Acetobacter is one of the important industrial bacteria.Acetobacter cells are elliptical to rod-shaped;Single, paired or chained;Terminal or peripheral hairs;Moving or not moving;gram-negative;Obligate aerobic bacteriaAnd oxidize various organic substances into organic acids and other oxides.Generally, it grows vigorously on ethanol or other oxidizable yeast boiling liquid or yeast digestion liquid culture medium.[1]
AcetobacterIts important feature is that it can oxidize ethanol to acetic acid, and can oxidize acetic acid and lactic acid to COtwoAnd HtwoO。Some strains can synthesize cellulose, whose cellulose forms the matrix outside the cell wall, while bacteria are buried in a layer of tangled filaments of fiber strands.When these kinds of bacteria grow at restLiquid mediumThey will form a layer of cellulose film on the surface.[2]
Acetobacter is a prokaryote, which proliferates in the form of dichotomy.Acetobacter is widely distributed in nature. It grows in the soil of orchard, on the surface of grapes or rancid food, and in unsterilized vinegar, fruit wine and beer.The metabolic type belongs to heterotrophic aerobic type, so oxygen is always required to participate in the fermentation process.Acetobacter is often used to brew vinegar in fermentation engineering.[3]
More than two centuries ago, it was found that alcoholic beverages with low alcohol content, such as beer and wine, were placed in the air for a little longer, and the liquid surface grew into a film, and the alcohol gradually became acetic acid.If a little of this film is transplanted into beer or wine, the wine will easily become vinegar, so the pole organism that forms the film is called "vinegar mother".In 1822, PerSON called this kind of living film the film yeast（Mycoderma）, the meaning of this word refers to mucinous film or bacterial film, but does not describe the relationship between film and acetic acid fermentation.In 1837, KUtzing found that this vinegar mother was composed of small dot organisms (now called bacteria).But two years later, German chemist Liebig believed that "vinegar mother" was a kind of lifeless protein precipitation.At that time, the famous Dutch wine chemist Muster did not find thatash contentTherefore, he also believed that "vinegar mother" was a compound of protein and cellulose, so Liebig's theory was supported. In 1852, Thom SON found that "vinegar mother" contained 94.53% water, 5.134% organic matter and 0.336% ash through accurate analysis of "vinegar mother", so Milder's analysis result was subsequently rejected.In 1864, Pasteur published two papers to reintroduce the idea of Kitting.He believed that acetic acid fermentation was caused by a tiny bacterium, Mycoderma aceti, but did not describe the morphology of the bacteria.It was not until 1878 when Hansen studied the natural fermentation of beer that he found that there were at least two kinds of bacteria that caused this fermentation, one was film yeast acetate and the other was film yeast pasteurizer（Mycoderma pasteurianum）。Later it was renamed Acetobacter（Bacteriumaceti）And Pasteurella（Bacterium pasteurianum）。In 1900, he added Klebsiella according to the discovery of others（hutzingianum）。He also conducted a detailed study on the morphology of these three acetic acid bacteria, which laid the foundation for the classification of modern acetic acid bacteria.[4]
classification
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Acetobacter
Acetobacter is a kind of gram-negative and aerobic bacteria. Since its discovery, its classification status and name have been confused, which has been changed and revised many times.
Early studies classified Acetobacter as a genus, Acetobacter（Acetobacter）。 Frater divided the bacteria of this genus into four subgroups according to different biochemical standards: glucose peroxides subgroup（peroxydans）Gluconobacter oxyoxidans subgroup（oxydans）Gluconobacter hemioxidans subgroup（mesOxydans）And Gluconobacter weakly oxidans subgroup（suboxydαns）。Later, Leifson (1954) divided acetic acid into two genera: (1) Acetobacter（Acetobacter）The bacteria of this genus have periflagella and can oxidize acetic acid;(2) The pole flagellum of Aeeomnas bacteria can not oxidize acetic acid.As α i et al. (1958)（Acetomonas）Renamed Glucobacillus（Gluconobacter）。[4]
Berger's Manual for Bacterial Identification (8th edition, 1974) accepted the classification of two genera of Acetobacter, namely Acetobacter (not moving or moving with peripheral flagella) and Glucobacillus (not moving or moving with polar flagella).And the genus Glucobacillus is classified as Pseudomonas（Pseudomonadaceae）Acetobacter does not belong to any family, but is a separate genus.[4]
Berger's Handbook of Bacterial Systematics (9th Edition, 1984) finally classified these two genera into a new family Acetobacter（Acetobacteraceae）The unique feature of this family of bacteria is that it can oxidize alcohol into acetic acid.Acetobacter includes four species: Acetobacter acetate（Acetobeαcer αceti）Acetobacter hansonii（Acetobacter hansenii）, Acetobacter liquefaciens（Acetobacter liquefaciens）And Acetobacter pasteureus（Acetobacter pasteurianuma）；There is only one species of Gluconobacter, namely Gluconobacter oxydans（Gluconobαcter oxydans）。The areal pole of two genera of Acetobacter family is that Acetobacter can oxidize alcohol to acetic acid and acetic acid to HtwoO and COtwoGlucobacillus can only oxidize alcohol to acetic acid.[4]
In view of the continuous deepening of the taxonomic research on acetic acid bacteria, in 1993, Berger's Handbook of Bacterial Systematics (9th Edition) was published on the basis of Berger's Handbook of Bacterial Identification (9th Edition).The manual still retains the two genera of Acetobacter, but increases the number of families.The manual has increased the number of species of Acetobacter by 3 (making the number of species of this genus reach 7), namely: Diazotrophic Acetobacter（A. diazotrophicus）The bacteria can grow in 30% D-glucose medium;Acetobacter methanol（A. methanolicus）, methanol can be used as carbon source;Acetobacter gelatinosus（A. xylinum）The bacteria isolated from vinegar can produce a thick, leathery cellulose bacterial gum film on the liquid surface.Acetobacter gelatinosus and Acetobacter acetate are very similar, but the difference is that the former canthree-It grows on alcohol culture medium while the latter cannot.The manual also increases the number of species of Gluconobacter by 2 (making the number of species of this genus reach 3), that is, Gluconobacter freundii（C. frateurii）Gluconobacter asai（G. asai）Among them, Gluconobacillus freundii can use ribose alcohol as carbon source, while Gluconobacillus shallowly needs nicotinic acid for growth.[4]
According to the classification of Yoshinori Asai in Japan, Acetobacter can be divided into two categories according to the optimal temperature and characteristics for its development: one category is called Acetobacter when the optimal temperature for development is above 30 ℃, and the oxidized alcohol is acetic acid;One kind of bacteria that can oxidize glucose to gluconic acid at a temperature below 30 ℃ is called glucose oxidizing bacteria.[5]
(1) Acetobacter.At a relatively high temperature of 39-40 ℃, it can develop, and the suitable temperature for proliferation is above 30 ℃. The main role is to oxidize alcohol to acetic acid, and also to oxidize glucose to generate a small amount of gluconic acid, and can continue to oxidize acetic acid to carbon dioxide and water.[5]
(2) Glucose oxidizing bacteria.This genus can develop at relatively low temperature (7-9 ℃), and the optimal temperature for proliferation is below 30 ℃.Its main function is to oxidize glucose toGluconic acidIt can also oxidize alcohol to generate a small amount of acetic acid, but cannot oxidize acetic acid to carbon dioxide and water.[5]
Cell morphology
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Acetobacter
Cells are oval to rod-shaped, straight or slightly curved, and exist in single, pair or chain.Some strains often appear in the degenerative type.Moves or does not move with peripheral or lateral flagella.The colony is gray white, most strains do not produce pigment, and a few strains produce brown water-soluble pigment, or the colony is pink because of the porphyrin contained in the cells.The model species is Acetobacter aceti.[2]
Growth requirements
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Acetobacter is an aerobic bacterium, which will form a biofilm on the liquid surface during the static liquid culture, but will not form a biofilm when glucose is oxidized into bacteria.In the environment with high concentration of ethanol and acetic acid, Acetobacter is very sensitive to hypoxia, and interruption of oxygen supply will cause cell death[6]
Acetobacter
The suitable temperature for the growth and reproduction of acetic acid bacteria is 28 ℃ - 33 ℃.Acetobacter was not heat-resistant and died after 10 minutes at 60 ℃.The optimal pH for Acetobacter growth is 3.5-6.5, and the general Acetobacter can tolerate acetic acid up to 7% - 9%.Acetobacter has a high tolerance to alcohol, and the alcohol concentration can reach 5% - 12% (volume fraction), but the tolerance to salt is very poor. When the salt concentration exceeds 1% - 1.5%, the activity stops.In production, salt is added when acetic acid fermentation is completed. In addition to regulating the taste of vinegar, it is also an effective measure to prevent acetic acid bacteria from continuing to act and oxidize acetic acid into carbon dioxide and water.[6]
Most suitable for acetic acid bacteriacarbon sourceIt's glucose, fructose, etcHexose, followed by sucrose and maltose.Acetobacter could not use starch and other polysaccharides directly.Alcohol is also a suitable carbon source, and some acetic acid bacteria can also use glycerol mannitol and other polyols as carbon sources.Protein hydrolysates, urea, ammonium sulfate, etc. are suitable for human beings as nitrogen sources of acetic acid bacteria. As for minerals, phosphorus, potassium and magnesium are necessary.Since the raw material for preparing vinegar is generally grain, that is to say, substitute raw materials are used. The content of starch, protein and minerals is also very rich, and the nutritional components can meet the needs of acetic acid bacteria. Except for a few vinegar making processes, it is generally no longer necessary to add nitrogen sources, minerals and other nutrients.[6]
Enzymatic characteristics
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Acetobacter has quite strongAlcohol dehydrogenase、Aldehyde dehydrogenaseIsooxygenase system activity, so in addition to oxidizing alcohol to acetic acid, it also has the ability to oxidize other alcohols and sugars to generate corresponding acids, ketones and other substances, such as:butyrate, gluconic acid, xylic acid, arabic acid, pyruvic acidSuccinic acid, lactic acid and other organic acids, as well as the oxidation of glycerol to diketone, and the oxidation of mannitol to fructose.Acetic acid bacteria also have the ability to produce esters. The flavor of vinegar can be doubled by adding strains that produce more aromatic esters for fermentation.The existence of the above substances plays an important role in forming the flavor of vinegar.[6]
Separation identification
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Acetic acid is a volatile acid with the smell of vinegar. When its sodium salt, calcium salt and other solutions are co heated with ferric chloride solution, a reddish brown precipitate will be generated, and the original liquid will become colorless. This can be used to identify the isolated bacteria.The ability of acetic acid bacteria to convert ethanol to acetic acid can be titrated with 0.1mol/L NaOH.[1]
Common strains
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Acetobacter
In the factory, in order to improve the production and quality, and avoid the contamination of miscellaneous bacteria, the fermentation is generally conducted by artificial pure inoculation.Due to the different types of acetic acid bacteria, their ability to oxidize alcohol is also different.In actual production, it is very important to select strains. It is better to select strains with fast oxidation of alcohol, no decomposition of acetic acid, strong acid resistance and good product flavor.In the production of vinegar, the commonAcetobacter striatum（A.aceti）, Acetobacter schneideri（A. schutzenbachii）Acetobacter orlandii（A.orleanense）、Acetobacter gelatinosum（A.xytinum）, Acetobacter fetida（A.rancens）, turbid variety (Chinese family AS 1.41), Acetobacter pasteurella（A. pasteurianus）, Bacillus pasteuricus subsp. (Hu Niang 1.01), etcAcetobacter rancens , turbid variety, Acetobacter pasteurellae and subspecies pasteurellae.[5]
A. orleanense
Acetobacter orleans is the main strain producing vinegar from wine in the region of orleans, france.It can produce a small amount of esters and has weak ability to produce acetic acid, but has strong acid resistance. It can produce 5.26% grape vinegar from glucose.[5]
Acetobacter schrenckii（A.schutzenbachii）
Acetobacter Schneideri is a well-known strain of fast brewing vinegar in foreign countries, and also one of the more important strains in the vinegar industry, with acid production up to 115g/L (calculated by acetic acid).The optimum growth temperature is 25-27.5 ℃, and acetic acid will not be produced at 37 ℃, so there is no further oxidation of acetic acid.[5]
Acetobacter fetida（A.rancens）
This is one of the strains used in vinegar production in China.It forms a wrinkled membrane on the liquid surface, the bacterial membrane rises along the container wall, and the liquid is not turbid.Generally, it can produce 60-80g/L acid, and some strains can produce 20g/L.Gluconic acid can further oxidize acetic acid into carbon dioxide and water.[5]
Acetobacter film climbing（A.ascendens）
Harmful bacteria for brewing wine and grape vinegar.It can often be separated from the fermented grains of vinegar, forming a fragile bacterial membrane on the liquid surface, rising very high along the container wall, and the liquid is very turbid, which is not suitable for vinegar brewing.[5]
Acetobacter gelatinosum（A.xytinum）
A special acetic acid bacteria forms a leather like thick cellulose like film on the liquid surface.Propagating in the mash of liquor can cause liquor rancidity, viscosity, weak acid generating ability and re decomposition of acetic acid. It is a harmful bacterium for vinegar production.[5]
Acetobacter spp. AS 1.41
The strain has extensive requirements for the culture medium. It grows well in rice starter and other culture mediums, and is specialized and gas loving. It can oxidize alcohol to acetic acid, and make alcohol turbid in the air. There is a film on the surface, which has a sour taste. It can also oxidize acetic acid to carbon dioxide and water. The suitable temperature for reproduction is 31 ℃, and the fermentation temperature is generally controlled at 36-37 ℃.
Hu Niang 1.01 Acetobacter
Acetobacter sp. Huniang 1.01 is a subspecies of Acetobacter pasteureus.The strain was isolated from Dandong instant vinegar in 1972, and was put into use in the experimental plant of Shanghai Brewing Science Research Institute and Shanghai Vinegar Factory. Now it has been used in the production of liquid vinegar by many vinegar factories throughout the country.Its cells are 0.3-0.55 μ m, specialized aerobic bacteria, and the colonies on yeast extract glucose light wine agar medium are milky white;An opaque film was formed on the surface of alcohol culture.[5]
Acetobacter distributes in flowers, fruits, palm oil, wine, beer, apple juice, vinegar and orchard soil.[2]Acetobacter oxydans producing more acetic acid exists in un sterilized vinegar, yellow rice wine, beer (traditional fermentation), fruit wine, distiller's grainsDaquEtc.[1]
Main purpose
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Acetobacter is one of the important industrial bacteria.[1]The brewing industry uses it to produce vinegar.[7]
Main hazards
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It can cause pink disease of pineapple and rot disease of apple and pear.[2]It is a harmful bacterium in the brewing industry.[7]
