CN103194409A - Bacillus aceticus and application thereof in preparing apple vinegar - Google Patents
Bacillus aceticus and application thereof in preparing apple vinegar Download PDFInfo
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- CN103194409A CN103194409A CN2013101142147A CN201310114214A CN103194409A CN 103194409 A CN103194409 A CN 103194409A CN 2013101142147 A CN2013101142147 A CN 2013101142147A CN 201310114214 A CN201310114214 A CN 201310114214A CN 103194409 A CN103194409 A CN 103194409A
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- bacillus aceticus
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Abstract
The invention discloses a newly separated bacillus aceticus, and a process method for fermenting apple vinegar by immobilizing the bacillus aceticus with husks. The immobilized bacillus aceticus can be used in batches, and the continuous batch fermenting time can be over 90d, and the corresponding acid conversion rate can be over 90 percent, so that the fermenting production operation can be simplified, the production efficiency can be improved, the production capacity can be improved, and cells can be easily separated in the production tail and can be recycled.
Description
Technical field
The invention belongs to the food fermentation technical field, the method that relates to the new bacillus aceticus bacterial strain that separates of a strain and behind adsorption of immobilization on the husk, carry out the fermentation of apple fruit vinegar.
Background technology
Cider vinegar more and more is subjected to human consumer's favor with its unique local flavor and many nourishing functions.Apple vinegar beverage by the former vinegar allotment of apple forms presented rising tendency faster in recent years especially.Apple vinegar beverage is of a great variety in the market, real actually rare based on the apple vinegar beverage of fermentation.In May, 2012, the standard of first apple vinegar beverage on the 1st came into effect, and this standard clearly must not propose and used non-apple fermentation such as grain to produce or modulation apple vinegar beverages such as the vinegar of synthetic, acetic acid, oxysuccinic acid, citric acid.
Acetic fermentation is the key technique of brewageing cider vinegar, and the local flavor of cider vinegar mainly comes from this link.At present, China produces cider vinegar aspect zymamsis, though comparatively ripe free cell fermentation technique is arranged, lacks the apple acetic acid bacteria strain of stable performance, the existing vinegar unstrained spirits of the usefulness that has, and what have uses compound acetic bacteria, and the usefulness that also has is the bacterial classification of seed selection voluntarily.At present, liquid state fermentation is produced most acetic bacterias that use and is AS1.41, and its alcohol-tolerant ability is less than 8%, high acid amount is 70～90g/L, the alcohol transformation efficiency is about 70%, wherein also has quite a few acetic acid to be broken down into carbonic acid gas and water, and the total acidity that obtains at last is generally about 5%.
Ethanol content must be controlled than low value in the apple fruit vinegar beverage, and this just requires the acetic fermentation of apple fruit vinegar will compare thoroughly.Reach this requirement, it is only adopting the batch fermentation mode, and technology is also simple relatively.But free bacillus aceticus cell causes the acetic acid conversion rate to reduce greatly in the later stage in acetic fermentation stage along with the decline of cell viability, and for residual ethanol is changed into acetic acid as far as possible, just must continue to prolong fermentation time, so not only production efficiency is low, but also cause the flavour substances heavy losses of apple fruit vinegar, finally influence product quality and style.Adopt the immobilization fermentation can head it off, the bacillus aceticus method for immobilizing cell mainly contains absorption method and entrapping method at present, just in the at present existing immobilized patent of bacillus aceticus, mainly be based on calcium-alginate-immobilized, but because bacillus aceticus belongs to aerobic microbiological, in order to obtain enough dissolved oxygens, the bacillus aceticus cell preferably is directly exposed in the fermented liquid during the fermentation, otherwise can influence mass transfer and the acetic fermentation speed of dissolved oxygen, and influence the bacillus aceticus cell activity.
Summary of the invention
The objective of the invention is to solve problems of the prior art, one aspect of the present invention relates to a kind of bacillus aceticus, it is characterized in that its preserving number is CCTCC M 2012551, is preserved in Chinese typical culture collection center.
The present invention also relates to the application of above-mentioned bacillus aceticus in fermentation apple fruit vinegar on the other hand.
In a preferred embodiment of the present invention, the technology that it is characterized in that described fermentation apple fruit vinegar comprises the steps: husk carrier sterilization pre-treatment, to activate good bacillus aceticus then and insert fermentor tank with sterilising treatment good carrier and sterilized apple fruit wine matrix, in 25 ~ 32 ℃ of fermentation 1 ~ 5 d, carry out immobilization fermentation 3 ~ 8d then and prepare the apple fruit vinegar.
The present invention relates to a kind of processing method of immobilization fermentation apple fruit vinegar on the other hand, it is characterized in that comprising the steps:
Step 1: carrier pre-treatment: with husk oven dry earlier, cooling, the paddy skin is suitably broken, and preferably, each husk becomes 2 to 3 to be advisable; With edible ethanol to the husk pre-treatment of sterilizing;
Step 2: will weigh above-mentioned bacillus aceticus and carry out actication of culture;
Step 3: the carrier husk that the bacillus aceticus after will activating and step 1 are handled well and sterilized hard cider insert fermentor tank, adsorb in 25 ~ 32 ℃ of fermentation 1 ~ 5 d, liquid in the fermentor tank is emitted, re-enter hard cider and carry out immobilization fermentation 3 ~ 8 d.
In a preferred embodiment of the present invention, the inoculum size of described bacillus aceticus is 1% ~ 5%(v/v).
In a preferred embodiment of the present invention, the load rate of described carrier husk Dichlorodiphenyl Acetate bacilli-cell is greater than 1 * 10
6Individual/mg.
In a preferred embodiment of the present invention, described hard cider alcohol concn is 2 ~ 8%(v/v).
Microorganism information
The bacillus aceticus that arrives involved in the present invention (
AcetobacterSp
.CCTCC M 2012551) XZJ001 separates from red bayberry fruit wine distiller's wort first, be preserved in Chinese typical culture collection center on December 27th, 2012, be called for short CCTCC, preserving number is M 2012551, and the preservation address is Chinese Wuhan City, Hubei Province Wuhan University (postcode is 430072).
Embodiment
The bacillus aceticus that arrives involved in the present invention (
AcetobacterSp
.CCTCC M 2012551) XZJ001 separates from red bayberry fruit wine distiller's wort first, be preserved in Chinese typical culture collection center on December 27th, 2012, be called for short CCTCC, preserving number is M 2012551, and the preservation address is Chinese Wuhan City, Hubei Province Wuhan University (postcode is 430072).
The Physiology and biochemistry of bacillus aceticus is identified:
(1) produces the acetic acid qualitative experiment
With fresh bacterial classification inoculation in the bacillus aceticus proliferated culture medium, 30 ℃ of shaking tables were cultivated after 2 days, get nutrient solution 5 mL in vitro, sodium hydroxide solution is neutralized to pH 7.0, add 2 ~ 3 mixings of 100 g/L ferric chloride Solutions, observe liquid color and whether become yellowish red color, boil at flame, observation has or not reddish-brown precipitation to generate, and whether clear liquid bleach.Proliferated culture medium: yeast extract 10 g/L, glucose 10 g/L add 30 mL/L raw spirits behind 4.5,121 ℃ of sterilizations of pH 20min.
(2) oxidation of ethanol experiment
With fresh bacterial classification inoculation oxidation of ethanol substratum, cultivate after 2 ~ 3 days and observe, if substratum produces the positive reaction of sour flavescence person.The oxidation of ethanol culture medium prescription is: yeast extract 10 g/L, and 20 mL/L concentration are the 0.4 g/L tetrabromophenol sulfonphthalein aqueous solution, pH 6.8 ~ 7.0, add 40 mL/L raw spirits behind 121 ℃ of sterilization 20min.
(3) hydrogen peroxide enzymatic determination
Get a ring and grow in the culture of PYG agar slant, be applied on the clean slide glass, Dropwise 5 drips the H of 100 mL/L thereon then
2O
2, if there is bubble to produce then positive reaction.PY basic medium: peptone 5 g/L, pancreatin solution casein 5 g/L, yeast extract 10 g/L, salts solution 40 mL/L.
PY salts solution composition: Calcium Chloride Powder Anhydrous 0.2 g/L, magnesium sulfate heptahydrate 0.48 g/L, dipotassium hydrogen phosphate 1.0 g/L, potassium primary phosphate 1.0 g/L, sodium bicarbonate 10.0 g/L, sodium-chlor 2.0 g/L.
PY salts solution method for making: calcium chloride and magnesium sulfate heptahydrate mixed dissolution in 300 mL distilled water, are added 500 mL water again, slowly add other salts while stir.Add 200 mL distilled water after continuing to be stirred to whole dissolvings, stock in 4 ℃ standby after the mixing.Add 10 g/L glucose in the PY basic culture solution and be the PYG substratum.
(4) litmus milk experiment
With fresh bacterial classification inoculation litmus milk enrichment, observe litmus milk after 1,3,7 days in 37 ℃ of cultivations and produce acid and coagulation reaction.Litmus milk enrichment: be that to add 4 mL concentration in the 100 g/L skimmed milks be the reindeer moss of 25 g/L in 100 mL concentration, packing test tube, milk height 4-5 cm.
(5) gelatine liquefication experiment
With fresh bacterial classification inoculation gelatine liquefication experiment substratum, place 37 ℃ of cultivations, do contrast with two nonvaccinated test tube substratum.Cultivate after 2 ~ 3 days, will inoculate with control tube and place refrigerator respectively, if control tube solidifies, inoculated tube liquefaction is recorded as the positive, liquefies simultaneously or solidifies then negative result.
The gelatin-based basal culture medium: peptone 10 g/L, yeast extract 10 g/L, glucose 1 g/L, salts solution 40 mL/L(and PY basic medium are together), gelatin 120 g/L, 7.0,121 ℃ of sterilizations of pH 20min.
(6) produce the hydrogen sulfide experiment
The preparation of lead acetate reagent strip: common filter paper bar is cut into the wide paper slip of about 0.5～0.6 cm, and length is decided according to test tube and substratum height.Be that the lead acetate of 50～100 g/L soaks into paper slip with concentration, place oven for drying then, put into culture dish or in vitro, the sterilization back is standby.
Behind fresh bacterial classification inoculation product hydrogen sulfide experiment substratum test tube, hang in the inoculated tube with aseptic nipper gripping one lead acetate paper slip.The lower end does not contact liquid level, upper end tampon jam-pack near media surface.A test tube of not inoculating is made as blank, also will hang the lead acetate paper slip, places 37 ℃ of cultivations, and the paper slip blackening is positive reaction then.
Produce hydrogen sulfide experiment substratum (halfcystine substratum): Tryptones 10 g/L, beef extract 3 g/L, yeast extract 5 g/L, sodium-chlor 5 g/L, halfcystine 0.4 g/L, glucose 2 g/L, the packing test tube, every pipe nutrient solution height 4-5 cm, pH 7.2-7.4,121 ℃ of sterilization 20 min.
(7) produce the indoles experiment
Fresh bacterial classification inoculation is produced indoles experiment substratum, place 37 ℃ of cultivations.Cultivate after 2-3 days, get nutrient solution and add test tube, add the high reagent of 3-5 mm in the nutrient solution surface, if then positive reaction of redness appears in the liquid layer interface along tube wall.
Reagent: Paradimethylaminobenzaldehyde 0.08 g, dehydrated alcohol 7.6 mL, dense HCl 1.6 mL,
Produce indoles experiment substratum: the 10 g/L tryptone aqueous solution, transfer pH 7.2～7.5, packing test tube, 121 ℃ of sterilization 20min.
The physio-biochemical characteristics of bacterial classification are as shown in table 1, can produce acetic acid and hydrogen sulfide, the catalase positive, and the litmus milk experiment is positive, and energy oxidation ethanol can not carry out gelatine liquefication, does not produce indoles.According to morphological specificity and physiological and biochemical property, according to " uncle's Jie Shi Bacteria Identification handbook can identify that bacterial classification is genus acetobacter, binding molecule biological assay result, tentatively determine strain X ZJ001 be bacillus aceticus (
Acetobacter pomorum).
Table 1 bacillus aceticus XZJ001 Physiology and biochemistry identification experiment result
| Catalase | Gelatine liquefication | Produce H 2S | Litmus milk | Produce indoles | Produce acetic acid | Oxidation of ethanol |
| + | - | + | + | - | + | + |
"+": expression is positive, "-": expression is negative.
Example 1:
1) carrier pre-treatment: with husk 85 ℃ of 2.5 h oven dry earlier, cooling.Get husk 300g, the paddy skin is suitably broken, become 2 to 3 to be advisable with each husk.Press solid-liquid ratio 1:7 with edible ethanol and stir immersion heating in water bath to 40 ℃ 3h, constantly stir.Filtered through gauze adds water 2 h that reflux then in the essential oil extraction flask, use filtered through gauze after pouring out again, and then in 80 ℃ of oven dry, cooling back dress plastics bag is stand-by.
2) the above-mentioned husk of 30 g sterilising treatment is put into 10 L fermentor tanks, insert the bacillus aceticus liquid of 200 mL enlarged culturing, importing 6 L alcoholic strengths then is 2%(v/v) hard cider, carry out adsorption of immobilization fermentation in 30 ℃, dissolved oxygen is controlled all the time at 2.0 ~ 5.0 mg/L between yeast phase, fermentation 5d obtains the apple fruit vinegar, and the transformation efficiency of acetic acid reaches 93%, and the cell loading rate of carrier Dichlorodiphenyl Acetate bacillus is 1.5 * 10
6Individual/the mg carrier.
3) adopt contamination-free manner that the liquid in the above-mentioned fermentor tank is emitted, the immobilization bacillus aceticus is retained in the jar, re-entering 7 L alcoholic strengths is the hard cider of 4.5% (v/v), carry out immobilization fermentation in 28 ℃, dissolved oxygen is controlled all the time at 2.0 ~ 5.0 mg/L between yeast phase, 5 d that ferment obtain acetic acid, and the transformation efficiency of acetic acid reaches more than 90%.Repeat aforesaid operations, carried out 18 batches of fermentations, change sour rate all between 90% ~ 95%, the immobilized cell active duration reaches 96 d.
Example 2:
1) with husk earlier: 90 ℃ of 2.0 h oven dry, cooling.Get husk 300g, suitably broken, become 2 to 3 to be advisable with each husk.Press solid-liquid ratio 1:5 with edible ethanol and stir immersion heating in water bath to 40 ℃ 3h, constantly stir.Filtered through gauze adds water 2 h that reflux then in extraction flask, use filtered through gauze after pouring out again, and then in 70 ℃ of oven dry, cooling back dress plastics bag is stand-by.
2) the above-mentioned husk of 25 g sterilising treatment is put into 10 L fermentor tanks, insert the bacillus aceticus liquid of 300 mL enlarged culturing, importing 5 L alcoholic strengths then is 2%(v/v) hard cider, carry out adsorption of immobilization fermentation in 30 ℃, dissolved oxygen is controlled all the time at 2.0 ~ 5.0 mg/L between yeast phase, 5 d that ferment obtain acetic acid, and the transformation efficiency of acetic acid reaches 92%, and the cell loading rate of carrier Dichlorodiphenyl Acetate bacillus is 6.1 * 10
6Individual/the mg carrier.
3) adopt contamination-free manner that the liquid in the above-mentioned fermentor tank is emitted, the immobilization bacillus aceticus is retained in the jar, re-entering 5 L alcoholic strengths is the hard cider of 4.0% (v/v), carry out immobilization fermentation in 30 ℃, dissolved oxygen is controlled 2.0 ~ 5.0 mg/L all the time between yeast phase, 5 d that ferment obtain the apple fruit vinegar, and the transformation efficiency of acetic acid reaches 95%.Repeat aforesaid operations, carried out 20 batches of fermentations, change sour rate all between 90% ~ 96%, the immobilized cell active duration reaches 115 d.
The above only is the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any variation or replacement of expecting without creative work all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (7)
1. a bacillus aceticus is characterized in that its preserving number is CCTCC M 2012551, is preserved in Chinese typical culture collection center.
2. the application of the described bacillus aceticus of claim 1 in fermentation apple fruit vinegar.
3. application according to claim 2, it is characterized in that comprising the steps: with husk carrier sterilization pre-treatment, to activate good bacillus aceticus then and insert fermentor tank with sterilising treatment good carrier and sterilized apple fruit wine matrix, in 25 ~ 32 ℃ of fermentation 1 ~ 5 d, liquid in the fermentor tank is emitted, re-enter hard cider then and carry out immobilization fermentation 3 ~ 8d and prepare the apple fruit vinegar.
4. the processing method of an immobilization fermentation apple fruit vinegar is characterized in that comprising the steps:
Step 1: carrier pre-treatment: with husk oven dry earlier, cooling, the paddy skin is suitably broken, and preferably, each husk becomes 2 to 3 to be advisable; With edible ethanol to the husk pre-treatment of sterilizing;
Step 2: the described bacillus aceticus of claim 1 is carried out actication of culture;
Step 3: the carrier husk that the bacillus aceticus after will activating and step 1 are handled well and sterilized hard cider insert fermentor tank, adsorb in 25 ~ 32 ℃ of fermentation 1 ~ 5 d, liquid in the fermentor tank is emitted, re-enter hard cider and carry out immobilization fermentation 3 ~ 8 d.
5. processing method according to claim 4, the inoculum size that it is characterized in that described bacillus aceticus is 1% ~ 5%(v/v).
6. processing method according to claim 4 is characterized in that the load rate of described carrier husk Dichlorodiphenyl Acetate bacilli-cell is greater than 1 * 10
6Individual/mg.
7. processing method according to claim 4 is characterized in that described apple wine alcohol concn is 2 ~ 8%(v/v).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328022A (en) * | 2014-10-31 | 2015-02-04 | 天地壹号饮料股份有限公司 | Peracid hawthorn vinegar as well as plant drink containing hawthorn vinegar and preparation method of peracid hawthorn vinegar |
| CN106434435B (en) * | 2016-09-09 | 2019-04-26 | 锦州医科大学 | One plant of acetobacter and the application in acceleration green starch separation sedimentation |
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| CN1526811A (en) * | 2003-09-19 | 2004-09-08 | 李玉文 | Prepn of apple vinegar |
| CN101586072A (en) * | 2008-10-06 | 2009-11-25 | 北京晨钟荣福科技有限公司 | Production technology for apple vinegar |
| CN102994359A (en) * | 2012-12-04 | 2013-03-27 | 天地壹号饮料股份有限公司 | Process method for fermenting fruit vinegar by adsorbing immobilized bacillus aceticus |
| CN102994362A (en) * | 2012-12-06 | 2013-03-27 | 陕西科技大学 | Method for fermenting apple vinegar |
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2013
- 2013-04-03 CN CN201310114214.7A patent/CN103194409B/en active Active
Patent Citations (4)
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| CN1526811A (en) * | 2003-09-19 | 2004-09-08 | 李玉文 | Prepn of apple vinegar |
| CN101586072A (en) * | 2008-10-06 | 2009-11-25 | 北京晨钟荣福科技有限公司 | Production technology for apple vinegar |
| CN102994359A (en) * | 2012-12-04 | 2013-03-27 | 天地壹号饮料股份有限公司 | Process method for fermenting fruit vinegar by adsorbing immobilized bacillus aceticus |
| CN102994362A (en) * | 2012-12-06 | 2013-03-27 | 陕西科技大学 | Method for fermenting apple vinegar |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328022A (en) * | 2014-10-31 | 2015-02-04 | 天地壹号饮料股份有限公司 | Peracid hawthorn vinegar as well as plant drink containing hawthorn vinegar and preparation method of peracid hawthorn vinegar |
| CN104328022B (en) * | 2014-10-31 | 2016-08-17 | 天地壹号饮料股份有限公司 | A kind of peracid Hawthorn Vinegar and the plant beverage comprising this Hawthorn Vinegar and preparation method thereof |
| CN106434435B (en) * | 2016-09-09 | 2019-04-26 | 锦州医科大学 | One plant of acetobacter and the application in acceleration green starch separation sedimentation |
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