CN103540553A - Method for mixing and fermenting compound strain to prepare mare milk vinegar and prepared mare milk vinegar - Google Patents
Method for mixing and fermenting compound strain to prepare mare milk vinegar and prepared mare milk vinegar Download PDFInfo
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- CN103540553A CN103540553A CN201310529793.1A CN201310529793A CN103540553A CN 103540553 A CN103540553 A CN 103540553A CN 201310529793 A CN201310529793 A CN 201310529793A CN 103540553 A CN103540553 A CN 103540553A
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Abstract
The invention discloses a method for mixing and fermenting a compound strain to prepare mare milk vinegar and prepared mare milk vinegar. According to the method, fresh mare milk in South Mountain regions of Xinjiang Urumuqi City is used as a raw material; the compound strain is formed by combining self-screened lactobacillus casei WWMR-1 CGMCC (China General Microbiological Culture Collection Center) No.8244 with K1uyveromyces marxianus and acetic acid bacteria on the basis of a single-factor test according to a volume ratio of 3:5:10, and is used for mixing and fermenting; a response surface method is used for optimizing and determining optimal preparation process parameters of conditions of alcohol fermentation and acetic acid fermentation of the mare milk vinegar; the invention provides the method for mixing and fermenting the compound strain to prepare the mare milk vinegar and the prepared mare milk vinegar so as to provide important and real development values to sufficiently develop and utilize Xinjiang mare milk resources.
Description
Invention field
The present invention relates to microbial fermentation technology field, concrete, the present invention relates to a kind of technical field that adopts microbial strains mixed fermentation to prepare mare's milk vinegar.
Background technology
Mare's milk is the rarity in Xinjiang Minority cooking culture.The nutritive value of mare's milk is very high, compares and contains rich in protein, lactose, VITAMIN and mineral substance, and contain the indispensable amino acid of human body and lipid acid with other domestic animals milk.In mare's milk, be rich in multiple nutrients material, the utilization that is easily absorbed by the body, is particularly suitable for infant and patient the old and the weak drinks, and has the effect that qi-restoratives was kept fit, moisturized skin makeup, relieves heat and thirst.The bright mare's milk impurity in grassland is few, and the contained methyl alcohol of kumiss, isopropylcarbinol, the primary isoamyl alcohol composition that with it, spawn are extremely low, and the heavy metals such as lead, mercury are not enough to 1/10th of national standard, and the content of formaldehyde is few especially.
At present, the domestic food that occurs on the market take that mare's milk is raw material has koumiss, mare's milk powder, kumiss, mare's milk beer etc., but rarely has report about mare's milk vinegar product.Mare's milk vinegar is as a kind of novel mare's milk drink, not only retained nutrition abundant in mare's milk, more gives the nourishing function of its vinegar, is the development project of being potential very much, and has great marketing space.The microbial strains that research and development have a practicality obtains stable mare's milk vinegar fermentation preparation technology and has vital role.
Summary of the invention
For having no relevant in prior art, utilize composite bacteria mixed fermentation to prepare the present situation that mare's milk vinegar develops, geo-advantage based on Xinjiang region various nationalities' long-term drinking mare's milk, make full use of ferment microflora's resource in the different places of production of Xinjiang mare's milk, exploitation is applicable to the complex microorganism bacterial classification preparation of mare's milk vinegar.The object of the invention is to provide a kind of method that adopts composite bacteria mixed fermentation to prepare mare's milk vinegar and preparation thereof.
Technical scheme of the present invention:
By take the fresh mare's milk in Xinjiang Urumqi South Mountain area, it is raw material, on the basis of single factor experiment, utilize on self-sizing lactobacterium casei basis, associating Kluyveromyces marxianus bacterium and acetic bacteria form composite bacteria mixed fermentation, adopt the optimization of response surface method to determine the zymamsis of mare's milk vinegar and the best preparation technology parameter of acetic fermentation condition, determine and provide a kind of composite bacteria mixed fermentation to prepare the method for mare's milk vinegar and the mare's milk vinegar of preparation to there is important reality exploitation value for fully developing Xinjiang mare's milk resource.
Concrete, the invention provides a kind of composite bacteria mixed fermentation and prepare the composite bacteria of mare's milk vinegar, (Kluyveromyces marxianus} and acetic bacteria (Acetic acid bacteria) form according to volume ratio meter 3:5:10 for composite bacteria Cheesecake Bacterium lacticum (Lactobacillus casei) WWMR-1, Kluyveromyces marxianus bacterium.
The lactobacterium casei that the present invention selects (Lactobacillus casei) WWMR-1, according to the singularity of the geographical environment in Xinjiang, by sampling the self-control sour camel milk gathering from Manasi, xinjiang county herdsman family, filter out a collection of well-grown microorganism strains, from sour camel milk, carry out the cultivation of microbial strains, isolation and screening, therefrom optimize the bacterial strain that a strain is numbered WWMR-1, bacterial classification bacterium colony after cultivating is neat in edge, the circle of smooth surface oyster white projection, the bacterium colony of diameter 0.5-3mm, carry out gramstaining, dyeing qualification result is Gram-positive rod-shaped bacterium, this bacterium is negative in catalase test, catalase is negative, and can not reduce nitrate, not liquefy gelatin, edwardsiella hoshinae and H
2s, do not move, can not ferment pectinose, melibiose, raffinose, rhamnosyl and wood sugar, can be 15 ℃ of growths, there is inoculation after growth stable, lag period is short, adapts to soon, fermentation initial stage acid producing ability is stronger.Through microbiology classification and identification, be lactobacterium casei (Lactobacillus casei).
The lactobacterium casei (Lactobacillus casei) that the concrete bacterium numbering adopting of the present invention is WWMR-1, this bacterium is through MRS substratum separation and Culture, in modified MRS culture medium, can produce again molten calcium circle and carry out preliminary screening, it is tentatively defined to lactic bacterium strains; This bacterial strain is streak culture 24h on MRS culture medium flat plate, can form that colony edge is neat, the circle of smooth surface oyster white projection, the bacterium colony of diameter 0.5-3mm; Carry out gramstaining, dyeing qualification result is Gram-positive rod-shaped bacterium; This strain culturing condition: 37 ℃ of culture temperature, 37 ℃ of the suitableeest culture temperature; Preferred growth is in MRS media surface, MRS nutrient media components extractum carnis 10.0g/L, yeast extract paste 5.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, citric acid hydrogen diamine 2.0g/L, tween-80 1.0ml/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate heptahydrate 0.2g/L, seven water manganous sulfate 0.05g/L, distilled water 1.0L, solid medium adds agar 20.0g/L; Bacterial strain colony diameter size on solid medium is 0.5-3mm, positive rounded, lateral projections; Neat in edge, smooth surface; Bacterium colony is opaque, is creamy white; There is certain toughness; Gram-positive microorganism.
In conjunction with colonial morphology, the physio-biochemical characteristics of above-mentioned this bacterial classification, be tentatively defined as lactobacterium casei (Lactobacillus casei).With reference to < < uncle Jie Shi systematic bacteriology identification handbook > > (< < Bergey, s Manual of Systematic Bacterio-logy > >) the conventional bacterial system of the 9th edition and < < is identified in volume > > etc. lactobacterium casei (Lactobacillus casei) (CGMCC No.8244) bacterial strain is carried out to morphology mensuration, Physiology and biochemistry detects, 16S rDNA homology in conjunction with this bacterial classification is analyzed, Phylogenetic Analysis result, determine that bacterium numbering is that WWMR-1 bacterial strain is lactobacterium casei (Lactobacillus casei, this bacterial classification was preserved in the international depositary institution of budapest treaty microorganism before the applying date: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101, preservation date is on 09 23rd, 2013, preserving number is CGMCC No.8244.Through mentioned microorganism, learn comprehensive evaluation and be attributed to lactobacterium casei (Lactobacillus casei).
Meanwhile, the invention provides the zymotechnique of bacterial strain lactobacterium casei (Lactobacillus casei) WWMR-1CGMCC No.8244: under aseptic condition, this bacterial strain of picking, 37 ℃, 135r/min cultivates 18-24h.
The present invention selects outsourcing bacterial classification: Kluyveromyces marxianus bacterium (Kluyveromyces marxianus), and this bacterial strain has that acid resistance is strong during the fermentation, and producing alcohol ability will be strong, and environmental compatibility is strong, the attribute that variability is little.Adopt 28 ℃ of the culture condition culture temperature of this bacterial classification, 28 ℃ of the suitableeest culture temperature; Preferred growth is in YPD media surface, and YPD nutrient media components adopts yeast extract paste 10g/L, peptone 20g/L, and glucose 20g/L, solid medium adds 20g/L agar powder.Meanwhile, the invention provides the zymotechnique of bacterial strain Kluyveromyces marxianus mattress (Kluyveromyces marxianus): under aseptic condition, this bacterial strain of picking, 30 ℃, 135r/min cultivates 48h.
Outsourcing one strain acetic bacteria of the present invention (Acetic acid bacteria), it is high that this bacterial strain has acid producing ability, the feature that fermentation period is short.This strain culturing condition: 30 ℃ of culture temperature, 30 ℃ of the suitableeest culture temperature; Preferred growth is in AS1.41 substratum, media surface, AS1.41 nutrient media components glucose 10g/L, yeast extract paste 10g/L, calcium carbonate 15g/L, agar 20g/L, dehydrated alcohol 20ml/L.Meanwhile, the invention provides the zymotechnique of acetic bacteria (Acetic acidbacteria): under aseptic condition, this bacterial strain of picking, 30 ℃, 135r/min cultivates 48h.
Meanwhile, the method for horse vinegar is prepared in the composite bacteria mixed fermentation that the invention provides a kind of employing as above provides, and concrete preparation method's step is as follows:
(1) preparation of seed fermentation liquid and female fermented liquid: seed fermentation liquid: bacterial classification lactobacterium casei (Lactobacillus casei) WWMR-1CGMCC No.8244, Kluyveromyces marxianus bacterium (Kluyveromyces marxianus) and the acetic bacteria (Acetic acid bacteria) preserved at 4 ℃ are inoculated in respectively in MRS substratum, YPD substratum and AS1.41 substratum and are activated under gnotobasis, be placed in respectively 37 ℃ and cultivate 24h, 28 ℃ of cultivation 48h and 30 ℃ of cultivation 48h, in order to later stage use; Female fermented liquid: the good mare's milk of pre-treatment is sub-packed in fermenting container, under aseptic condition, the milk-acid bacteria having activated, yeast and acetic bacteria seed fermented liquid are accessed in fermenting container by 5% inoculum size, be placed in respectively 37 ℃ and cultivate 24h, 28 ℃ of cultivation 48h and 30 ℃ of cultivation 48h, in order to later stage use.
(2) mare's milk pre-treatment: fresh mare's milk is removed to impurity and the foreign matter in mare's milk by four layers of filtered through gauze, then mare's milk is preheating to 65 ℃, homogeneous 5min under 20MPa condition; After homogeneous, mare's milk is carried out to pasteurize, place and be cooled to room temperature, treat fermentation use.
(3) zymamsis: milk-acid bacteria prepared by step (1) and saccharomycetes to make fermentation liquid add after mixing according to volume ratio 3:5 meter in mare's milk prepared by step (2), add glucose, sugaring amount is added according to 8g/100ml, at 32 ℃, 135r/min, cultivate 80h, obtaining alcoholic strength is the mare's milk alcohol fermentation liquid of 7% (V/V).
(4) acetic fermentation: according to volume ratio meter, in the alcohol fermentation liquid that the inoculum size of acetic bacteria prepared by step (1) (Acetic acid bacteria) by 10% prepared in step (3), requiring alcohol initial alcohol degree is 8% (V/V), optimum leavening temperature is 33 ℃, 135r/min, cultivate 5 days, obtaining acidity is the mare's milk acetic acid fermentation liquid of 120 ° of T.
(5) aftertreatment: to fermentation termination, acidity, at 120 ° of T, is carried out pasteurize until product, carries out sterile filling after cooling and prepares horse vinegar.
In the present invention, alcoholic strength is measured and is adopted distillation to obtain with Ebullioscope, measuring after ethanol aqueous solution; Acid test adopts determination of acid-basetitration; Pol is measured and is used hand-held saccharometer to measure; Sensory evaluation, physical and chemical index are measured with reference to GB/T15038-2006.
The product quality characteristic of the horse vinegar that the present invention is prepared by technique scheme:
(1) Oranoleptic indicator: color and luster: oyster white, micro-Huang has the intrinsic color and luster of mare's milk; Fragrance: mare's milk taste perfume (or spice) is strongly fragrant, acetic acid taste is mellow has the distinctive fragrance of mare's milk vinegar; Flavour: tart flavour is tasty and refreshing, free from extraneous odour; Figure: emulsion is allowed a little precipitation.
(2) physical and chemical index: total acid: 120 ° of T, reducing sugar content≤4g/L, V
0content is 68.72mg/100mL.
(3) microbiological indicator: total number of bacterial colony (cfu/mL)≤100; Coliform (MPN/100mL)≤3; Pathogenic bacterium: do not detect.
By implementing the concrete summary of the invention of the present invention, can reach following technique effect:
1. lactobacterium casei (Lactobacillus casei) the WWMR-1CGMCC No.8244 of horse vinegar is prepared in fermentation provided by the invention, this bacterial strain can be when low pH value activity stronger, Heat stability is good, has a broad antifungal spectrum, produces bacteriocin; Producing is during the fermentation mainly the organic acids such as lactic acid, pH value is reduced and suppress the growth of some spoilage organism and pathogenic bacterium, and improve quality and the local flavor of food.
2. lactobacterium casei (Lactobacillus casei) the WWMR-1CGMCC No.8244 that adopts the present invention to set, Kluyveromyces marxianus bacterium (Kluyveromyces marxianus) and acetic bacteria (Acetic acid bacteria) form composite bacteria mixed fermentation and prepare horse vinegar, milk-acid bacteria and yeast mixed fermentation not only promote the growth of bacterium number separately, and can regulate and control the variation of meta-bolites, aspect raising flavour and fragrance, playing an important role, the mare's milk vinegar of preparation is as a kind of novel mare's milk drink, micro-Huang has the intrinsic color and luster of mare's milk and fragrance, acetic acid taste is mellow, not only retain nutrition abundant in mare's milk, more given the nourishing function of its vinegar.
Accompanying drawing explanation
Fig. 1 is shown as and adopts composite bacteria mixed fermentation to prepare the process flow sheet of horse vinegar.
Fig. 2 is shown as the colonial morphology figure of lactobacterium casei preservation strain.
Fig. 3 is shown as the phyletic evolution of lactobacterium casei preservation strain and grows tree graph.
Fig. 4 is shown as inoculum size and the response surface figure of fermentation time interaction on zymamsis impact.
Fig. 5 is shown as inoculum size and the response surface figure of leavening temperature interaction on zymamsis impact.
Fig. 6 is shown as leavening temperature and the response surface figure of fermentation time interaction on zymamsis impact.
Fig. 7 is shown as difference and connects the impact of bacterium amount on acidity.
Fig. 8 is shown as the impact of different initial alcohol degrees on acidity.
Fig. 9 is shown as the impact of different fermentations temperature on acidity.
Figure 10 is shown as the impact of different fermentations time on acidity.
Figure 11 is shown as the response surface figure of alcoholic strength and inoculum size reciprocal effect acidity.
Figure 12 is shown as the response surface figure of leavening temperature and alcoholic strength reciprocal effect acidity.
Figure 13 is shown as the response surface figure of leavening temperature and inoculum size reciprocal effect acidity.
Embodiment
Below, for embodiment, the present invention is described, still, the present invention is not limited to following embodiment.
The main plant and instrument adopting: LD2X-50KB vertical electric pressure steam sterilizer (Shanghai Shen An Medical treatment device factory); HR40-IIA2 Biohazard Safety Equipment (Qingdao Haire Special Electrical Appliances Co., Ltd); DHP-781 electro-heating standing-temperature cultivator (Huangshi, Hebei province medical apparatus and instruments factory); DZKW-172 electric-heated thermostatic water bath (Beijing bright Medical Instruments factory); CH2176J microcomputer electromagnetic oven (Glanz, Zhongshan city living electric apparatus Manufacturing Co., Ltd); SXKW digital display control-temperature electric heating cover (Beijing forever bright Medical Instruments factory).
All raw and auxiliary materials, reagent and the instrument of selecting in the present invention, equipment are all well known selecting, but do not limit enforcement of the present invention, and other reagent more well known in the art and equipment are all applicable to the enforcement of the following embodiment of the present invention.
Embodiment mono-: screening, separation and purification and the evaluation of lactobacterium casei (Lactobacillus casei) WWMR-1CGMCC No.8244
Primary dcreening operation: get Hogormag-A traditional fermented camel milk product 1mL and put into the test tube that 9mL stroke-physiological saline solution is housed, fully mix on vortex mixer.Fermentation sour camel milk is diluted with 10 times of levels, and extent of dilution is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6.With pipettor, draw above-mentioned each 1mL of extent of dilution sample liquid, adopt tilt-pour process to pour modified MRS culture medium into, flat board is placed in 37 ℃ of incubators and cultivates 24h.Observing and record colony characteristics picking has the single bacterium colony of different shape of transparent circle, carries out gramstaining, and picking G+ bacteria falls, and in microscope oil Microscopic observation cellular form, and doubtful bacterial classification is numbered.
The purifying of bacterial classification: the bacterial strain of doubtful product transparent circle is connect and is beneficial to MRS nutrient agar, in 37 ℃ of incubator constant temperature culture 24h, repeat purified lactic acid bacterium 3～4 times.Lactic bacterium strains after purifying is inoculated in modified MRS inclined-plane, and after 37 ℃ of incubator constant temperature culture 24h, 4 ℃ save backup.
The present invention is according to the singularity of the geographical environment in Xinjiang, from sour camel milk, carry out cultivation, the isolation and screening of microbial strains, obtain a collection of bacterium, and therefrom separate lactobacterium casei (Lactobacillus casei), culture presevation is numbered CGMCC No.8244.
The present invention specifically provides a kind of lactobacterium casei, and called after WWMR-1 is numbered CGMCC No.8244, and it can lactic acid producing.With reference to < < uncle Jie Shi systematic bacteriology identification handbook > > (< < Bergey, s Manual of Systematic Bacterio-logy > >) the conventional bacterial system identification handbook > > of the 9th edition and < < etc. carries out morphology mensuration to lactobacterium casei (the numbering CGMCC No.8244 of bacterial classification) bacterial strain, Physiology and biochemistry detects, determine that lactobacterium casei (Lactobacillus casei) (numbering CGMCC No.8244) bacterial strain is the member in lactobacterium casei (Lactobacillus casei).This bacterial strain was preserved in the international depositary institution of budapest treaty microorganism before the applying date: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101, preservation date is on 09 23rd, 2013, and preserving number is CGMCC No.8244.
This strain culturing condition: 37 ℃ of culture temperature, 37 ℃ of the suitableeest culture temperature; Preferred growth is in MRS media surface, MRS nutrient media components extractum carnis 10.0g/L, yeast extract paste 5.0g/L, glucose 20.0g/L, sodium acetate 5.0g/L, citric acid hydrogen diamine 2.0g/L, tween-80 1.0ml/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate heptahydrate 0.2g/L, seven water manganous sulfate 0.05g/L, distilled water 1L, solid medium adds agar 20.0g/L.Bacterial strain colony diameter size on solid medium is 0.5-3mm, positive rounded, side and projection; Neat in edge, smooth surface; Bacterium colony is opaque, is creamy white; There is certain toughness; Gram-positive microorganism.
The evaluation of bacterial strain:
(1) Physiology and biochemistry is identified: this bacterium is through MRS substratum separation and Culture, then in modified MRS culture medium, the molten calcium circle of meeting generation carry out preliminary screening, and it is tentatively defined to lactic bacterium strains; This bacterial strain is streak culture 24h on MRS culture medium flat plate, can form that colony edge is neat, the circle of smooth surface oyster white projection, the bacterium colony of diameter 0.5-3mm.Carry out gramstaining, dyeing qualification result is Gram-positive rod-shaped bacterium.This bacterium is negative in catalase test, catalase is negative, and can not reduce nitrate, not liquefy gelatin, edwardsiella hoshinae and H
2s, do not move, pectinose, melibiose, raffinose, rhamnosyl and wood sugar can not ferment, can be 15 ℃ of growths, through the analysis of 16SrRNA homology, Phylogenetic Analysis and cell fatty acid proximate analysis result, all show, the bacterial strain that is numbered WWMR-1 is accredited as lactobacterium casei (Lactobacillus casei) through Physiology and biochemistry.Referring to accompanying drawing 2.
(2) 16S rDNA sequence alignment and Phylogenetic Analysis: the 16S rDNA sequence that order-checking is obtained and the nucleotide sequence in GenBank database carry out BLAST analysis, therefrom obtain close 16S rDNA sequence, with Maximum Likelihood method constructing system evolutionary tree in MEGA5.0.The similarity of the 16S rDNA sequence of bacterial strain WWMR-1 and lactobacterium casei (Lactobacillus casei) is up to 99%,
The relative genus sequence of downloading in the 16S rDNA sequence of this bacterial strain and GenBank is carried out to Phylogenetic Analysis, adopt MEGA5.0 software building phylogenetic tree, result is referring to shown in accompanying drawing 3, bacterium numbering is that the bacterial strain of WWMR-1 is under the jurisdiction of lactobacterium casei (Lactobacillus casei), nearest with Lactobacillus casei parent source relation, homology similarity is 99%, determine that bacterial strain WWMR-1 is for lactobacterium casei, the colonial morphology that is WWMR-1 in conjunction with the above-mentioned bacterium numbering providing, physio-biochemical characteristics, biology specific name is lactobacterium casei (Lactobacillus casei).
The 16S rDNA sequence of bacterial strain WWMR-1 is as follows, and concrete sequence is referring to attached sequence table:
Embodiment bis-: composite bacteria is mixed with the zymotechnique of horse vinegar
Referring to accompanying drawing 1, adopt the composite bacteria mixed fermentation as above providing to prepare the method for horse vinegar, concrete preparation method's step is as follows:
(1) preparation of seed fermentation liquid and female fermented liquid: seed fermentation liquid: bacterial classification lactobacterium casei (Lactobacillus casei) WWMR-1CGMCC No.8244, Kluyveromyces marxianus bacterium (Kluyveromyces marxianus) and the acetic bacteria (Acetic acid bacteria) preserved at 4 ℃ are inoculated in respectively in MRS substratum, YPD substratum and AS1.41 substratum and are activated under gnotobasis, be placed in respectively 37 ℃ and cultivate 24h, 28 ℃ of cultivation 48h and 30 ℃ of cultivation 48h, in order to later stage use; Female fermented liquid: the good mare's milk of pre-treatment is sub-packed in fermenting container, under aseptic condition, the milk-acid bacteria having activated, yeast and acetic bacteria seed fermented liquid are accessed in fermenting container by 5% inoculum size, be placed in respectively 37 ℃ and cultivate 24h, 28 ℃ of cultivation 48h and 30 ℃ of cultivation 48h, in order to later stage use.
(2) mare's milk pre-treatment: fresh mare's milk is removed to impurity and the foreign matter in mare's milk by four layers of filtered through gauze, then mare's milk is preheating to 65 ℃, homogeneous 5min under 20MPa condition; After homogeneous, mare's milk is carried out to pasteurize, place and be cooled to room temperature, treat fermentation use.
(3) zymamsis: milk-acid bacteria prepared by step (1) and saccharomycetes to make fermentation liquid add after mixing according to volume ratio 3:5 meter in mare's milk prepared by step (2), add glucose, sugaring amount is added according to 8g/100ml, at 32 ℃, 135r/min, cultivate 80h, obtaining alcoholic strength is the mare's milk alcohol fermentation liquid of 7% (V/V).
(4) acetic fermentation: according to volume ratio meter, in the alcohol fermentation liquid that the inoculum size of acetic bacteria prepared by step (1) (Acetic acid bacteria) by 10% prepared in step (3), requiring alcohol initial alcohol degree is 8% (V/V), optimum leavening temperature is 33 ℃, 135r/min, cultivate 5 days, obtaining acidity is the mare's milk acetic acid fermentation liquid of 120 ° of T.
(5) aftertreatment: to fermentation termination, acidity, at 120 ° of T, is carried out pasteurize until product, carries out sterile filling after cooling and prepares horse vinegar.
Embodiment tri-: the selecting of substratum
The activation medium of lactobacterium casei (Lactobacillus casei) WWMR-1CGMCC No.8244 adopts:
MRS substratum: glucose 20g, peptone 10g, extractum carnis 10g, yeast extract paste 5g, citric acid diamines 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, sal epsom 0.58g, manganous sulfate 0.25g, tween-80 1mL, distilled water 1000mL.
YPD substratum: yeast extract paste 10g, peptone 20g, glucose 20g, distilled water 1000mL.
Acetic bacteria (Acetic acid bacteria) adopts AS1.41 substratum: glucose 10g, yeast extract paste 10g, calcium carbonate 15g, agar 20g, dehydrated alcohol 20ml, distilled water 1000mL.
The physiological and biochemical test substratum of lactobacterium casei (Lactobacillus casei) WWMR-1CGMCC No.8244:
(1) modified MRS culture medium: add 0.5% aseptic CaCO3 after MRS medium sterilization, mix.
(2) PY basic medium: peptone 0.5g, Tryptones 0.5g, yeast extract paste 1.0g, salts solution 4.0mL, anhydrous CaCl20.2g, MgSO47H2O0.48g, K2HPO41.0g, KH2PO41.0g, NaHC0310.0g, NaCl2.0g, distilled water 100mL.
(3) sugar-fermenting substratum: extractum carnis 3g, peptone 10g, sodium-chlor 5g, trial sugar, glycosides or alcohol 5g, 1.6% purpurum bromocresolis spirituous solution 1mL, distilled water 1000mL.
(4) nitrate reduction substratum: extractum carnis 3g, peptone 5g, saltpetre 1g, distilled water 1000mL.
Nitrate reduction reagent (griess reagent) configuration: first liquid is dissolved in Sulphanilic Acid 0.5g in 5mol/L acetum 100mL.Second liquid is dissolved in d-naphthylamines 0.5g in 5mol/L acetum 100mL.
(5) gelatine liquefication substratum: peptone 5g, beef extract 3g, gelatin 120g, distilled water 1000mL.
(6) indole (indoles) substratum: peptone 20g, sodium-chlor 5g, distilled water 1000mL.Dissolve each composition, adjust pH to 7.4, packing test tube, 115 ℃ of sterilizing 20min.
Ou-Bo (Ehrlich-Bohme) family name reagent: get Paradimethylaminobenzaldehyde 1g, be first dissolved in 95mL95% ethanol, then slowly add concentrated hydrochloric acid 20mL, mix.
(7) produce H
2s substratum: extractum carnis 3g, yeast extract paste 3g, peptone 15g, ferrous sulfate 0.2g, Sulfothiorine 0.3g, sodium-chlor 5g, agar 5g, distilled water 1000mL.
(8) catalase (H
2o
2) substratum: 3% hydrogen peroxide (H
2o
2) solution, face used time preparation.
(9) LB liquid nutrient medium: yeast extract paste 5g, peptone 10g, sodium-chlor 10g, distilled water 1000mL; LB solid medium: add 1.5% agar on the basis of liquid medium within.
(10) nutrient agar: peptone 12g, sodium-chlor 5g, extractum carnis 3g, yeast extract paste 3g, agar 12g, distilled water 1000mL.
Embodiment tetra-: composite bacteria is mixed with the zymamsis condition single factor experiment of horse vinegar
Choose respectively the bacterium amount that connects (milk-acid bacteria %: yeast %) be 2:6,3:5,4:4,5:3,6:2, cultivation and fermentation temperature is 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃, fermentation time is 24h, 48h, 72h, 96h, 120h, and sugared addition is 4g, 6g, 8g, 10g, 12g; Carry out single factor fermentation test, by mensuration alcoholic strength, acidity, pol index, carry out single factor and investigate.
1. difference connects the impact of bacterium amount on zymamsis
Table 1 difference connects the impact of bacterium amount on zymamsis
Saccharomycetic content be affect zymamsis important factor it.As shown in Table 1, when the shared ratio of yeast is less, the ethanol content of fermented liquid is lower.Alcohol accumulation volume raises with the increase of adding yeast ratio, when meeting bacterium amount (milk-acid bacteria %: yeast %) ratio is 3:5, the ethanol content of fermented liquid is the highest, pol and acidity are all lower, illustrate that a large amount of sugar and acid have all changed into alcohol, therefore select to connect bacterium amount (milk-acid bacteria %: yeast %) ratio 3:5 is the most appropriate.
2. the impact of different fermentations temperature on zymamsis
The impact of table 2 different fermentations temperature on zymamsis
Temperature has a great impact the growth and breeding of thalline, only in suitable temperature range, could grow and play fermentative action.According to the literature, leavening temperature is the principal element that affects fruit wine local flavor.According to table 2, can find out, when temperature is 30 ℃, fermented liquid ethanol content reaches and is up to 1.2% (V/V), but when excess Temperature surpasses 30 ℃, breeding and the metabolic capacity of yeast decline on the contrary, and the accumulation of alcohol also reduces gradually, therefore select 30 ℃ for optimum fermentation temp the most.
3. the impact of different fermentations time on zymamsis
The table 3 different fermentations time is on spilling the impact of essence fermentation
Along with the prolongation of fermentation time, yeast is fully grown, and alcoholic strength also raises gradually.As shown in Table 3, when fermentation time is 72h, be a flex point, 24h raises gradually to the ethanol content of 72h indirect fermentation liquid, and when fermentation time is when 72h arrives 120h, the alcohol accumulation volume of fermented liquid obviously reduces, and therefore selecting fermentation time is 72h.
4. the impact of different sugar addition on zymamsis
The impact of table 4 different sugar addition on zymamsis
The fermentation capacity of bacterial strain strengthens along with the increase of carbon source within the specific limits, add sucrose and provide abundant carbon source for yeast and milk-acid bacteria, strengthened the fermentation capacity of bacterial strain, made ethanol content, the pol of fermented liquid, acidity is all higher than former groups of single factor experiment results.As shown in Table 4, sugaring amount is between 4-8g/100mL time, alcoholic strength accumulation volume increases gradually, but when sugaring amount surpasses 8g/100mL, fermentation capacity, parallel downward trend gently, may be too high because add sucrose content, the sugared excessive concentration in horse Ruzhong, suppressed the fermentation of yeast and milk-acid bacteria, thereby alcoholic strength is declined, the reason that pol and acidity are higher.Therefore, comprehensive above reason selection sugaring amount is that 8g/100mL is the most appropriate.
Embodiment five: composite bacteria is mixed with the zymamsis conditional response curved design optimization Test of horse vinegar
On the single factor experiment result basis providing at embodiment tri-, according to the center combination experimental design principle of Box-Behnken respectively on affecting three factors of zymamsis alcoholic strength (Y): inoculum size (milk-acid bacteria %: yeast %) (A), fermentation time (B), leavening temperature (C), utilize response surface software Design-Expert8.05 software, according to Box-Behnken experiment unitized design, carry out Three factors-levels experiment.The factor of response surface test and level code value are as table 5.
Factor and the level code value of the test of table 5 response surface
1. the optimization of zymamsis response surface is analyzed: comprehensive single factor experiment result, adopts Box-Behnken response surface analysis method to be optimized it.Response surface test design and response value the results are shown in Table 6.
The test of table 6 response surface and response value
According to table 6 testing data, carry out multiple regression equation matching, can set up take fermented liquid alcoholic strength to the fit equation of inoculum size (A), fermentation time (B), leavening temperature (C) as:
Y=6.5—0.57A+0.14B+0.56C-0.45AB-0.9AC+0.22BC-1.04A
2-0.26B
2-1.31C
2
Table 7 response surface test-results and variance analysis
Note: " * " represents significantly (0.01<p<0.05); " * * " represents extremely significantly (p<0.01)
In regression equation, the significance of each variable on index (response value) impact, is checked to judge by F, and the value of probability P is less, and the significance degree of relevant variable is higher.As shown in Table 7, when model F=68.44, P < 0.0001, illustrates that model is extremely significant.When item F=1.12 is intended in mistake, P=0.4404>0.05, illustrates that model loses plan item not remarkable.Coefficient of determination R
2=0.9888, correction coefficient R
2 adj=0.9742, show to there is good degree of fitting between the measured value of acidity and predictor.The foundation that it can be said that bright model is significance, and the coefficient of determination and correction coefficient be all greater than 0.8, illustrates that the fitting degree of this model is better, and test operation is accurately credible, therefore can utilize this model to carry out analysis and prediction to alcoholic fermentation process.
As can be seen from Table 7, once item all shows utmost point conspicuous level to the Y value impact of zymamsis with quadratic term, mutual AB, an AC show as conspicuous level to the impact of zymamsis Y value, and a mutual BC does not have the impact of conspicuous level on the impact of kumiss Y value.Test-results can show that each factor on the impact of fermented liquid alcoholic strength (A>C>B): inoculum size ratio > leavening temperature > fermentation time.
2. response surface map analysis: in order to investigate each mutual impact on fermented liquid alcoholic strength, in the changeless situation of other factors, utilize Design-Expert8.05 software to carry out computing to regression equation, make the response surface figure of mutual.Three-dimensional response surface figure can explain the impact on response value between each variable and variable more intuitively, specifically referring to accompanying drawing 4 to accompanying drawing 6.
By accompanying drawing 4 to accompanying drawing 6, can be found out, along with the increase of inoculum size ratio, alcoholic strength is the rear downward trend that first rises, and fermentation time is not obvious on alcoholic strength impact, its curve shows as level and smooth straight line, and fermentation time and the interaction connecing between sharp amount ratio are not remarkable; Alcoholic strength is the rear downward trend that first rises, the significant interaction of leavening temperature and inoculum size ratio with the rising of leavening temperature and inoculum size ratio; Along with the rising of leavening temperature, alcoholic strength is the trend that first rises and reduce afterwards, and the two interaction is not remarkable.
Embodiment six: the acetic fermentation condition acetic fermentation single factor experiment of preparation horse vinegar
Access a certain amount of acetic bacteria in alcohol fermentation liquid, and detect fermented liquid acidity since 2d.
Choosing respectively yeast connects bacterium amount (4%, 6%, 8%, 10%, 12%), initial alcohol degree (5% (V/V), 6% (V/V), 7% (V/V), 8% (V/V), 9% (V/V)), leavening temperature (24 ℃, 27 ℃, 30 ℃, 33 ℃, 36 ℃), four factors of fermentation time (2d, 3d, 4d, 5d, 6d) and carries out acetic fermentation single factor experiment.
1. difference connects the impact of bacterium amount Dichlorodiphenyl Acetate fermentation: as shown in Figure 7, it is very large on the impact of fermented liquid acidity that different acetic bacterias connects bacterium amount, acidity presents rising trend along with connecing the increase of bacterium amount, and the transformation efficiency of alcohol is obviously increased, and produces acid amount the highest when connecing bacterium amount and being 10%.When connecing bacterium amount and increase to 12%, acidity has downtrending after 3d, and the nutritive substance in fermented liquid is mainly used in the breeding of thalline, and the transformation efficiency of alcohol has been reduced.So in general, the suitableeest bacterium amount that connects is 10%.
2. the impact of initial alcohol degree Dichlorodiphenyl Acetate fermentation: by drawing the analysis of Fig. 8, acidity increases when initial alcohol degree raises within the specific limits, but on the contrary can inhibited reaction speed when initial alcohol degree too high levels.When initial alcohol degree reaches 7% (V/V), fermented liquid is keeping higher acidity, and when alcoholic strength higher or lower than 7% (V/V), the accumulation of acidity obviously reduces.In general, the suitableeest initial alcohol volume fraction is chosen in 7% (V/V).
3. the impact of leavening temperature Dichlorodiphenyl Acetate fermentation: acetic fermentation process is obviously subject to the impact of temperature.Within the specific limits, the growth and breeding of acetic bacteria.When temperature is lower, acetic bacteria growth metabolism is suppressed, and produces slow acid; And acetic bacteria activity is suppressed when temperature is higher, easily early ageing is degenerated.As can be seen from Figure 9, leavening temperature acidity between 27 ℃ to 33 ℃ increases along with the rising of temperature, and fermentation and acid ability also strengthens, and 33 ℃ of acidity while fermenting 5d reach the highest 120 ° of T, and according to test, optimum leavening temperature is 33 ℃.
4. the impact of fermentation time Dichlorodiphenyl Acetate fermentation: from Figure 10, from 2d～5d acidity along with the growth of fermentation time is rising trend, the highest 120 ° of T of acidity during 5d, fermentation time is during to 6d, acidity is slightly reduced to 119 ° of T.So the suitableeest fermentation time is chosen in 5d.
Embodiment seven: the acetic fermentation condition acetic fermentation response curved design optimization Test of preparation horse vinegar
On the single factor experiment basis providing at embodiment five, according to Box-Benhnken center combination test design principle, take acidity as response value (Y), alcoholic strength (A), connect bacterium amount (B), leavening temperature (C) for factor.The test of design Three factors-levels response surface analysis, level of factor is encoded as following table 8.Utilize response surface software Design-Expert8.05 software, the optimum fermentation parameter of Analysis deterrmination.
1. the optimization of mare's milk vinegar acetic fermentation response surface is analyzed: comprehensive single factor experiment result, and adopt Design-Expert8.05 to analyze, test-results and variance analysis are in Table 9 and table 10.According to table 9 testing data, carry out multiple regression equation matching, obtain mare's milk vinegar acetic fermentation process Partial Linear Models: Y=117.60+1.87A-0.88B+3.25C-0.75AB-2.50AC+2.00BC-10.93A2-4.93B2-2.17C2
The factor profit level code value of table 8 response surface test
Table 9 response surface test design table and result
Table 10 response surface test-results and variance analysis
Note: " * " represents significantly (0.01<p<0.05); " * * " represents extremely significantly (p<0.01)
Mare's milk vinegar acetic fermentation response surface test-results is analyzed, and as shown in Table 10, when model F=65.02, P < 0.0001, illustrates that model is significant.When item F=0.11 is intended in mistake, P=0.9506 > 0.05, illustrates that model loses plan item not remarkable.The coefficient of determination R of model
2=0.9687, correction coefficient R
2 adj=0.9730, show to there is good degree of fitting between the measured value of acidity and predictor.Therefore, can utilize this model Dichlorodiphenyl Acetate fermentation condition to carry out analysis and prediction.
Analysis by table 10 draws, once item shows utmost point conspicuous level with the Y value impact of the equal Dichlorodiphenyl Acetate fermentation of quadratic term, the impact of mutual AC Dichlorodiphenyl Acetate fermentation Y value shows as conspicuous level, and the impact of a mutual AB, BC Dichlorodiphenyl Acetate fermentation Y value does not have the impact of conspicuous level.
2. response surface map analysis: in order to investigate each mutual impact on mare's milk acetic acid degree, in the changeless situation of other factors, utilize Design-Expert8.05 software to carry out computing to regression equation, make mutual response surface chart, specifically referring to accompanying drawing 11 to accompanying drawing 13.
From accompanying drawing 11 to accompanying drawing 13, the interaction connecing between two factors of bacterium amount and initial alcohol degree is not remarkable, acidity raises along with connecing the increase within the specific limits of bacterium amount, and initial alcohol degree is larger on the impact of fermentation and acid amount, initially spills to spend that to produce acid amount when low on the low side; From accompanying drawing 11, the interaction between two factors of leavening temperature and initial alcohol degree is more remarkable.From figure accompanying drawing 12, leavening temperature and the interaction connecing between two factors of bacterium amount are more remarkable, and when inoculum size is fixed on zero level, acidity increases along with the rising of temperature, reach after maximum value and start to reduce.
Embodiment eight: composite bacteria is mixed with the fermentation condition optimization of horse vinegar
Respectively zymamsis alcoholic strength is got to maximum value, by software automatic analysis, can obtain best zymamsis condition theoretical value is: inoculum size (milk-acid bacteria %: yeast %) 2.8:6.2, fermentation time 80.8h, leavening temperature are 31.68 ℃, and the alcoholic strength of surveying is 6.96782% (V/V).Consider that actually operating is convenient, choose inoculum size (milk-acid bacteria %: yeast %) 3:5, fermentation time 80h, leavening temperature are 32 ℃, carry out 3 parallel tests, alcoholic strength is 7.0% (V/V), 6.9% (V/V) and 7.0% (V/V).By software analysis, draw the best theoretical processing condition of acetic fermentation: connect bacterium amount 9.8%, alcoholic strength 7.98% (V/V), 32.8 ℃ of leavening temperatures, consider the facility of actually operating simultaneously, top condition is modified to acetic bacteria and connects bacterium amount 10%, initial alcohol degree 8% (V/V), 33 ℃ of leavening temperatures, through 3 times, repeat experiment, the actual mare's milk acetic acid degree obtaining is 119 ° of T, 119 ° of T, 120 ° of T, approach with theoretical prediction value, illustrate that two equations and practical situation matching are good, the resulting Optimized model of response surface analysis is reliable, mathematical model is feasible to optimizing mare's milk vinegar fermentation processing condition, there is practical value.
On the basis of single factor experiment, utilize response surface software, respectively zymamsis and acetic fermentation are carried out to single factor experiment, and utilize response surface software analysis, show that zymamsis optimal conditions of fermentation is respectively: inoculum size (milk-acid bacteria %: yeast %) 3:5,32 ℃ of leavening temperatures, fermentation time 80h, sugared addition is 8g/100ml.Alcoholic strength average out to 7.0% (V/V) with this understanding.Acetic fermentation top condition: connect bacterium amount 10%, initial alcohol degree 8% (V/V), 33 ℃ of leavening temperatures, empirical tests tests measured actual acidity and theoretical value difference is less, prove that two mathematical models are feasible to optimizing mare's milk vinegar fermentation processing condition, for comprehensive utilization mare's milk provides theoretical foundation.
The mare's milk vinegar of preparing by above-described embodiment campaign is creamy white, and nutritious, milk is strong, and mouthfeel is simple and honest, and acid is refreshing soft, and the fragrance that mare's milk has and vinegar perfume (or spice) merge preferably, unique flavor.Concrete product typicalness is as follows:
(1) Oranoleptic indicator: color and luster: oyster white, micro-Huang has the intrinsic color and luster of mare's milk; Fragrance: mare's milk taste perfume (or spice) is strongly fragrant, acetic acid taste is mellow has the distinctive fragrance of mare's milk vinegar; Flavour: soft, the soft tasty and refreshing free from extraneous odour of tart flavour; Figure: emulsion is allowed a little precipitation.
(2) physical and chemical index: total acid: 120 ° of T, reducing sugar content≤4g/L, VC content is 68.72mg/100mL.
(3) microbiological indicator: total number of bacterial colony (cfu/mL)≤100; Coliform (MPN/100mL)≤3; Pathogenic bacterium: do not detect.
Claims (4)
1. lactobacterium casei (Lactobacillus casei) WWMR-1 who is applicable to mare's milk vinegar fermentation, is characterized in that, the culture presevation of lactobacterium casei (Lactobacillus casei) WWMR-1 is numbered CGMCC No.8244.
2. a method for mare's milk vinegar is prepared in composite bacteria mixed fermentation, it is characterized in that, concrete preparation method's step is as follows:
(1) preparation of seed fermentation liquid and female fermented liquid: seed fermentation liquid: bacterial classification lactobacterium casei (Lactobacillus casei) WWMR-1CGMCC No.8244, Kluyveromyces marxianus bacterium (Kluyveromyces marxianus) and the acetic bacteria (Acetic acid bacteria) preserved at 4 ℃ are inoculated in respectively in MRS substratum, YPD substratum and AS1.41 substratum and are activated under gnotobasis, be placed in respectively 37 ℃ and cultivate 24h, 28 ℃ of cultivation 48h and 30 ℃ of cultivation 48h, in order to later stage use; Female fermented liquid: the good mare's milk of pre-treatment is sub-packed in fermenting container, under aseptic condition, the milk-acid bacteria having activated, yeast and acetic bacteria seed fermented liquid are accessed in fermenting container by 5% inoculum size, be placed in respectively 37 ℃ and cultivate 24h, 28 ℃ of cultivation 48h and 30 ℃ of cultivation 48h, in order to later stage use;
(2) mare's milk pre-treatment: fresh mare's milk is removed to impurity and the foreign matter in mare's milk by four layers of filtered through gauze, then mare's milk is preheating to 65 ℃, homogeneous 5min under 20MPa condition; After homogeneous, mare's milk is carried out to pasteurize, place and be cooled to room temperature, treat fermentation use;
(3) zymamsis: milk-acid bacteria prepared by step (1) and saccharomycetes to make fermentation liquid add after mixing according to volume ratio 3:5 meter in mare's milk prepared by step (2), add glucose, sugaring amount is added according to 8g/100ml, at 32 ℃, 135r/min, cultivate 80h, obtaining alcoholic strength is the mare's milk alcohol fermentation liquid of 7% (V/V);
(4) acetic fermentation: according to volume ratio meter, in the alcohol fermentation liquid that the inoculum size of acetic bacteria prepared by step (1) (Acetic acid bacteria) by 10% prepared in step (3), requiring alcohol initial alcohol degree is 8% (V/V), optimum leavening temperature is 33 ℃, 135r/min, cultivate 5 days, obtaining acidity is the mare's milk acetic acid fermentation liquid of 120 ° of T;
(5) aftertreatment: to fermentation termination, acidity, at 120 ° of T, is carried out pasteurize until product, carries out sterile filling after cooling and prepares horse vinegar.
3. the method for mare's milk vinegar is prepared in composite bacteria mixed fermentation as claimed in claim 2, it is characterized in that, described MRS substratum, YPD substratum and AS1.41 substratum are respectively:
(1) MRS substratum: glucose 20g, peptone 10g, extractum carnis 10g, yeast extract paste 5g, citric acid diamines 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, sal epsom 0.58g, manganous sulfate 0.25g, tween-80 1mL, distilled water 1000mL;
(2) YPD substratum: yeast extract paste 10g, peptone 20g, glucose 20g, distilled water 1000mL;
(3) AS1.41 substratum: glucose 10g, yeast extract paste 10g, calcium carbonate 15g, agar 20g, dehydrated alcohol 20m1, distilled water 1000mL.
4. the mare's milk vinegar that as claimed in claim 2 prepared by composite bacteria mixed fermentation method.
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