CN102433268A - 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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- CN102433268A CN102433268A CN2011102289741A CN201110228974A CN102433268A CN 102433268 A CN102433268 A CN 102433268A CN 2011102289741 A CN2011102289741 A CN 2011102289741A CN 201110228974 A CN201110228974 A CN 201110228974A CN 102433268 A CN102433268 A CN 102433268A
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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 a strain Pasteur bacillus aceticus and an application thereof, particularly relate to a strain Pasteur bacillus aceticus 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 to be main raw material with the citrus, a kind of nutritious, the acid-tasting drink that local flavor is good that utilize that the modern biotechnology brew forms.It contains rich organic acid, can soften vegetable fibre and the metabolism that promotes sugar; Regulating blood fat, cardiovascular, anti-oxidant, the Green Tea Extract of protection are prevented and treated arteriosclerosis, senile dementia etc.Acetic acid bacteria strain is one of principal element of decision fruit vinegar output and quality.
Yet utilizing citrus to be processed into the bitter taste problem that is occurred 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 it is the verivate of the triterpenoid of representative---limonoids that the bitter substance in the organge fruit vinegar mainly contains with obacalactone, Nomilin etc.Limonoids is claimed limonoid (Limonoids) again, is one type of triterpene compound with furan nucleus.Limonoid has highly oxidized triterpene alcohols inductor structure, combines 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 characteristic, mainly exist with free aglycon and 2 kinds of forms of glycoside.Limonoids in the citrus is main with obacalactone and Nomilin, and Nomilin is very low because of content, and is lower than obacalactone to the bitter taste influence.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, are used in liquid fermentation method mostly and produce on the technology of vinegar.Because existing common bacillus aceticus only has the function that the ethanol that fermentation for the first time in the fruit vinegar production is produced is reduced into acetate, can not the generation effect to the bitter substance that exists in the citrus fruit juice, so products obtained therefrom has intensive bitter taste characteristic.
Summary of the invention
The purpose of this invention is to provide the new Pasteur's bacillus aceticus of a strain.
Pasteur's bacillus aceticus 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 septic citrus and acetic acid, citrus juice, make citrus wine certainly; Finishing screen is selected the bacillus aceticus of a strain excellent property; According to " description in the outstanding Bacteria Identification handbook of uncle (the 8th edition) and " the common bacteria system identification handbook ", combining form characteristic, cultural characteristic, physiological and biochemical property, 16S rDNA sequencing result are accredited as acetobacter (AcetobacterBeijerinck) Pasteur's bacillus aceticus (pasteurianus-Acetobacter).
The cultural characteristic of this bacterial strain is: colonial morphology is rounded on solid medium, and smooth surface has convexity, and periphery of bacterial colonies has tangible dissolution of calcium carbonate circle; Liquid medium within top fermentation, substratum become muddy, and tangible acetic acid flavor is arranged.
The 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.Extreme strict aerobic, can the zymamsis in the system vinegar raw material 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 accomplished zymamsis after, get into the acetic fermentation process again through sterilization once, sterilising temp is 80 ℃, 0.5 hour time.
Said fermented product can be leavened prod itself, through the leavened prod that diluted or purified leavened prod.
Because bacillus aceticus provided by the present invention; It is edible not only can the zymamsis in the system vinegar raw material to be generated acetic acid fermentation liquid; Simultaneously can produce different obacalactone lytic enzyme, the different obacalactone that exists in the citrus fruit juice decomposed, thereby realize that organge fruit vinegar removes bitter taste.
Embodiment
From septic orange peel and vinegar unstrained spirits, citrus juice, make and filter out acetic bacteria the citrus wine certainly, carry out tests such as Physiology and biochemistry, 16S rDNA, finally confirm its kind.
The substratum of LSYY001.2 Pasteur bacillus aceticus (Acetobactuer pasteurium) is selected for use:
The acetic bacteria solid medium: citrus sugar 1%, yeast extract paste 1%, CaCO3 2%, absolute ethyl alcohol 6%vol, agar 2%, natural pH value.
Acetic bacteria liquid nutrient medium: citrus sugar 1%, yeast extract paste 1%, absolute ethyl 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 absolute ethyl alcohol 6%vol before the inoculation.
Strain separating: sample → enrichment culture → dilution coating → picking has the single bacterium colony → purifying cultivation → qualitative test → quantitative test → cultural characteristic → morphological specificity → physiological and biochemical property → strain identification of variable color circle.
The multiplication culture of LSYY001.2 Pasteur bacillus aceticus (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%, absolute ethyl alcohol 6%vol), behind 30 ℃, 150r/min shaking culture 48h, selects pH value obviously to descend, and the confession plate isolation that has acetic acid to distinguish the flavor of.
The strain separating purifying: dilution multiplication culture liquid, (10-1～10-10) separate application is cultivated 3d～5d, is selected the big bacterium colony of transparent circle and carry out being stored in 4 ℃ of refrigerators behind the slant culture 2d for 30 ℃ on the solid medium flat board 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 were cultivated 3d, the centrifugal 20min of nutrient solution 4000r/min drew the nutrient solution that 5mL does not have thalline to remove thalline; Being neutralized to the pH value with 0.1mol/L sodium hydroxide is 7.0; Neutral solution is boiled again, add 8% ferric chloride Solution(38 number droplet, 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 TP, it is that physiology is becomed privileged the characteristic contrast that obtaining bacterial strain is carried out strain, find that G3 bacterial strain product acetic acid amount is higher, and the characteristic of debitterize has appearred in products obtained therefrom.Therefore with the G3 bacterial strain called after LSYY001.2 that obtains.And through the genetic stability test, LSYY001.2 is gone down to posterity 5 times, observe the cultural characteristic and the individual morphology of the back 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.
Table one: each bacterial strain acetate yield
| 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 two: each bacterial strain acetic fermentation product mouthfeel performance
| Strain number | G1 | G2 | G3 | M1 | Z1 | Z2 |
| The fruit vinegar mouthfeel | Bitter taste | Bitter taste | No bitter taste | Bitter taste | Bitter taste | Bitter taste |
Observe and microscopy through morphological specificity, visible LSYY001.2 gramstaining, reaction is negative.No gemma is rod-short, single or paired, one-tenth chain alignment.Colonial morphology is rounded on solid medium, and smooth surface has convexity, and periphery of bacterial colonies has tangible dissolution of calcium carbonate circle; Liquid medium within top fermentation, substratum become muddy, and tangible 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 16S rDNA sequence that higher homology bacterial strain is arranged with it that bacterial strain LSYY001.2 and Application of B last retrieve; And utilize Mega 4.1 softwares to make genealogical tree with N-J method (neighbor-joining); And constructing system grows tree, confirms the classification position of bacterial strain.Can find out that LSYY001.2 and Pasteur's bacillus aceticus (pasteurianus-Acetobacter) are very approaching on phylogenetic tree, the gene order similarity is very high, can confirm that LSYY001.2 is Pasteur's bacillus aceticus (pasteurianus-Acetobacter).
Utilize LSYY001.2 Pasteur bacillus aceticus 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.After the zymamsis fermenting process of pulp is accomplished; The high-temperature steam that in pulp liquid, feeds 100 ℃ was sterilized 1 hour; Be cooled to 40 ℃ again, add the YY001.2 acidfast bacilli and carry out acetic fermentation, fermentation time is 6 months, and looks the fermentation situation during the fermentation and stir gently; Become clear state up to fermented liquid, at last to fermented liquid filter, can.
Claims (4)
1. a strain Pasteur bacillus aceticus (Acetobactuer pasteurium), name was called LSYY001.2, had been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on 06 14th, 2011, and preserving number is CGMCC № 4954.
2. Pasteur's bacillus aceticus is at the application method of producing on the 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, can, sterilization, finished product packing process; Wherein the acetic fermentation time is 6 months, it is characterized in that: described bacillus aceticus is the LSYY001.2 bacillus aceticus.
3. application method as claimed in claim 2, its characteristic also is: after the citrus pulp is accomplished zymamsis, get into the acetic fermentation process again through once sterilizing, sterilising temp is 80 ℃, 0.5 hour time.
4. by a kind of organge fruit vinegar product of claim 1 to 3 gained.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106167781A (en) * | 2016-08-25 | 2016-11-30 | 湖北工业大学 | Acetobacter pasteurianus that a kind of 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 |
| CN113969245A (en) * | 2021-10-25 | 2022-01-25 | 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 |
| CN116814509A (en) * | 2023-08-23 | 2023-09-29 | 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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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106167781A (en) * | 2016-08-25 | 2016-11-30 | 湖北工业大学 | Acetobacter pasteurianus that a kind of temperature capacity is excellent and purposes |
| 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 |
| CN113969245A (en) * | 2021-10-25 | 2022-01-25 | 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 |
| 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 |
| CN116814509A (en) * | 2023-08-23 | 2023-09-29 | 仙乐健康科技股份有限公司 | Ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii |
| 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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