PRODUCTION OF SPIRIT VINEGAR BY THE QUICK PROCESS WITH A PURE CULTURE OF ACETOBACTER RANCENS BEIJERINCK

Transcription

1 Vol. 71,1965] 41 PRODUCTION OF SPIRIT VINEGAR BY THE QUICK PROCESS WITH A PURE CULTURE OF ACETOBACTER RANCENS BEIJERINCK By Heikki Suomalainen, D.Sc, A. J. A. Keranen, M.Sc, and Jaakko Kangasperko, M.Sc. (Research Laboratories of the State Alcohol Monopoly (Alko), Helsinki, Finland^ Received 25th August, 1004 Spirit vinegar has been manufactured by the quick vinegar process for about two decades at the Rajamaki Factories of the Finnish State Alcohol Monopoly, with the use of a pure culture of acetic acid bacteria. The culture was originally isolated from shavings used in a vinegar factory. Cultivation is successfully continued on 5 Balling unhopped barley wort containing 2.5% of ethyl alcohol. According to Frateur's system and Bergey's Manual, the bacterium has been classified as Acetobocter rancens Beijerinck. The high ability to form 2-ketogluconic acid is noted as a distinctive characteristic. Introduction AT the Rajamaki Factories ol the Finnish species and strains, and has expressed doubt ^ it *^* ***g & oiblcteria stainless-steel vinegar generators to trial production, capacity 70 cu.m., are filled with rolls of birchwood shavings. The culture of bacteria is sprayed on the surface^: the layer of shavings, a low concentration ot acid and alcohol being kept in the mash. Excessive development of heat during the growth period after bo^bp" " by a heat exchanger. The fermentation, proceeding to 10-11% of acid, normally lasts 5-6 days. The highest concentration obtained is 14-3% and tiie highest daily risj observed only occasionally, is l-7%.20 Gas chromatographic analysis has shown that ethyl acetate is the only aroma compound (about 200 mg./l.) is formed during production, while the main part (up to 1500 mg./l.) is formed during Storage.22 The pure culture of bacteria was originally isolated from shavings used in a spirit vinegar factory. Cultivation is successfully continued on 5 Balling unhopped barley wort containing 2-5% of ethyl alcohol.80 According to Frateur's system3 and Bergey's Manual2 the bacterium has been classified as Acetobacter rancens Beijerinck.21 The identification of our strain was confirmed by the late Dr. J. L. ShimweU (Research Department, British Vinegars Ltd., Frome, Somerset). He has stated that a rampant mutability exists in Acetobacter observable when the inverted plate was examined through the bottom with a magnification of x 40 Although, by maintaining your strata ^2 ^^2^?eYy^a«y ^«been maintaining a mixture (and so has everyone eyse^ a change of medium would probably favour one of the constituent cell-types, and f nave not time to pick off these separate colony form9 and 'identify* each of them by Frateur's criteria. However, when, on previous occasions I^done ^** ^*^Jg 3 dig^t 'species.' Thus you will see that any attempt to give precise species (or even varietal) names to your g^ lam cm ft A rflbcen4_this merely representing the combination of Frateur's biochemical properties produced by the combined efforts of the different mutants present Without wanting to get involved in the difficult and intensively debated question regarding classification of acetic add bacteria, we were naturally interested in determining whether the strain used by us really can be heterogeneous and, in particular, whether it contains cells of different biochemical properties. As already mentioned, we have successfully used this strain in industrial vinegar fermentation for almost twenty years without observing any changes. During this time we have used pure cultures

2 42 suomalainen et al.: vinegar production by pure culture Acetobacter \J. Inst. Brew. cultivated from our original stock culture of vinegar bacteria to inoculate generators which were in process, or to start generators which were out of use, whenever the necessity has arisen. Experimental Microscopy. Hundreds of colonies from cells of our pure culture separated by surface plating either on wort agar containing alcohol (8 Balling wort + 1-5% ethanol -f- 2-5% agar) or on yeast extract agar (0-5% Difco yeast extract + 1-5% ethanol + 2-5% agar), were examined through a binocular micro scope in transmitted light. No distinct differences could be observed in their appearance. Isolation and identification o/loo colonies. In order to investigate more systematically the possible heterogeneity of our pure culture, cells from the stock culture were inoculated, either on wort containing alcohol (5 Balling wort +2-5% ethanol) or on wort agar slopes. They were left to grow for 48 hr. at 30 C. Surface platings were performed in petri dishes, both from wort and from slopes on wort agar, and the colonies were again left to grow for 48 hr. at 30 C. From both platings, 50 colonies, as dissimilar in appear ance as possible, were isolated and replated on wort agar slopes. Tests were then made of the properties of these 100 isolated colonies, including stab transfers from each slope into wort agar in petri dishes. All the giant colonies grown had a similar appearance, with a glossy surface. The biochemical properties of the isolated cultures were tested mainly according to Frateur's system3 and partly also following Bergey's Manual.2 When using Frateur's system, a mass of bacteria was transferred by means of a platinum wire from each slope into petri dishes, with 20 separate colonies per dish, and the biochemical characteristics were tested. Comparison tests were performed with the strains Acetobacter ratteens Beijerinck ATCC 7839, A. turbidans Cosbie, ToSid and Walker ATCC 9325, A. mesoxydans ATCC and A. suboxydans Kluyver and de Leeuw ATCC 621 and 621H. Over-oxidation of acetic acid. -Dr. Shimwell has justifiably criticized Frateur's test,81 according to which over-oxidation of acetic acid, and of lactic acid, is indicated by precipitation of CaCO8 either on ethanol- CaCOa-agar or on Ca-lactate agar. Shimwell pointed out that both of these tests ultimately depend on the ability of an over-oxidizing Acetobacter to convert calcium acetate, rather than acetic acid, to calcium carbonate. Calcium acetate, however, is too high in ph for many acetic acid bacteria to attack, especially the strains capable of making high-strength vinegar, with 8% acetic acid or more. Therefore, in order to observe over-oxidation of acetic acid, we used a method, recommended by Shimwell and successfully applied by us,m as follows: Make up 1% Difco (or similar) yeast extract- 1-5% ethanol in 2 to 2-5% agar with bromcresol green as ph indicator. Dispense about 8-ml portions of this medium into tall, narrow-necked, screw-capped bottles (about 25-ml. capacity) and, after autoclaving, make them into slopes. Heavily inoculate the surface with the culture and then incubate at C. with loosened cap. If the strain is an over-oxidizer of ethanol the colour will change at first from blue-green to yellow because of the formation of acetic acid. After continued incubation (up to 10 days) the colour will change back to blue as the acetic acid is converted to carbon dioxide and water. When following the change of the bromcresol green colour in petri dishes at 30 C. we observed that within 24 hr. the colour around the colonies had already turned yellowish owing to the formation of acetic acid; the yellow colour grew stronger during the following 48 hr. After 5 days all the medium surrounding the colonies was again clearly green and even partly blue. After 8 days, each colony was surrounded by a bright blue colour, indicating over-oxidation. Oxidation of glycerol to dihydroxyacetone. The growth plates were prepared with 0-5% yeast extract {Difco) +2% glycerol +2% agar.3 After incubation for 1-4 days at 30 C., Fehling's solution at room temperature was pipetted on to the colonies in petri dishes; dihydroxyacetone would have caused the known brick-red colour of cuprous oxide, at least after 1 hr. All the experiments gave negative results.* Hromatka12 has paid special attention to the formation of dihydroxy-acetone from It may be mentioned that when we previously10 arrived at a positive result in a corresponding experiment, it was due to misinterpretation.

3 Vol. 71,1965] suomalainen et al.: vinegar production by pure culture Acetobacter 43 glycerol by Acetobacter strains, used in sub merged fermentation and determined as A. suboxydans. Bearing this in mind and also considering the influence of adaptation on the oxidation ability,12 we repeated the experiment, using A. ratteens ATCC 7839 and A. suboxydans ATCC 621 and 621H as refer ence strains. After 24 hr. growth at 30 C. on yeast extract agar containing 2% glycerol, both suboxydans strains, when tested accord ing to Frateur's system, with Fehling's solution at room temperature, changed from orange to the brick-red colour of cuprous oxide. Our own pure culture as well as the rancens collection strain gave a clearly different, faintly brownish red colour. In order to identify dihydroxyacetone by paper chromatography, 1-cm. discs of agar with the bacteria colony were cut out and shaken in a test tube with 2 ml. water. 10 pi. of our strain and of rancens and 5 /xl. of both suboxydans comparison strains were pipetted on Whatman No. 1 paper and chromatographied by the ascending technique. In accordance with Hromatka and Steiner,u ethyl acetate:acetic acid:water (10:1*3:1) was used as solvent system and silver nitrate as colouring agent. The same result was obtained as in the test with Fehling's solu tion: both the suboxydans strains formed dihydroxyacetone, whereas our own pure culture and the rancens collection strain did not. Neither our strain nor rancens would oxidize glycerol to dihydroxyacetone, even when transferred twice more on yeast extract agar containing glycerol. This was V g. I A suboxydans i 1. Paper chromatogram showing oxidation of glycerol (G) to dihydroxyacetone (D) by Acetobacter suboxydans, and lack of oxidation by our pure culture. found with Fehling's solution as well as when paper chromatography and silver nitrate colouring was used (Fig. 1). The adaptability of our strain to form dihydroxyacetone from glycerol was investi gated also in Roux-bottles on yeast extract wort containing either 2% glycerol, or 2% glycerol and 2-5% ethanol, or 2% glycerol, 2*5% ethanol and 1*5% acetic acid. In all cases the results were negative, while the comparison strains of suboxydans, when tested in the last medium, again gave a positive result. Our strain grew surprisingly well on yeast-extract wort, or agar, containing glycerol; in fact it grew markedly better than on the customary wort containing 2-5% ethanol. In yeast-extract wort containing glycerol the bacteria precipitated on the bottom of the Roux bottles, but in the wort containing ethanol the growth was mainly on the surface of the medium. Thus, it was confirmed by paper chromatographic identi fication, and by test after adaptation, that our pure culture strain did not oxidize glycerol to dihydroxyacetone. Formation of 2-ketogluconic acid from glucose. Compared to the tested strains of A. rancens ATCC 7839, A. turbidans ATCC 9325, A. mesoxydans ATCC and A. suboxydans ATCC 621, our pure culture caused the highest formation of 2-keto gluconic acid from glucose. In accordance with Frateur, 10% glucose +3% calcium carbonate +0-5% yeast extract +2% agar was used as growth medium. 2-Ketogluconic acid was identified by paper chromatography in the same way as dihydroxyacetone: about 2-5-cm. discs of agar with the bac teria colony, grown in a petri dish on glucosecalcium carbonate-yeast extract agar, were cut out and shaken in a test tube with 2 ml. 2-N oxalic acid. 50 pi. were pipetted on Whatman No. 1 paper and chromatographed by the ascending technique with n-butanol: propionic acid: water (10:5:7) as solvent system, and treated with aniline-oxalic add according to Frateur, Simonart and Coulon.6 The ability of our strain to produce add was proved by the red colour which appeared the following day and the amount of 2- ketogluconic add steadily increased at least until the fourth day. Of the reference strains, A. turbidans was the best producer of 2- ketogluconic acid,5 although the red colour did not appear until the third day. When testing the formation of ketogluconic add

4 44 suomalainen et al.: vinegar production by pure culture Acelobacter [J. Inst. Brew. according to Frateur it may be noted that both 2-ketogluconic acid and 6-ketogluconic acid are able to reduce Fehling's solution at room temperature. Oxidation of glucose to gluconic acid. The gluconic acid, formed by oxidation of glucose on glucose-calcium carbonate-yeast extract agar, dissolves the calcium carbonate sur rounding the colonies in a petri dish and the medium thus becomes transparent. Positive results were obtained with our pure culture after 48 hr. in all cases investigated. Maltose gave negative results in a corresponding test, even after a prolonged duration of culture. Other tested properties of our strain of bacteria included positive catalase reaction, formation of only traces of fructose from mannitol, formation of acid from xylose and arabinose, and lack of growth in Hoyer's mineral nutrient solution with ammonia as the only source of nitrogen. From these properties, it is obvious that the bacteria belong to Frateur's oxydans group and should be classified as Acetobacter ratteens Beijerinck. No variations in the biochemical charac teristics could be observed in 100 isolated colonies, showing that at least the majority of the cells have remained unchanged. Discussion As previously80 mentioned, our pure culture bacteria, Acetobacter rancens, grows on the surface of alcoholic wort in thin, dry furrows and does not seem suitable for submerged fermentation. Hence, it is not surprising that Hromatka8'10.12 determines the strains of bacteria, which have been used in sub merged spirit vinegar production, as belong ing to the suboxydans group of Frateur's system; at least in one case the strain seemed to be Acetobacter suboxydans var. hoyerianum.9 Shimwell14 has also reported that he isolated in a vinegar acetifier a pure culture of the working bacteria belonging to the group mesoxydans in Frateur's classification. It has been successfully used on a laboratory scale for the production of malt, spirit and wine vinegar by all the different known acetification processes. Shimwell called this strain A. operans and a British patent was granted for its use in the manufacture of vinegar,14 but it is not known whether the strain has ever been used in industrial production. In a later communication,19 however, Shimwell again referred to the possibility of using pure culture for large scale vinegar production. It should be added that Frateur and Simonart* from an industrially working Frings vinegar genera tor, producing vinegar of up to 11%, have isolated strains with biochemical properties corresponding to those of A. rancetis, A. mesoxydans and A. xylinum. According to our knowledge, the Aceto bacter rancens we have been using is the only pure culture which is inoculated and con tinuously used in the quick vinegar process on an industrial scale. At least, none is mentioned in the reviews of Haeseler,6 Allgeier & Hildebrandt,1 or Janke.13 The pure culture, with its advantages, has already been used in industrial production for 20 years and, as far as it is possible to determine, its properties have not changed and the strain has remained free from infection under industrial conditions. However, this does not necessarily rule out the possibility that mutations could occur also in our strain of acetic acid bacteria. Note added in proof. The late Dr. J. L. Shimwell read the manuscript of this paper and referred to it in a letter of August 26th, Dr. Shimwell, who was familiar with much of the microbiological part of our work, accepted our opinion regarding our strain of bacteria, although he suggested that different colony forms might be present. Z. Drazic and V. Johanides as well as D. Salopek and I. Bach from the Institute of Food Science and Technology, Zagreb, reported quite recently (Abstracts of Papers of the 2nd Conference of Yugoslav Microbiologist; Microorganisms in Industrial Use, Zagreb 30th September 3rd October 1964) that they have used Acetobacter rancens (strain A-160) for the production of acetic acid by continuous cultivation in a two-stage laboratory fermenter. Salopek and Bach, when studying the stability of the strain during cultivation, found a number of different colony types. After isolation it was found that the colony variation did not involve detectable biochemical differences from the parent culture. References 1. Allgeier, R. J., & Hildebrandt, F. M., Adv. appl. Microbiol., 1000, 2, Bergey's Manual of Determinative Bacteriology. 7th Edn. Baltimore: Williams & Wilklns Company, Frateur, J., La Cellule, 1060, 53, 285.

5 Vol. 71,1966] suomalainen el at.: vinegar production by pure culture Acelobacter Frateur, J., & Simonart, P.. IX Congresso Internationale Industrie Agrarie, Roma Relation*. Vol. II, C.P Frateur, J., Simonart, P., & Coulon, T., Antonie van Leeuwenhoek, 1954,20, Haescler, G., in Ullmanns Encyklop&die der ttchnischen Chemie, Vol. 6, 3rd Edn. Ed. by Foerst, W. MQnchen-Berlin: Urban & Schwarzcnberg, 1055, pp Hais, I. M., in Handbuch der Papierchromatographie, Vol. I. Ed. by Hais, I. M., & Macck, K. Jena: VEB Gustav Fischer Verlag p Hromatka, O., personal communication. 0. Hromatka, O., & Leutncr, U., Branntweinwirtschaft, Hromatka, O., & Polesofsky, W., Entymologia. 1002, Hromatka, O., & Staincr, J., Branntweinwirtscha/ , Hromatka, O., & Staincr, }., Entymologia. 1963, Jankc. A., in Encylopedia of Plant Physiology, Vol. XII, Part 1. Ed. by Ruhland. W. Bcrlin-GOttingen-Heidclberg: Springer Ver lag, I960, pp Shimwell. J. L., this Journal. 1054, Shimwell. J. L., British Patent 781, Shimwell, J. L., Antonie van Leeuwenhoek , Shimwell, J. L.t & Carr, J. G., Antonie van Leeuwenhoek, 1060, Shimwell. J. L., & Carr, J. G., Antonie van Leeuwenhoek, 1061, 27, Shimwcil. J. L., & Carr, J. G.. Nature, Land., Suomalainen, H.. Brauwissenscha/t, Suomalainen, H., Brauwissenscha/t, 1062, 15, Suomalainen. H., & Kangaspcrko, J., Z. Lebensm. Untersuch. Forsch., , 353.