CN102433268B - Acetobacter pasteurium and its application method in producing citrus vinegar - Google Patents
Acetobacter pasteurium and its application method in producing citrus vinegar Download PDFInfo
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- CN102433268B CN102433268B CN 201110228974 CN201110228974A CN102433268B CN 102433268 B CN102433268 B CN 102433268B CN 201110228974 CN201110228974 CN 201110228974 CN 201110228974 A CN201110228974 A CN 201110228974A CN 102433268 B CN102433268 B CN 102433268B
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Abstract
The invention discloses a acetobacter pasteurium and its application method in producing citrus vinegars and resulting citrus vinegar products. The invention provides acetobacter pasteurium named LSYY001.2, preserved in CGMCC on 14th June, 2010, preservation number: CGMCC No.4954. Method of the invention for producing citrus vinegar comprises: selecting materials, peeling citruses, removing segment membranes, crushing, filtering, adding yeasts for alcoholic fermentation, adding LSYY001.2 for acetic acid fermentation, then filtering. The acetobacter pasteurium provided by the invention not only ferments alcohol for producing acetic acid, but simultaneously produces iso-limonin hydrolase, thus realizing removing bitter of the citrus vinegar.
Description
Technical field
The present invention relates to strain Acetobacter pasteurianus and an application thereof, particularly relate to a strain Acetobacter pasteurianus and utilize this bacterial strain to produce the method for tangerine vinegar and the fruit vinegar product of gained.
Background technology
Organge fruit vinegar is take citrus as main raw material, a kind of nutritious, the acid-tasting drink that local flavor is good that utilize that the modern biotechnology brew forms.It contains abundant organic acid, can soften vegetable fibre and promote sugared metabolism; Regulate blood fat, cardiovascular, anti-oxidant, the Green Tea Extract of protection, prevent and treat arteriosclerosis, senile dementia etc.Acetic acid bacteria strain is one of principal element that determines fruit vinegar output and quality.
Yet the bitter taste problem of utilizing citrus to be processed into to occur in the fruit vinegar process has become a global problem, becomes the bottleneck of restriction China organge fruit vinegar processing industry development.
Result of study shows that the bitter substance in the organge fruit vinegar mainly contains derivative---the limonoids of the triterpenoid take obacalactone, Nomilin etc. as representative.Limonoids claims again limonoid (Limonoids), is the triterpene compound that a class has furan nucleus.Limonoid has highly oxidized triterpene alcohols inductor structure, is combined with the furans of C-17 position, forms the lactone ring of C-3 position or C-6 position, has C-14, the C-15 position to the epoxide feature, mainly exist with free aglycon and 2 kinds of forms of glycoside.Limonoids in the citrus is take obacalactone and Nomilin as main, and Nomilin is very low because of content, and is lower than obacalactone on the bitter taste impact.Obacalactone bitter taste threshold value is very low, is 1.0mg/L in the aqueous solution, is 3.4mg/L in the orange juice, and 60% eater can feel bitter taste.
During China's vinegar was produced at present, only have 2 strains: AS1.41 and Shanghai commonly used made 1.01, mostly are used in liquid fermentation method and produce on the technique of vinegar.Because existing common bacillus aceticus only has the function that the ethanol that for the first time fermentation in the fruit vinegar production is produced is reduced into acetic acid, can not the generation effect to the bitter substance that exists in the citrus fruit juice, so products obtained therefrom is with strong bitter taste feature.
Summary of the invention
The purpose of this invention is to provide the new Acetobacter pasteurianus of a strain.
Acetobacter pasteurianus provided by the present invention (Acetobactuer pasteurium) name is called LSYY001.2, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 06 14th, 2011, preserving number is CGMCC № 4954.
This bacillus aceticus derives from the rotten citrus and acetic acid, citrus juice, certainly make citrus wine, finishing screen is selected the bacillus aceticus of a strain excellent property, according to " the outstanding Bacteria Identification handbook of uncle (the 8th edition) and " description in the common bacteria system identification handbook, combining form feature, cultural characteristic, physiological and biochemical property, 16S rDNA sequencing result are accredited as acetobacter (AcetobacterBeijerinck) Acetobacter pasteurianus (pasteurianus-Acetobacter).
The cultural characteristic of this bacterial strain is: colonial morphology is rounded on solid medium, and smooth surface has projection, and periphery of bacterial colonies has obvious dissolution of calcium carbonate circle; Liquid medium within top fermentation, substratum become muddy, and obvious acetic acid flavor is arranged.
Cell ellipse or the quarter butt of this bacterial strain, 0.8～1.2 μ m * 1.5～2.5 μ m, becomes short chain in pairs once in a while at Dan Sheng; Cell end point or flat.Gram-positive.Motion, inferior single flagellum or two flagellums of extremely giving birth to.Do not produce gemma.Extremely strictly aerobic, the zymamsis in the vinegar raw material processed can be generated acetic acid, can produce different obacalactone enzyme simultaneously, the different obacalactone that exists in the citrus fruit juice is decomposed, thereby realize that organge fruit vinegar removes bitter taste.
Another object of the present invention provides a kind of method of producing organge fruit vinegar, may further comprise the steps: material choice, orange peeling, remove the capsule clothing, smash, filter, add yeast and carry out zymamsis, add bacillus aceticus again and carry out acetic fermentation, filtration procedure, wherein the acetic fermentation time is 6 months, it is characterized in that: described bacillus aceticus is the YY001.2 bacillus aceticus.
The method of described production organge fruit vinegar, comprise that also the citrus pulp is finished zymamsis after, by once the sterilization enter again the acetic fermentation process, sterilising temp is 80 ℃, 0.5 hour time.
Described fermented product can be leavened prod itself, the leavened prod through diluting or purified leavened prod.
Because bacillus aceticus provided by the present invention, it is edible not only the zymamsis in the vinegar raw material processed can be generated acetic acid fermentation liquid, simultaneously can produce different obacalactone lytic enzyme, the different obacalactone that exists in the citrus fruit juice be decomposed, thereby realize that organge fruit vinegar removes bitter taste.
Embodiment
From the orange peel that rots and vinegar unstrained spirits, citrus juice, certainly make and filter out acetic bacteria the citrus wine, carry out the tests such as Physiology and biochemistry, 16S rDNA, finally determine its kind.
The substratum of LSYY001.2 Acetobacter pasteurianus (Acetobactuer pasteurium) is selected:
Acetic bacteria solid medium: citrus sugar 1%, yeast extract paste 1%, CaCO32%, dehydrated alcohol 6%vol, agar 2%, natural pH value.
Acetic bacteria liquid nutrient medium: citrus sugar 1%, yeast extract paste 1%, dehydrated alcohol 6%vol, natural pH value.
The acetic bacteria storage medium: citrus sugar 1%, yeast extract paste 1%, CaCO30.5%, agar 1.8%, natural pH value adds dehydrated alcohol 6%vol before the inoculation.
Strain separating: sample → enrichment culture → dilution spread → picking has single bacterium colony of variable color circle → purifying cultivation → qualitative test → quantitative test → cultural characteristic → morphological specificity → physiological and biochemical property → strain identification.
The multiplication culture of LSYY001.2 Acetobacter pasteurianus (Acetobactuer pasteurium):
The sample of getting collection is placed in the 250mL triangular flask that fills 50mL proliferated culture medium (citrus sugar 1%, yeast extract paste 1%, dehydrated alcohol 6%vol), behind 30 ℃, 150r/min shaking culture 48h, select the pH value obviously to descend, and the confession plate isolation of acetic acid flavor is arranged.
The strain separating purifying: dilution multiplication culture liquid, (10-1～10-10) coat respectively on the solid medium flat board cultivates 3d～5d, selects the large bacterium colony of transparent circle and carry out being stored in 4 ℃ of refrigerators behind the slant culture 2d for 30 ℃ with the diluent of different concns.
Acetic bacteria qualitative test: the bacterial strain that filters out is inoculated in respectively in the acetic bacteria liquid nutrient medium, after 30 ℃ of thermostat containers are cultivated 3d, the centrifugal 20min of nutrient solution 4000r/min is to remove thalline, draw 5mL without the nutrient solution of thalline, be neutralized to the pH value as 7.0 take 0.1mol/L sodium hydroxide, solution with neutralization boils again, adds 8% ferric chloride Solution number droplet, and forming the reddish-brown precipitation person is acetic bacteria.
The acetic bacteria quantitative test: the acetic bacteria that filters out is inoculated in respectively in the acetic bacteria liquid nutrient medium, and after 30 ℃ of thermostat containers were cultivated 3d, employing alkali formula volumetry (with acetometer) was surveyed it and is produced the acid amount, and therefrom the high bacterial strain of screening product acid amount carries out multiple sieve.
Filter out 5 strain bacterium from rotting the orange peel, it is numbered G1～G3; From rice vinegar, filter out 3 strain bacterium, it is numbered M1; From citrus juice, filter out 5 strain bacterium, it is numbered Z1～Z2.Use above-mentioned substratum and various test method, obtaining bacterial strain is carried out strain physiology become privileged Characteristic Contrast, find that G3 bacterial strain product acetic acid amount is higher, and the feature of debitterize has appearred in products obtained therefrom.Therefore with the G3 bacterial strain called after LSYY001.2 that obtains.And by the genetic stability test, LSYY001.2 is gone down to posterity 5 times, observe cultural characteristic and the individual morphology of the rear bacterial strain that at every turn goes down to posterity.Wherein, colonial morphology is respectively for consistent, and calcium dissolving circle is obvious, and individual morphology is all normal, does not morph, is out of shape.Simultaneously it being produced acidity can compare, and record each generation product acid amount is under the same conditions cultivated with slant medium, and after 5 generations of propagating, its total acid content slightly descends, and the range of decrease is all less than 1%, and its genetic stability is good.
| Strain number | G1 | G2 | G3 | M1 | Z1 | Z2 |
| Total acid (g.L -1) | 0.51 | 37.12 | 32.11 | 28.21 | 22.32 | 26.53 |
Table one: each bacterial strain acetate yield
| Strain number | G1 | G2 | G3 | M1 | Z1 | Z2 |
| The fruit vinegar mouthfeel | Bitter taste | Bitter taste | Without bitter taste | Bitter taste | Bitter taste | Bitter taste |
Table two: each bacterial strain acetic fermentation products taste performance
Observe and microscopy by morphological specificity, visible LSYY001.2 gramstaining, reaction is negative.Without gemma, be rod-short, single or paired, one-tenth chain alignment.Colonial morphology is rounded on solid medium, and smooth surface has projection, and periphery of bacterial colonies has obvious dissolution of calcium carbonate circle; Liquid medium within top fermentation, substratum become muddy, and obvious acetic acid flavor is arranged.
Select positive colony and serve Hai Shenggong and check order, check order and be listed in the EUBI website contrast is 99% with tropical bacillus aceticus similarity through sequence.Utilize Clustalx1.81 software and GenBank gene order to do the maximum homology comparative analysis with the 16SrDNA sequence that higher homology bacterial strain is arranged with it that application Blast retrieves bacterial strain LSYY001.2, and utilize Mega 4.1 softwares to make genealogical tree with N-J method (neighbor-joining), and phylogenetic tree construction, determine the classification position of bacterial strain.Can find out, LSYY001.2 and Acetobacter pasteurianus (pasteurianus-Acetobacter) are very approaching on phylogenetic tree, the gene order similarity is very high, can determine that LSYY001.2 is Acetobacter pasteurianus (pasteurianus-Acetobacter).
Utilize the LSYY001.2 Acetobacter pasteurianus to produce the method for organge fruit vinegar, may further comprise the steps: material choice, orange peeling, remove the capsule clothing, smash, filter, add yeast and carry out zymamsis and acetic fermentation process.After citrus carried out the epidermis clean, the citrus fruit making beating is crushed into liquid glucose, adds yeast and carry out zymamsis, make the sugar part in the pulp change alcohol into.The zymamsis of pulp, after fermenting process is finished, the high-temperature steam that passes into 100 ℃ in pulp liquid was sterilized 1 hour, be cooled to again 40 ℃, add the YY001.2 acidfast bacilli and carry out acetic fermentation, fermentation time is 6 months, and look during the fermentation the fermentation situation and stir gently, until fermented liquid becomes clear state, at last to fermented liquid filter, can.
Claims (2)
- One strain Acetobacter pasteurianus ( Acetobacter pasteurium), name is called LSYY001.2, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 06 14th, 2011, and preserving number is CGMCCNo.4954.
- 2. one kind is utilized Acetobacter pasteurianus claimed in claim 1 in the application of producing on the organge fruit vinegar, its feature may further comprise the steps: material choice, orange peeling, remove the capsule clothing, smash, filter, add yeast and carry out zymamsis, add bacillus aceticus again and carry out acetic fermentation, filtration, can, sterilization, finished product packing process, wherein the acetic fermentation time is 6 months.
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| CN106167781B (en) * | 2016-08-25 | 2019-07-30 | 湖北工业大学 | A kind of Acetobacter pasteurianus that temperature capacity is excellent and purposes |
| CN109486731A (en) * | 2019-01-10 | 2019-03-19 | 江西科技师范大学 | A kind of resistance to acetate ethanol bacterium and its application |
| CN113969245B (en) * | 2021-10-25 | 2022-07-29 | 爱康食品(青岛)有限公司 | Sushi vinegar acetic acid bacteria, method for fermenting sushi vinegar and application of sushi vinegar to sushi |
| CN116814509B (en) * | 2023-08-23 | 2023-12-22 | 仙乐健康科技股份有限公司 | Ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii |
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| CN1428414A (en) * | 2001-12-25 | 2003-07-09 | 福建农林大学 | Method for preparing tangerine fruit vinegar and its fruit vinegar beverage |
| CN1392240A (en) * | 2002-05-28 | 2003-01-22 | 湖南省农业科学院农产品加工研究所 | Process for producing orange fruit vinegar |
| CN101270329A (en) * | 2008-05-15 | 2008-09-24 | 天津科技大学 | Method for preparing high concentration fruit vinegar with liquid state submerged fermentation |
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