is preserved CCTCC (China Center for Type Culture Collection) in Wuhan university, Wuhan, China on Dec.31, 2010 and has a preservation number of CCTCCNO: M2020374 (Hstrain). The invention also discloses a meat product color fixative prepared from the strain and a preparation method of the color fixative. The preparation method comprises the following steps of: carrying out fermented culture on the strain, then collecting thallus, adding a diluting agent and mixing to obtain the meat product color fixative. The lactic acid bacteria strain for protecting the metal product color has stronger color fixation capacity, not only can keep the original flavors of products, but also can keep bright color of fresh met or fermented meat.
Description
Technical field
The present invention relates to food processing field, be specifically related to a kind of lactobacillus strain that is used to protect the meat product color.
Background technology
The color and luster of meat product and stability are important organoleptic qualities, and it directly determines human consumer's purchase to be intended to, and therefore how making meat keep bright red is the fresh-keeping important subject of meat.
The redness of animal muscle mainly is by the myohaemoglobin in the muscle cell (Myoglobin, Mb; 70% ~ 80%) and the oxyphorase in the capillary blood vessel (Hemyoblobin, Hb; 20%-30%) constituting, is the myohaemoglobin colour generation more than 90% in the ketoboidies muscle after butchering bloodletting, so the content of myohaemoglobin has certain positive correlation with yellowish pink, and Mb content is many more, and yellowish pink is dark more.The color and luster of fresh meat depends primarily on three kinds of different chemical existence forms and stability, i.e. the deoxidation myohaemoglobin (Mb-H of myohaemoglobin in the meat (Mb)
2O), oxymyoglobin (MbO
2) and MetMb (Metmyoglobin, content MetMb) and distribution.The colour generation effect of Mb comes from its intramolecular protoheme prothetic group, the different group of the valence state of iron ion and bonded in the protoheme prothetic group, and their actings in conjunction make Mb and verivate thereof different on absorption spectrum, thereby show distinct colors.At the during storage of fresh meat, three kinds of forms of Mb are the phase co-conversion constantly, and their relative content is determining the color of muscle.
All there be deficiency and defective in various degree in the method that existing fresh meat protects look with technology.People have researched and developed multiple fresh meat color protecting method at present, are with the amino acid (like Histidine and halfcystine) of imidazolyl etc. like nitrite treatments, NO or CO modified atmosphere packaging, hyperoxia modified atmosphere packaging, interpolation natural antioxidants (like tea-polyphenol, carnosine, vitamin E etc.), interpolation.But the nitrite (or nitrate salt) that has only that is extensively adopted is handled, or methods such as CO modified atmosphere packaging and hyperoxia modified atmosphere packaging.Meat processing enterprise generally adopts the chromogenic reagent of this nitrite (or nitrate salt) as the fermentation meat both at home and abroad, obtains meat product ideal color and luster and local flavor, extends the shelf life.But the safety issue of nitrate salt and nitrite is perplexing the mankind always: excessive nitrite can make normal oxyphorase (ferrous iron) become MetMb (ferric iron) after getting into blood, loses function of carrying oxygen, causes histanoxia; Nitrate salt and nitrite can with multiple aminocompound (mainly from the protein degradation production) reaction, produce strong carcinogenic N-nitroso compound, like NSC 223080 etc., the substitute of therefore developing the nitrite chromogenic agent is necessary.
Likewise, CO and high oxygen are stealthily substituted and are contained in meat and protect look and also have weak point in using.CO combines with Mb to form carbonyl myoglobin, presents stable shiny red.The binding ability of Mb and CO is higher about 240 times than the bonding force of itself and oxygen, and therefore, the CO of trace just can keep the bright red of muscle, and the carbonyl myoglobin of formation is closely similar with the absorption spectrum of Oxyhemoglobins, and stability is higher, is difficult for oxidation, and color and luster is stablized.Yet use the CO controlled atmosphere to have toxicity problem; After although the international standard ISO that Norway meat expert detects according to toxic gas uses the toxicity of CO gas to detect in to controlled atmosphere cooling meat; Reach a conclusion: there is not any poisonous harm in lower concentration CO mixed gas (0.5%-1.0%) to the human consumer; But and after causing the case of poisoning from occurring on the world market that edible CO protects the coloured gold marlin; People query to the application of CO color protection technology, utilize CO to carry out fresh meat and protect look and still deposit dispute, the further investigation of still needing.
The principle of hyperoxia modified atmosphere packaging is in sealing bag, to feed high oxygen concentration, promote the formation of oxymyoglobin, thereby make muscle present bright red, but high-concentration oxygen meeting accelerate fat oxidation influences meat.In a word, there is the place of discussion in the use of modified atmosphere packaging, the use mixed gas of how arranging in pairs or groups, and how rationally using CO etc. all is the focus of research.
Except above-mentioned several kinds of meat colour protecting agents; In recent years; Many reports that the fermented meat prods color influenced about mikrobe are arranged, and promptly using microbe is transformed into red verivate (Arihara K, Kushida H with myohaemoglobin (Mb); Kondo Y, et al. Conversion of metmyoglobin to bright red myoglobin derivatives by
Chromobacterium violaceum, Kurthiasp., and
Lactobacillus fermentumJCM1173. J. Food Science, 1998 (53): 38-42).Discover that lactobacillus fermentum JCM1173 can be transformed into MetMb (Met-Mb) NO-Mb of shiny red.Morita (Morita H, Yoshikawa H, Sakata R, et al. Synthesis of nitric oxide from the two equivalent guanidino nitrogens of L-arginine by
Lactobacillus fermentum. J Food Sci; 1997 (12): 7812-7815) 10 strain lactobacillus fermentums are discovered; They all can be transformed into NO-Mb with Met-Mb in the MRS substratum; Wherein the conversion capability of IFO3956 is the strongest, thereby infers that lactobacillus fermentum IFO3956 possibly contain bacterium nitricoxide synthase (NOS).Moler (Moler J K S; Jensen J S; Skibsted L H; Et al. Microbial formation of nitrite-cured pigment, nitrosylmyoglobin, from metmyoglobin in model systems and smoked fermented sausages by
Lactobacillus fermentumStrains and a commercial starter culture. Eur. Food Res. Technol.; 2003; 216:463-469) lactobacillus fermentum JCM1173 and IFO3956 are applied in the no nitre Fermented Sausages production, have obtained stable butcher's meat color.
Except that milk-acid bacteria, also there is research to report staphylococcic color development effect.It is red that Morita discovers that several kinds of staphylococcuses can make Fermented Sausages produce ideal; And the color that forms in some cases is than also stable (the Morita H of nitrite cured meat; Sakata R; Nagata Y. Nitric oxide complex of iron and bacterial influence on its formation. J Food Sci., 1996 (61): 1021-1023; Morita H, Sakata R, Nagata Y. Nitric oxide complex of iron (II) myoglobin converted from metmyoglobin by Staphylococcus xylosus. J Food Sci, 1998 (63): 352-355).
The compound ferment that Chinese patent " a kind of Pediococcus pentosaceus bacterial strain and starter and the application of this starter in meat product supported with this bacterial strain " (patent No. ZL03126227.9) is processed Pediococcus pentosaceus and staphylococcus xylosus is used for the production process of meat product such as fermented sausages; Color development is respond well; And with rich flavor, improved the flavor quality of product..
More than report and patent all are confined to utilize milk-acid bacteria or staphylococcus to the color development effect of type meat product of fermenting, but do not see about fresh meat and protect the look documents and materials.
Along with people's is to the understanding of food safety and the improve of requirement, and people hope to have a kind of novel colour protecting agent can replace nitrous acid and modified atmosphere packaging, and it can keep the original local flavor of meat, can safeguard or prolong fresh meat chromatic colour again.Utilizing the meta-bolites of generally regarded as safe (GRAS) food-grade microorganisms to carry out fresh meat protects look and has bigger development potentiality.
Summary of the invention
The objective of the invention is to provides a kind of lactobacillus strain of protecting the meat product color according to the above-mentioned deficiency that exists in the prior art, called after H bacterial strain.
Another object of the present invention provides the application of above-mentioned lactobacillus strain.
The present invention realizes above-mentioned purpose through following technical scheme:
A kind of lactobacillus strain that is used to protect the meat product color, said bacterial strain are lactobacillus salivarius H strain (lactobacillus salivarius H strain)
Lactobacillus salivariusH strain, depositary institution are Chinese typical culture collection center (CCTCC), and deposit number is CCTCC NO:M 2010374, and the preservation address is a China. Wuhan. and Wuhan University, preservation date is on December 31st, 2010.
The above-mentioned lactobacillus strain that is used to protect the meat product color, colonial morphology is: bacterium colony is rounded, protuberance, oyster white, smooth surface, colony diameter 1～2mm; The mycelia thickness does not have peculiar pigment; No gemma, gram-positive microorganism; Cell is rod-short, and is single or become double row; Do not move amphimicrobian; Main biochemical character: catalase is negative, and nitrite reductase is positive, and nitricoxide synthase is positive, and high ferro myohemoglobin reductase is positive, no amino acid decarboxylase enzymic activity; Glucose, lactose, sucrose, semi-lactosi, SANMALT-S, N.F,USP MANNITOL, fructose and the experiment of trehalose fermentation and acid are positive; The experiment of gluconate fermentation and acid is negative; Do not produce hydrogen peroxide, do not produce hydrogen sulfide, not gelatin hydrolysate and starch, not hydrolysis l-arginine; The qualitative positive of lactic acid, the V-P reaction negative; Biological safety check H bacterial strain no pathogenicity.
The above-mentioned lactobacillus strain that is used to protect the meat product color, its 16S rRNA sequence is shown in SEQ ID NO:1.
A kind of meat product colour protecting agent is characterized in that comprising the above-mentioned lactobacillus strain that is used to protect the meat product color.
The preparation method of above-mentioned meat product colour protecting agent may further comprise the steps:
(1) fermentation culture of bacterial classification: select cell age at 12 ~ 18 hours H bacterial strain seed, inoculum size is 3% ~ 5% (V/V or V/W), 37 ℃ of fermentation culture of liquid modified MRS culture medium, fermentation time 18 ~ 24 hours;
Aforesaid liquid modified MRS culture medium preparation: glucose 20g, peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract 5g, dibasic ammonium citrate 2g, potassium hydrogenphosphate 2g, sodium acetate 5g, MgSO
47H
2O 0.58 g, MnSO
44H
2O 0.25g, tween 80 (being Tween 80) 1mL, lime carbonate 15g, zero(ppm) water 1000mL, pH6.4 ~ 7.0,121 ℃, sterilization 15 ~ 30min.
(2) collect thalline after fermentation is accomplished, add thinner, mix promptly obtaining the meat product colour protecting agent.
The preferred sterilized water of said thinner, pure water or zero(ppm) water.
The mass ratio of said thalline and thinner is preferably 1 ~ 10:10 ~ 19.
The application of said meat product colour protecting agent in protection meat product color, the especially application in fresh or the refrigerated meat product course of processing.Adopt infusion method or spraying process with colour protecting agent attached to fresh or refrigerated meat product surface; Said infusion method is meat product to be placed in the colour protecting agent soak, and dries the final vacuum packing, places 20 ℃ of preservations again; Said spraying process is that colour protecting agent is joined in the lactic acid solution, is sprayed onto the meat product surface, sprays dosage and is about 50 ~ 100ml colour protecting agent/1000g meat product.
The present invention has following beneficial effect:
It is of the present invention that to be used to protect the lactobacillus strain of meat product color be safe bacterial strain, no pathogenicity, and have the stronger look ability of protecting, and can keep the original local flavor of product, can keep fermented meat or fresh meat chromatic colour again.Can avoid food-safety problem, also can as modified atmosphere packaging, may not cause CO to poison and perhaps produce the fats oxidn problem because of using the nitrite chromogenic agent to produce.
Description of drawings
Fig. 1. screening H bacterial strain colonial morphology among the embodiment 1;
Fig. 2. H strain morphology under the oily mirror;
Fig. 3. H strains separation, screening process figure;
Fig. 4. the 16S rRNA analyses and comparison of H bacterial strain are figure as a result, 1:FJ390111 (plant lactobacillus) wherein, 2:AB470238 (rice wine milk bacillus); 3:AB470231 (streptococcus acidi lactici), 4:H bacterial strain, 5:AY137588 (lactobacillus salivarius); 6:EU694139 (bacillus bulgaricus); 7:EF468098 (Lactobacterium acidophilum), 8:AB375445 (lactobacillus curvatus), 9:AF182720 (lactobacillus fermentum);
Fig. 5. each is handled pork and places the color comparison after 1 day, and wherein A is the blank group of sterile distilled water, and B is for adding NaNO
2Positive controls, C is for adding H bacterial strain group (H bacterial strain group), the D group is added lactobacillus fermentum J;
Fig. 6. each is handled pork and places the color comparison after 2 days, and wherein A is the blank group of sterile distilled water, and B is for adding NaNO
2Positive controls, C is for adding H bacterial strain group (H bacterial strain group), the D group is added lactobacillus fermentum J;
Fig. 7. each is handled pork and places the color comparison after 3 days, and wherein A is the blank group of sterile distilled water, and B is for adding NaNO
2Positive controls, C is for adding H bacterial strain group (H bacterial strain group), the D group is added lactobacillus fermentum J;
Fig. 8. different microbial inoculums are handled the brightness value (L value) of pork and are schemed over time;
Fig. 9. different microbial inoculums are handled the red scale value (a value) of pork and are schemed over time;
Figure 10. different microbial inoculums are handled the yellow value degree (b value) of pork and are schemed over time;
Figure 11. each handles the spectral scan of pork placement after 1 day figure as a result;
Figure 12. each handles the spectral scan of pork placement after 2 days figure as a result;
Figure 13. each handles the spectral scan of pork placement after 3 days figure as a result;
Figure 14. each is handled cold fresh meat and places the color comparison after 0 day, and wherein A is the blank group of sterile distilled water, and B is for adding NaNO
2Positive controls, C is for adding H bacterial strain group (H bacterial strain group);
Figure 15. each is handled cold fresh meat and places the color comparison after 2 days, and wherein A is the blank group of sterile distilled water, and B is for adding NaNO
2Positive controls, C is for adding H bacterial strain group (H bacterial strain group);
Figure 16. each is handled cold fresh meat and places the color comparison after 4 days, and wherein A is the blank group of sterile distilled water, and B is for adding NaNO
2Positive controls, C is for adding H bacterial strain group (H bacterial strain group);
Figure 17. each is handled cold fresh meat and places the color comparison after 6 days, and wherein A is the blank group of sterile distilled water, and B is for adding NaNO
2Positive controls, C is for adding H bacterial strain group (H bacterial strain group);
Figure 18. the red scale value of the cold fresh meat of different treatment (a value) is schemed over time;
Figure 19. the yellow value degree of the cold fresh meat of different treatment (b value) is schemed over time.
Embodiment
Explain technical scheme of the present invention with embodiment below, but do not limit the present invention in any way.
1. the initial gross separation of bacterial strain, screening
(1) preparing culture medium
Modified MRS solid medium: glucose 20g, peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract 5g, dibasic ammonium citrate 2g, potassium hydrogenphosphate 2g, sodium acetate 5g, MgSO
47H
2O 0.58 g, MnSO
44H
2O 0.25g, tween 80 (being Tween 80) 1mL, lime carbonate 15g, agar 20g, zero(ppm) water 1000mL, pH6.8,121 ℃, 15 min sterilize.
(2) sample separation, screening bacterial strain
Sample thief 1.0g joins mixing in the 10.0ml sterilized water from the fermentation ham, and this gets 10 times of diluted liquid 1.0ml for 10 times of dilutions, adds mixing dilution in the sterilized water of 9.0mL again, and this is dilution 10
2Doubly, method progressively is diluted to 10 according to this
10Doubly, get diluent 1.0ml then in petridish and add 20ml, 50 ℃ modified MRS solid medium; Shake up, in 37 ℃ of incubators, cultivate 18 ~ 24h, dissolve the bigger single bacterium colony of calcium circle in each substratum of picking; Repeat this step; Each step purifying is all wanted microscopy observation, after the cellular form unanimity, single strain is lined in the modified MRS solid medium and cultivates 24h, seals up for safekeeping in 4 ℃ or-20 ℃ of preservations with aseptic glycerine afterwards.
(3) screening can transform the bacterial strain of oxyphorase
Hemoglobin solutions is joined in the modified MRS solid medium that is cooled to 50 ℃, and the methemoglobin final concentration is 2-5mg/ml.The bacterial strain of step (2) gained is inoculated in the modified MRS solid medium that contains oxyphorase with diluting flat band method; Cultivate 24 ~ 48h in 37 ℃; Select the bacterium colony with obvious shiny red or maroon, further do line and separate, each step purifying all microscopy is observed; After the cellular form unanimity, single strain is lined in the modified MRS solid medium, aseptic glycerine is sealed up for safekeeping in 4 ℃ or-20 ℃ of preservations behind 37 ℃ of cultivation 24h.
2. obtained strains in the step 1 is carried out preliminary evaluation
Bacterial strain to step 1 gained carries out preliminary evaluation, comprises that gramstaining, milk-acid bacteria Physiology and biochemistry are identified, 16S rRNA checks order and the biological safety check, and concrete grammar is following:
(1) gramstaining
1) smear is fixed; 2) ammonium oxalate crystal violet dyed 1 minute; 3) tap water flushing; 4) adding iodine liquid covers and to be coated with face and to dye about 1 minute; 5) washing is inhaled the branch that anhydrates with thieving paper; 6) add 95% alcohol number droplet, and shake gently and decolour, 20 seconds after washings are inhaled the branch that anhydrates; 7) behind the luxuriant safflower dyeing 2min, the tap water flushing.Drying, microscopy.The Gram-positive thalline is purple, and negative thalline takes on a red color.
(2) the milk-acid bacteria Physiology and biochemistry is identified, comprises with the lower section:
(a) the katalase negative strain is selected in catalase test.Bacterium colony with on the transfering loop picking solid medium places clean tube, drips 3% superoxol 2ml, observations.0.5min the positive bacterial strain of interior generation bubble person, otherwise negative bacterial strain.
(b) lactic acid qualitative experiment.Get bacterium liquid 10ml, add 10% H
2SO
41ml adds 2% KMnO again
41ml.Get ammoniated silver nitrate solution wetted filter paper bar, horizontal riding on the test tube mouth, the heating in water bath test tube like the filter paper blackening, is the qualitative positive of lactic acid, otherwise negative.
(c) aerobic test: will divide bacterial strain to be inoculated in respectively and 9cm liquid MRS substratum will be housed in vitro, 37 ℃ leave standstill cultivate 12h after, observe growing state.All liquid is limpid, a large amount of bacterial sediments in the pipe end, and perhaps tube wall has cotton-shaped coagulating to be anerobes; Limpid 0.8 ~ 1.2cm is thick on the liquid level upper strata, and lower floor is muddy, and the pipe end is precipitated as little aerobic bacteria in a large number; The even from bottom to top muddiness of liquid is an amphimicrobian; Liquid is limpid, and it is aerobic bacteria that liquid level has mycoderm, collarium.
(d) starch hydrolysis experiment: Starch Agar is poured in the sterilization petridish, solidify the back and adopt the strok method inoculating strain, draw " ten " word or several lines; Cultivate 48h for 37 ℃, drip iodine liquid on flat board, make the iodine fluid power evenly be paved with whole plane; If can hydrolyzed starch, then starch is decomposed and does not show purple with iodine liquid around the lawn, forms achromatic region; According to the size of achromatic region, the size of this bacterial strain starch-splitting ability can be described also.
(e) gelatin liquification test: with inoculation in the gelatin-based basal culture medium, 37 ℃ of constant temperature culture, a nonvaccinated test tube is as contrast.Place refrigerator or cold water observation experiment result with what inoculate with nonvaccinated control tube.When solidifying like control tube, the inoculated tube positive reaction of liquefying is solidified as negative reaction; Wherein the gelatin-based basal culture medium comprises following component: peptone 1.0g, yeast extract 3g, glucose 0.1g, gelatin 12.0g, salts solution 4.0ml, zero(ppm) water 100ml, 7.0,121 ℃ of sterilizations of pH 20min.
(f) V-P test: test strain is inoculated in the glucose peptone water substratum 37 ℃ of constant temperature culture 24 ~ 48h.Get bacterium liquid 1mL, add 0.6ml 5% ɑ-naphthol solution and 0.2ml 40% Pottasium Hydroxide and mix, use forced oscillation, put into 37 ℃ of incubators again and be incubated 15min～30min, with fast reaction speed.Gradually take on a red color like substratum, promptly represent the V-P reacting positive, can be described as feminine gender when in 4h, not showing red; Wherein the glucose peptone water substratum comprises following component: Tryptones 5g, glucose 5g, potassium primary phosphate 5g, zero(ppm) water 1000ml, 7.5,121 ℃ of sterilizations of pH 20min.
(g) mobility inspection: with bacterial strain with the staight needle percutaneous puncture-inoculation in the semi-solid MRS substratum that contains 1% agar, cultivate 48h for 37 ℃, observations, if culture only is grown on the puncture line of inoculation, the edge ten minutes is clear, representes that then bacterial strain does not have mobility.By being the cloud diffusion around the puncture alignment, its edge fog is cloud like the culture growth, and the expression bacterial strain has mobility.
(h) glucide fermentation and acid test: various carbohydrates comprise glucose, Sunmorl N 60S, lactose, sucrose, semi-lactosi, sorbyl alcohol, SANMALT-S, N.F,USP MANNITOL fructose and trehalose; Be mixed with 1% sugar soln respectively; 115 ℃ of sterilization 20min add liquid glucose in the basic medium with aseptic technique.Be inoculated in and mix in the substratum of back, 37 ℃ of cultivations, nutrient solution is to produce the acid positive by the purple stain Huang, otherwise negative for producing acid; Basic medium comprises following component: peptone 10g, and sodium-chlor 5g, 1.6% purpurum bromocresolis solution 5ml, zero(ppm) water 1000mL, pH 7.4,121
oThe C 20min that sterilizes.
(3) 16SrRNA order-checking
The total genomic dna of extraction step 1 obtained strains increases with PCR.The PCR response procedures: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 45s, 51 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 8min.
PCR is reflected on the PCR appearance of TaKaRa and carries out.The PCR product is through 1% agarose gel electrophoresis, and electrophoresis finishes the back to be observed under Ultraviolet Detector.After the PCR product is purified, check order, require to survey logical by Shanghai Ying Jun Bioisystech Co., Ltd.With the result's online that connects after the assembly: http://www.NCBI.nlm.nih.gov/, utilize BLAST software, gene order and GenBank DB that mensuration is obtained carry out the sequence alignment analysis, carry out the homology comparison.
(4) biological safety check
With intestinal bacteria O78 (
Escherichia coliO78) as pathogenic strains, healthy Kunming (KM) small white mouse is an experimental animal.Mouse is divided into negative control group (I group): no H bacterial strain, do not have and attack malicious intestinal bacteria; The II group: no H bacterial strain, irritate stomach and attack malicious intestinal bacteria; III group: do not have and attack malicious intestinal bacteria, irritate stomach H bacterial strain; The IV group: irritate the H bacterial strain that stomach is lived earlier, the back is irritated stomach and is attacked malicious intestinal bacteria; The V group: irritate stomach earlier and attack malicious intestinal bacteria, the H bacterial strain that stomach is lived is irritated in the back.H bacterial strain and intestinal bacteria are all got and are cultured to logarithmic phase later stage bacterium liquid, are diluted to 10 with saline water
8Individual/ml.Regularly take the about 0.1g of stool in mice, select substratum to measure H bacterial strain and colibacillary quantity in the ight soil with M17 and EMB.Observed and recorded was respectively organized mouse healthy growth situation and was comprised morbidity (neat whether smooth, the outage of living of outward appearance, hair) every day, and diarrhoea (ight soil is had loose bowels) is with dead; Write down the food ration of each treated animal every day, changes of weight.
(5) detected result and analysis
(a) morphological feature
37 ℃ cultivate 48 hours after, H bacterial strain bacterium colony is rounded, protuberance, oyster white, smooth, diameter 1～2 mm, the mycelia thickness does not have peculiar pigment (as shown in Figure 1).
Dye with gram staining method, oily sem observation, gramstaining is purple, is no gemma gram-positive microorganism.Cell is rod-short, and is single or become double row, is typical bacillus form (as shown in Figure 2).
(b) physiological and biochemical property, the result sees table 1 and table 2.
The physiological and biochemical property of table 1 H bacterial strain
Annotate :+expression positive reaction ,-expression negative reaction
The diagnostic characteristics of table 2 glucide fermentation
Annotate :+expression positive reaction ,-expression negative reaction
(c) the 16S rRNA sequencing analysis of H bacterial strain
The 16S rRNA sequence of H bacterial strain such as SEQ ID NO:1, length: 1464 bp.
(d) can know H bacterial strain no pathogenicity through the biological safety check, the ability of certain inhibition intestinal bacteria O78 is arranged.
Physiological and biochemical property according to the H bacterial strain; Standard of perfection and 16S rRNA sequencing analysis according to the relevant milk-acid bacteria of Bergey ' s Bacteria Identification handbook; The result shows the homology of H bacterial strain and lactobacillus salivarius the highest (100%), from physiological and biochemical property and molecular systematics angle confirm the H bacterial strain be lactobacillus salivarius (
Lactobacillus salivarius), and H bacterial strain no pathogenicity, be the safety bacterial strain.
Embodiment 2H
Bacterial strain protect look performance shaker test
The H bacterial strain is protected look performance shaker test.Ideal has the bacterial strain that protects chromatic effect must expire simultaneously
Be enough to down condition: do not produce hydrogen peroxide, do not produce hydrogen sulfide, not hydrolysis l-arginine, glucose fermentation produce not aerogenesis and do not have the amino acid decarboxylase enzymic activity of acid; Nitrite reductase is positive, and nitricoxide synthase is positive, and high ferro myohemoglobin reductase is positive.Concrete testing sequence is following:
(1) preparing culture medium
Detect substratum 1: Carnis Bovis seu Bubali cream 0.5g, yeast extract paste 0.5g, tween 80 0.05ml, MnSO
44H
2O 0.01g, zero(ppm) water 1000ml, agar 15g, pH6.5.Behind 121 ℃ of sterilization 15min, add 10g through 115 ℃, the glucose of 20min sterilization.
4%MnO
2Detection substratum 1: on the basis of detecting substratum 1 prescription, add MnO
240g behind the 121 sterilization 15min, adds 10g through 115 ℃, the glucose of 20min sterilization.
Detect substratum 2: peptone 10g, NaCl 5g, Carnis Bovis seu Bubali cream 10g, halfcystine 0.5g, zero(ppm) water 1000mL, pH 7.0 ~ 7.4,112 ℃ of sterilization 20 ~ 30min.
Detect substratum 3: l-arginine 30g, glucose 0.5g, peptone 10g, yeast extract 5g, Trisodium Citrate 2g, potassium hydrogenphosphate 2g, sodium acetate 5g, MgSO
47H
2O 0.58 g, MnSO
44H
2O 0.25g, tween 80 1ml, zero(ppm) water 1000ml, pH6.8 ~ 7.0,121 ℃, 15 min sterilize.
Detect substratum 4: peptone 10g, sodium-chlor 5g, glucose 10g, 1.6% purpurum bromocresolis solution 5ml, zero(ppm) water 1000mL, 7.4,121 ℃ of pH, sterilization 15min.
Detect substratum 5: peptone 10g, Carnis Bovis seu Bubali cream 10g, yeast extract 5 g, K
2HPO
42g, dibasic ammonium citrate 2 g, sodium acetate 5 g, glucose 0.5 g, tween 80 1ml, MgSO
4.7H
2O 0.5 g, MnSO
40.25 g, CaCO
30.2g, Y factor 5mg, 1.6% Australia cresol purple ethanolic soln 0.625ml, described amino acid, zero(ppm) water 1000ml, pH5.5,121 ℃, sterilization 10min.
(2) the screening tool protects the bacterial strain of look ability, and screening index is: do not produce hydrogen peroxide, do not produce hydrogen sulfide, not hydrolysis l-arginine, glucose fermentation produce not aerogenesis and do not have the amino acid decarboxylase enzymic activity of acid.Concrete test procedure is following:
(a) hydrogen peroxide test.Bacterial strain is inoculated in above the detection substratum 1 with strok method, on detect substratum 1, pours into the thick 4%MnO of one deck 2ml then
21,30 ℃ of detection substratum cultivated 5 days, observe periphery of bacterial colonies MnO continuously
2The variation of black, black dissolving person is for producing H
2O
2The positive produces H
2O
2Otherwise negative, do not produce H
2O
2
(b) hydrogen sulfide production test.Inoculation in detecting substratum 2, is soaked into the sugar of lead that uses concentration as 80g/L then, 80 ℃ of oven dry, 121 ℃, behind the sterilization 15min, hang on inoculation in vitro, place incubated at room temperature, the positive reaction of paper slip blackening promptly produces hydrogen sulfide; Otherwise hydrogen sulfide is not produced in negative reaction.
(c) hydrolysis arginine test.Inoculation to having in the detection substratum 3 of being inverted the Du Shi tubule, and is added through 121 ℃, 20min sterilising liq, and whiteruss covers the substratum liquid level, cultivates 24h for 30 ℃.Whether Du Shi tubule in have bubble produce, have bubble to be produced as the positive if observing, produce ammonia, the hydrolysis l-arginine; Otherwise negative, do not produce ammonia, not hydrolysis l-arginine.
(d) glucose fermentation test.Inoculation to having in the glucose aerogenesis substratum of being inverted the Du Shi tubule, is cultivated 24h for 30 ℃.Whether whether observe has the color of bubble generation and nutrient solution to change in the Du Shi tubule.There is bubble to be produced as positive test in the Du Shi tubule, aerogenesis; Otherwise negative, aerogenesis not.Nutrient solution is positive by the purple stain Huang, produces acid; Otherwise negative, do not produce acid.
(e) amino acid decarboxylase enzyme test.Bacterial strain is inoculated into respectively is added with 0.5% dead drunk propylhomoserin, 0.25% Histidine, 0.25% Methionin, 0.25% arginic detection substratum 5 in vitro, 30 ℃ of cultured continuously and observed 7 days.Nutrient solution is the positive of purple or redness, produces amino acid decarboxylase; It is negative to be yellow person, does not produce amino acid decarboxylase.
(f) nitrite reductase is positive.
1. the extraction of the thick enzyme of nitrite reductase: get and be cultured to logarithmic phase later stage bacterium liquid 4 ℃ of centrifugal 20min collection thalline of 10000 rpm in refrigerated centrifuge, add the pH7.5 phosphate buffered saline buffer and clean thalline 2 ~ 3 times.Adopt the broken thalline of ultrasonic cell-break method (work/quiescent interval is 3s, 350W, 15min, ice bath), 4 ℃ of centrifugal 20 min of 10000 rpm in refrigerated centrifuge collect supernatant.
2. nitrite reductase enzyme mensuration alive (adopts Na
2S
2O
4-methyl viologen method):
Solution
: take by weighing 0.4g sulfanilamide (SN); Put into the 200mL volumetric flask that fills the 160mL deionized water, heating for dissolving on boiling water bath.Cooling back (filtering in case of necessity) adds 20mL hydrochloric acid, and (ρ=1.19g/mL), use the deionized water constant volume keeps in Dark Place.
Solution
: take by weighing 0.1g two hydrochloric acid-1-naphthodiamide (content is more than 98.5%); Put into the 100mL volumetric flask; Add constant volume behind the deionized water dissolving, keep in Dark Place.
Solution
: measure 445ml hydrochloric acid (ρ=1.19g/ml); Put into the 1000ml volumetric flask, add deionized water dilution constant volume.
Reaction solution is formed (2ml): 0.1 mol/L Tris-HCl 1ml, and pH 7.5; 0.01 mol/L NaNO
20.3 mL; 0.02 mol/L methyl viologen 0.1 mL; 0.12 mol/L Na
2S
2O
40.1mL; Enzyme liquid 0.5 mL.
Elder generation is with 0.1 mol/L Tris-HCl 1mL, 0.01mol/LNaNO during mensuration
20.3mL, 0.02 mol/L methyl viologen 0.1mL kind solution adds test tube, adds 0.5 mL enzyme liquid again, adds 0.12mol/L Na then
2S
2O
40.1mL and pick up counting, 30 ℃ of following insulation 20min take out and shake when colourless sucking-off 0.1mL reaction solution immediately, add in the 5.9mL deionized water, add solution
1mL adds solution again
0.6mL, mixing.Place room temperature shading place; Add solution
0.2mL mixing again, water is settled to 10mL behind the 3min.In 15min, use the 1cm cuvette, with the blank solution zeroing, survey its absorbancy at wavelength 538 nm places.Check in corresponding nitrite ion quality from typical curve, with methylate purpurine not is contrast.
3. enzyme activity determination: at 30 ℃, pH is that PM (min) transforms the 1nmoL nitrite (with NO under 7.5 the condition
2 -Meter) required enzyme amount (mL) is an enzyme unit (U) alive
(g) nitricoxide synthase is positive.
1. the extraction of the thick enzyme of nitricoxide synthase: get and be cultured to logarithmic phase later stage bacterium liquid 10000 rpm, 4 ℃ of centrifugal 20min collection thalline in refrigerated centrifuge, add pH 7.5 phosphate buffered saline buffers and clean thalline 2 ~ 3 times.Adopt the broken thalline of ultrasonic cell-break method (work/quiescent interval is 3s, 350W, 15min, ice bath), 10000 rpm, 4 ℃ of centrifugal 20min in refrigerated centrifuge collect supernatant.
2. nitricoxide synthase enzyme activity determination:
Reaction solution is formed the slow liquid 50mM of (1mL): pH 7.0 phosphoric acid, CaCl
21.0mM, FAD 10mM, FMN 10mM, H
4B 80 μ M, NADPH 1.0mM, enzyme liquid 0.5mL.
Reaction beginning when adding L-Arg 1.0mM is in 30 ℃ of following oscillatory reaction 30min.In 15min, use the 1cm cuvette, with the blank solution zeroing, survey its absorbancy at wavelength 340nm place, be blank with the culture that does not add L-Arg.Checking in corresponding N ADPH quality from typical curve, is contrast with what do not add L-Arg.
3. enzyme activity determination: at 30 ℃, pH is under 7.0 the condition, and it is the enzyme unit (U) that lives that PM (min) transforms the required enzyme amount (mL) of 1nmol NADPH.
(h) high ferro myohemoglobin reductase positive test.
1. the extraction of the thick enzyme of high ferro myohemoglobin reductase: get and be cultured to logarithmic phase later stage bacterium liquid 10000 rpm, 4 ℃ of centrifugal 20min collection thalline in refrigerated centrifuge, add pH 7.5 phosphate buffered saline buffers and clean thalline 2 ~ 3 times.Adopt the broken thalline of ultrasonic cell-break method (work/quiescent interval is 3s, 350W, 15min, ice bath), 10000 rpm, 4 ℃ of centrifugal 20min in refrigerated centrifuge collect supernatant.
2. high ferro myohemoglobin reductase enzyme activity determination:
Reaction solution is formed (1mL:5.0mmol/L Na
2EDTA 0.1mL, 50.0mmol/L sodium citrate buffer solution (pH 5.65) 0.1mL, 3.0mmol/L yellow prussiate of potash 0.1mL, 0.75mmol/L pig MetMb reductase enzyme substrate 0.2mL, zero(ppm) water 0.2mL, enzyme liquid 0.2mL, 1.0mmol/L NADH (pH 7.0) 0.1mL.Final mixture pH value is 6.4, and probe temperature is 30 ℃.
Reaction beginning when adding NADH, under the 580nm wavelength, every at a distance from 15s (amounting to 2min) record absorbancy.MbO
2Maximum in the absorbancy difference of 580nm wavelength with MetMb, both molar extinction coefficients are 1.2 * 10
4
3. enzyme activity determination: an enzyme activity unit (1U) is defined as, the required enzyme amount of PM reduction 1nmol MetMb, and MetMb reductase enzyme specific activity (U/g sample) is expressed as the enzyme activity unit that contains in reaction every gram sample of starting stage.
Result and analysis: bacterial strain H does not produce hydrogen peroxide, does not produce hydrogen sulfide, and not hydrolysis l-arginine, glucose fermentation produce acid not aerogenesis and no amino acid decarboxylase enzymic activity; Nitrite reductase is positive, and nitricoxide synthase is positive, and high ferro myohemoglobin reductase is positive.So bacterial strain H has the stronger look ability of protecting, and can be used for the research that meat protects look.
The bacterial strain that embodiment 1 method is separated, screened is prepared into the fresh meat colour protecting agent, and concrete preparation method is following:
(1) H strain cultures preparation, bacterial screening substratum be with solid MRS substratum as minimum medium, add 0.1%CaCO
3, the pH value is 6.4,121 ℃ of sterilization 20min, and is subsequent use.
(2) fermentation culture of H bacterial strain, fermention medium be with liquid nutrient medium as minimum medium, the pH value be 6.4,121 ℃ the sterilization 30min, subsequent use.Seed cell age 12 ~ 18 hours, inoculum size are 3% ~ 5% (V/V or V/W).Fermentation time 18 ~ 24 hours, leavening temperature are 37 ℃.
(3) centrifugal: the centrifugal 15min of fermented liquid 3000r/min, remove supernatant, collect thalline.
(4) thalline adds thinner, and 50 gram H strain fermentation compositions add 950 gram zero(ppm) water, and thorough mixing promptly obtains the fresh meat colour protecting agent.
The application of embodiment 4 colour protecting agents and protect chromatic effect relatively
1. materials and methods
Supply examination colour protecting agent bacterial classification: the H bacterial strain, separate the self-control butcher's meat from the laboratory; Lactobacillus fermentum JCM1173 (hereinafter to be referred as lactobacillus fermentum J) purchases the institute of microbiology in the Chinese Academy of Sciences.
2. meat treatment process method
Cube meat is divided into 4 groups at random, and 9 every group, the A group is the blank group of sterile distilled water; The B group is for adding NaNO
2(5 * 10
-5Mol/L) positive controls; The C group is for adding H bacterial strain group (H bacterial strain group); The D group is added lactobacillus fermentum J (J bacterial strain group).Place each treatment solution to soak 1min the cube meat that cuts, taking-up is dried, and packs with vacuum packaging bag, with being placed on 20 ℃ of incubators.Sampling every day is handled the mensuration that meat carries out each item index to each.The cube meat of different treatment is placed 20 ℃, observe result such as Fig. 5, Fig. 6, shown in Figure 7 respectively at sampling after depositing 1d, 2d, 3d.
(1) subjective appreciation
Please, outward appearance, color, local flavor and the overall acceptability etc. of different tests group cube meat are marked by forming evaluation group through 10 people of specialized training, and scoring rank 1-9 branch (l-4: the outward appearance defectiveness, be faint in color, British plain spirits or peculiar smell is arranged, acceptability is poor; 4.1-6: outward appearance is normal, and color is rose-red, and slight fragrance is arranged, and can accept; 6.1-9: outward appearance is good, and is bright-colored, aromatic flavour, acceptability is good).
As can beappreciated from fig. 5 handle meat 20 ℃ deposit 1 day after, each is organized color and has taken place than obvious variation.Wherein, H bacterial strain group presents more bright-coloured redness, and muscular tissue structure is obvious, and the juice stream vector is little.Other organize the yellowish pink depth is NaNO successively
2Group, J bacterial strain group and negative control group.The color of J bacterial strain group and the color of negative control group are observed very nearly the same on naked eyes.To the 3rd day (Fig. 7), though the juice stream vector of each group increases, can't see the structure of obvious muscle tissue, it is greyish white that yellowish pink becomes, and H bacterium group is appointed the redness that keeps certain than other three groups.
After taking pictures, take out each treatment group cube meat, to its color, outward appearance, smell is observed, and record, according to standards of grading each treatment group is evaluated, and the sense organ value that draws at last is as shown in table 3 below:
Table 3
Each is handled the meat placement after 1 day, compare NaNO with negative control group
2Group and H bacterium group present good color and luster, and its subjective appreciation value reaches 4.8 fens respectively and 4.5 minutes, next are J bacterium group (3.3 minutes) and blank groups (2.5 minutes).Along with the prolongation of shelf-time, the subjective appreciation value of each group processing meat is all on a declining curve.The 2nd day, NaNO
2The downtrending of group and blank group is comparatively obvious.Its overall acceptability dropped to 3.8,1.5 fens respectively by original 4.8,2.5 minutes, had descended 21% and 40% respectively.And the variation of H bacterium group is minimum, is merely 11%, and J bacterium group has descended 12%.The 3rd day, the subjective appreciation value of each group all descended on the 2nd day basis to some extent, but changed not quite, and except that negative control group, its excess-three group velocity of variation all maintains below 15%.
(2) color difference measurement
Sampling after the sample pressing, is used the colour-difference meter analysis immediately, replication five times, brightness value (L), red scale value (a) and the yellow value degree (b) of record sample.
Measure the three-phase color coordinates value of respectively organizing the fresh tangent plane of sample respectively with colour-difference meter, i.e. brightness value (L value), red scale value (a value) and yellow value degree (b value).Result such as Fig. 8, Fig. 9, shown in Figure 10.
From each figure, can find out that generally, the L value and the b value difference of each treatment group are different not obvious.Along with change of time, the variation that L value and b value take place is also little.And being the red degree of meat, a value has than evident difference.As can be seen from Figure 9, H bacterium group and blank group (zero(ppm) water group) contrast, the significant difference of a value (P 0. 05).Along with the prolongation of shelf-time, a value of each group all descends to some extent.Compare with positive control with the blank group, H bacterium group is smaller with J bacterium group a value rate of descent, and wherein the rate of descent of H bacterium is minimum, descends 4% in 3 days, and J bacterial strain group has descended 17%, and this and subjective appreciation result present dependency.Proof is handled meat with the H bacterial strain and on color keeps, is had good effect.
(3) spectral scan
Get meat appearance 10g, add the 90mL0.2mol/L phosphate buffer soln, 12000r/min homogeneous 2min.Then centrifugal (4 ℃, 12000r/min) 20min gets the filtering with microporous membrane of supernatant with Φ=0.45um with mixture.Filtrating is placed in the ice bath of dark place, measures in the 2h.The spectral absorption value of continuous recording pigment extract in the 400-650nm wavelength region subsequently.
Can find out that from the spectral scan result each is organized meat appearance and places after 1 day except the blank group, other treatment group all have typical NOMb absorption spectrum, promptly near 540nm and 580nm, respectively have the absorption at an absorption peak and 540nm place stronger, see Figure 11.NaNO wherein
2The absorption value of group is the highest, secondly is H bacterium group>J bacterium group.After 2 days, the NaNO2 group still keeps original NOMb absorption curve, has descended 47% but compare absorption value with 1d.Obvious variation has then taken place with the absorption curve that the J bacterium is organized in H bacterium group, all disappears at the absorption peak of 540nm and 580nm, and the absorption spectrum similar with the blank group occurred, and a milder absorption peak is promptly arranged at the 530nm place, sees Figure 12.Place after 3 days NaNO
2Group mild absorption peak occurs at the 530nm place, and the absorption peak of other groups is still similar with 2 days absorption peak, and absorption value all has corresponding rising, sees Figure 13.
1. materials and methods
Supply examination colour protecting agent bacterial classification: the H bacterial strain, separate self-control butcher's meat from the laboratory, be prepared into H bacterium fresh meat colour protecting agent by case study on implementation 3.
2. ice the fresh meat treatment process
With the pork precooling of just having butchered, to cut apart stripping and slicing and be divided into 3 groups at random, the A group is the blank group of sterile distilled water; The B group is for adding NaNO
2(5 * 10
-5Mol/L) positive controls; The C group is for adding H bacterial strain group (H bacterial strain group).Place each treatment solution to soak 1mim cube meat, take out and drain 1min, pack, immediately put into 4 ℃ of refrigerator storages 6 days, sampling every other day, result such as Figure 14, Figure 15, Figure 16 and shown in Figure 17 with vacuum packaging bag.After the sample pressing, use the colour-difference meter analysis, replication five times.
Ice fresh meat color in storage after the packing is red-purple, because vacuum packaging makes pork be in the low oxygen partial pressure state, fresh meat surface myohaemoglobin can't exist with reduced state; When taking the packing color development apart after half a hour, yellowish pink is promptly recovered bright red.In 6 days storages, NaNO
2The ice fresh meat that group and colour protecting agent are handled keeps tempting bright red always.Can know by Figure 18, store after 6 days, through H bacterium colour protecting agent and NaNO
2The ice fresh meat red value a value rate of descent of handling is less, is respectively 7.6% and 5.8%, and the ice fresh meat a value rate of descent that zero(ppm) water is handled is 16%.H bacterium colour protecting agent group keeps lower b value, between 4.7 ~ 5.0, but zero(ppm) water treatment group and NaNO
2Group b value is higher, between 5.0 ~ 5.7 and 5.9 ~ 6.2 (Figure 19) respectively.Less through the ice fresh meat juice stream vector of H bacterium fresh meat colour protecting agent processing in addition, muscular tissue structure is more obvious, has guaranteed that preferably the quality of meat products reaches the purpose that prolongs the commodity price phase again.Therefore H bacterium fresh meat colour protecting agent also has certain color-protecting function to the ice fresh meat.
Conclusion and discussion
Result of the present invention shows that a strain lactobacillus salivarius H strain that is obtained has potential and protects look color development ability.The fresh meat that the H bacterium is handled all is superior to lactobacillus fermentum J group on subjective appreciation still is color difference measurement, also be superior to traditional chromogenic reagent NaNO simultaneously
2Process result.
According to analysis, the H bacterial strain can produce nitrite reductase, nitricoxide synthase and high ferro myohemoglobin reductase, makes muscle present good colour.Explain that the H bacterial strain has fresh meat and protect chromatic effect preferably.
SEQUENCE?LISTING
< 110>Agricultural University Of South China
< 120>be used to protect the lactobacillus strain of meat product color
<130>
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 1464
<212> DNA
< 213>artificial sequence
<400> 1
acatgcaagt?cgaacgaaac?tttcttacac?cgaatgcttg?cattcaccgt?aagaagttga 60
gtggcggacg?ggtgagtaac?acgtgggtaa?cctgcctaaa?agaaggggat?aacacttgga 120
aacaggtgct?aataccgtat?atctctaagg?atcgcatgat?ccttagatga?aagatggttc 180
tgctatcgct?tttagatgga?cccgcggcgt?attaactagt?tggtggggta?acggcctacc 240
aaggtgatga?tacgtagccg?aactgagagg?ttgatcggcc?acattgggac?tgagacacgg 300
cccaaactcc?tacgggaggc?agcagtaggg?aatcttccac?aatggacgca?agtctgatgg 360
agcaacgccg?cgtgagtgaa?gaaggtcttc?ggatcgtaaa?actctgttgt?tagagaagaa 420
cacgagtgag?agtaactgtt?cattcgatga?cggtatctaa?ccagcaagtc?acggctaact 480
acgtgccagc?agccgcggta?atacgtaggt?ggcaagcgtt?gtccggattt?attgggcgta 540
aagggaacgc?aggcggtctt?ttaagtctga?tgtgaaagcc?ttcggcttaa?ccggagtagt 600
gcattggaaa?ctggaagact?tgagtgcaga?agaggagagt?ggaactccat?gtgtagcggt 660
gaaatgcgta?gatatatgga?agaacaccag?tggcgaaagc?ggctctctgg?tctgtaactg 720
acgctgaggt?tcgaaagcgt?gggtagcaaa?caggattaga?taccctggta?gtccacgccg 780
taaacgatga?atgctaggtg?ttggagggtt?tccgcccttc?agtgccgcag?ctaacgcaat 840
aagcattccg?cctggggagt?acgaccgcaa?ggttgaaact?caaaggaatt?gacgggggcc 900
cgcacaagcg?gtggagcatg?tggtttaatt?cgaagcaacg?cgaagaacct?taccaggtct 960
tgacatcctt?tgaccaccta?agagattagg?ctttcccttc?ggggacaaag?tgacaggtgg 1020
tgcatggctg?tcgtcagctc?gtgtcgtgag?atgttgggtt?aagtcccgca?acgagcgcaa 1080
cccttgttgt?cagttgccag?cattaagttg?ggcactctgg?cgagactgcc?ggtgacaaac 1140
cggaggaagg?tggggacgac?gtcaagtcat?catgcccctt?atgacctggg?ctacacacgt 1200
gctacaatgg?acggtacaac?gagtcgcaag?accgcgaggt?ttagctaatc?tcttaaagcc 1260
gttctcagtt?cggattgtag?gctgcaactc?gcctacatga?agtcggaatc?gctagtaatc 1320
gcgaatcagc?atgtcgcggt?gaatacgttc?ccgggccttg?tacacaccgc?ccgtcacacc 1380
atgagagttt?gtaacaccca?aagccggtgg?ggtaaccgca?aggagccagc?cgtctaaggt 1440
gggacagatg?attggggtga?agtc 1464
Claims (9)
1. lactobacillus strain that is used to protect the meat products color, it is characterized in that said lactobacillus strain be lactobacillus salivarius H strain (
Lactobacillus salivariusH strain), depositary institution is Chinese typical culture collection center, and the preservation address is a China. Wuhan. and Wuhan University, deposit number is CCTCC NO:M 2010374, preservation date is on December 31st, 2010.
2. the lactobacillus strain that is used to protect the meat products color according to claim 1 is characterized in that colonial morphology is: circle, protuberance, oyster white, smooth surface, colony diameter 1～2mm; The mycelia thickness does not have peculiar pigment; No gemma, gram-positive microorganism; Cell is rod-short, and is single or become double row; Do not move amphimicrobian;
Biochemical character: catalase is negative, and nitrite reductase is positive, and nitricoxide synthase is positive, and high ferro myohemoglobin reductase is positive, no amino acid decarboxylase enzymic activity; Glucose, lactose, sucrose, semi-lactosi, SANMALT-S, N.F,USP MANNITOL, fructose and the experiment of trehalose fermentation and acid are all positive; The experiment of gluconate fermentation and acid is negative; Do not produce hydrogen peroxide, do not produce hydrogen sulfide, not gelatin hydrolysate and starch, not hydrolysis l-arginine; The qualitative positive of lactic acid, the V-P reaction negative; This bacterial strain no pathogenicity of biological safety check.
3. the lactobacillus strain that is used to protect the meat products color according to claim 1, the 16S rRNA sequence that it is characterized in that said bacterial strain is shown in SEQ ID NO:1.
4. a meat products colour protecting agent is characterized in that comprising the described lactobacillus strain that is used to protect the meat products color of claim 1.
5. the preparation method of the said meat products colour protecting agent of claim 4 is characterized in that may further comprise the steps:
(1) fermentation culture of bacterial classification: select cell age at 12 ~ 18 hours seed, inoculum size is 3% ~ 5% (V/V or V/W), 37 ℃ of fermentation culture of liquid modified MRS culture medium, fermentation time 18 ~ 24 hours;
(2) collect thalline after fermentation is accomplished, add thinner, mix promptly obtaining the meat products colour protecting agent.
6. the preparation method of meat products colour protecting agent according to claim 5 is characterized in that said modified MRS culture medium compound method is: glucose 20g, peptone 10g; Carnis Bovis seu Bubali cream 10g, yeast extract 5g, dibasic ammonium citrate 2g; Potassium hydrogenphosphate 2g, sodium acetate 5g, MgSO
47H
2O 0.58 g, MnSO
44H
2O 0.25g, tween 80 1mL, lime carbonate 15g, zero(ppm) water 1000mL, 121 ℃ of sterilizations; Said thinner is sterilized water, pure water or zero(ppm) water.
7. preparation method according to claim 5, the weight ratio that it is characterized in that said thalline and thinner is 1 ~ 10:10 ~ 19.
8. the application of the said meat product colour protecting agent of claim 4 in protection meat products color.
9. application according to claim 8 is characterized in that adopting infusion method or spraying process that colour protecting agent is surperficial attached to meat products; Said infusion method is meat products to be placed in the colour protecting agent soak, and dries the final vacuum packing; Said spraying process is that colour protecting agent is joined in the lactic acid solution, is sprayed onto the meat product surface, and consumption is that every 1000g meat product sprays colour protecting agent 50 ~ 100ml.
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| CN108977391B (en) * | 2018-08-10 | 2020-06-12 | 青岛农业大学 | Lactic acid bacterial strain with color development and corrosion prevention functions on meat products |
| CN110591987B (en) * | 2019-11-01 | 2021-05-14 | 四川农业大学 | Lactobacillus salivarius358 and application thereof, silage additive and silage |
| CN113142292A (en) * | 2020-12-29 | 2021-07-23 | 湖州展旺食品科技有限公司 | Method for prolonging preservation period of cold fresh meat by using antibacterial lactobacillus composite preservation solution |
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