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This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Beech, F. W Title: The incidence and classification of cider yeasts General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. However, if you have discovered material within the thesis that you consider to be unlawful e.g. breaches of copyright (either yours or that of a third party) or any other law, including but not limited to those relating to patent, trademark, confidentiality, data protection, obscenity, defamation, libel, then please contact [email protected] and include the following information in your message: •Your contact details •Bibliographic details for the item, including a URL •An outline nature of the complaint Your claim will be investigated and, where appropriate, the item in question will be removed from public view as soon as possible. _' .: THE INCIDENcE AND CLASSIFICATION OF CIDER YEASTS by F.W.BEECH Thesis submitted for the Degree of Doctor of Philosophy of the University of Bristol MARCH 1957 CONTAINS PULLOUTS CONTENTS Page. IN'mOIlJCTION. 1 SECTION I. THE ISOLATION OF YEASTS FROM APPLE JUICES ANDCIDERS. Introduction. 6 1. Preparation of a Standard. Medium. (i) Literature Review. 8 (ii) Mediumused. 10 2. Preparation of Differential Media. (i) Literature Review. 11 (a) Antibiotics. .. 11 (b) Inhibitory compoundsother than antibiotics. 18 (ii) Experimental Work. 24- Compcundstested. 24 Test Organisms. 24- Medium. 27 Preparation ot the Inocula. 27 F~eparation ot Test Plates. 28 Inoculation ot Plates. 28 Incubation and Observation of Plates. 29 (iii) Results. 29 (a) Canpcunds suitable for the selective isolation of yeasts. 29 (b) Ccmpcundssuitable for the selective isolation of lactic and acetic acid bacteria. 32 (c) Compoundssuitable for the isolation of certain yeast species. 33 (d) Compcundssuitable for suppressing moulds. 33 Effect of solvents. 34 . Compoundsused for isolation media. 35 (iv) Discussion. 36 3. Sampling and Counting. (i) Idterature Review. 37 Fruit. , 37 Juices and Ciders. 38 (ii) Methods Used. 39 Sampling• . Fruit. 39 Juices. 40 Ciders. 40 Counting. U (iii) Results. 42. 4. Purification and Storage. Purification. 43 [Storage. (i) Literature Review. 44 (ii) Methods Used. 47 - i - Page 5. Sources of Yeast Isolates. SECTIONII. THEIDENTIFICATIONOF lEAST ISOLATES. Introduction. .50 Basal Media. .50 Inocula. 51 Estimation of Yeast Growth. 51 . A. IDENTIFICATIONOF SProlES. 1. Sporulation. (i) Literature Review. 52 (ii) Methods Used. 55 Media. 55 Inoculation of Sporulation Media. 58 Observe.tions. 58 (iii) Results. 59 (iv) Discussion. 60 2. MOIllhology.in Liquid Culture. 62 (i) Experimental Work. 63 (ii) Method Used. 64- 3. Morrhology on Solid Media•. (a) Appearance of the streak culture. 65 (b) Formation of pseudomycelia. 65 4. Fennentation of Standard Sugars. (i! Literature Review. 67 (ii Experimental Work. 69 (11i Method Used. 70 (i v Results. 72 (v Discu ssion. 72 5. Assimilation of Standard Sugars. 74- (i~ Literature Review. 74 (ii :Method.Used. 75 (iii Results. 77 (i v Discussion. 77 6. Assimilation of Ethanol as Sole Source of Carbon. (i) Method Used. 7. Hydrolysis of the Gl,ycosides, aesculin and arbutin. (i) Literature Review. 79 (iil Experimental Work. 80 . (iii :Method.Used. 82 , (iv Results. 82 (v) Discussion. 82 8. Assimilation of Nitrate. (i) Method Used. 84 (ll) Results. 85 - ii - Page 9. Formation of Pigments. (i) Literature Review. 85 ajCarotenoid Pigments. 86 bIron-containing Pi.gment s , 87 c Pigment produced as a result of nutrient ! deficiencie s. 88 (d) Pigment produced by mutants. 89 (ii) MethodUsed. 89 (iii) Results •. 91 10. Fonnation of Starch. (il Idterature Review. 94 (ii MethodUsed. 94 (iii Results. 96 11. Formation of Acid fran Glllcose. (il Idterature Review. 96 (ii MethodUsed. 97 (iii Results. 98 (iv Discussion. 99 B. IDENTIFICATION OF STRAINS OF lEAST SPECIES. 101 1. Formation of Giant Colonies. 101 (i) };IethodUsed. 101 2. Flocculation. (i) MethodUsed. 102 3. Assimilation of Single Carbon Canpounds. (i) Literature RevieW. 103 (ii} Experimental Work. 104 (iii MethodUsed. 105 (iv Results. 107 4. Assimilation of Single AminoAcids. (il Literature Review. 107 (ii Experimental Work. 108 (iii MethodUsed. llO 5. Vitamin Reqairements. il Literature Review. ll2 « 1i Experimental Work. 114 (iii Method Used. 115 6. Formation.~f Ammonia. (1) Experimental Work. ll8 (U) MethodUsed. 119 C. NEW BIOCIIE1JICAL TESTS APPLICABLE TO lEAST CLASSIFICATION. 1. Fomation of acids fioan glucose. (i) Experimental Work. 122 (ii) Results. 123 - iii - Page 2. Production of amino acids from amnoniumsulphate. (i) Experimental Work. 124- (ii) Results. 125 3. Classification of yeasts according to their "antibiotic" spectra. 126 AH'ENDIX I. Composition of Difeo dehydrated media. 127 SECTION III. CLASSIFICATION OF YEAST ISOLA.TES. Intrcxllction. 128 1. The use of pInched cards for collating results. 132 2. Results. 135 Gemls Saccharanyces. 138 Genus Pichia. 144 Genus Debaryan"yces. 147 Genus Hanseniaspora. 149 Genus Cryptococcus. 150 Genus Torulopsis. 155 Genus Brettenomyces. " 161 Genus Candida. 168 Gems K1oeckera. 182 Genus Rhodotoru1a. 183 3. Discussion. 187 SECTION IV. DISTRIIDTION OF YEASTS ISOLATED FROM .APPLE JUICES ANDClIERS. 1. Literature Review. 190 2. Results. 193 (i) Yeasts present on the fruit. 193 (ii) Yeasts isolated 'from juices and ciders processed on pilot-plant equipnent. (a) Juices. 195 (b) During fermentation. 196 (iii) Yeasts in juices and ciders processed an large scale e~ipment. (a) Juice. 199 (b) Effect of cider-making processes on the yeast flora. 200 (c) Yeasts developing during laboratory tests based on ciders processed on the large scale. 203 3. Practical applications in Cider-l-!ald.ng. 205 SJMM.ARY. 210 ACKNOWLEDGl!}.lENTS. 214 BIBLIOGRAPHY. 215 .AFISNDIX II. 232 - iv - INTRODUCTION Until the early 1920's, cider-making in Englandwas confined mainly to the farms of the West Cruntry where empirical methodswere employed. Now, most of the cider produced canes fran factories where the older methodshave proved ina.deqy.atefor maintaining cQ'ltrolled fermentations and for the production of ciders of optinllmvintage quality. This problemwas recognised , in the past by many Continental research workers, most of whansought improve-. mentby the use of pure yeast cultures, althrugh the actuel. methodemployed in each cruntry has also been conditioned by the type of fruit available and by the national. palate. Thus, in Germany, the apples are high in acid and contain little tannin; a dry cider is preferred, so that sane extra treatment must be given to impart a better flavcur to the final product. It is not surprising, therefofie, to find that pure yeast cultures -wereused for cider- making :fran an early date. Evenin 1903, Alwoodnoted that nearly all the large cider factories used the yeast cultures sent cut regularly fran the Royal Panological. School at Geisenheim. The cultures were added to the freshly expressed juices that were then allowed to ferment rapidly at 15-l8°C, racked at dryness and stored at low temperature for several. months. This treatment produceda vinous type of beverage more akin to a light white wine. Noattempt wasmadeto control the natural flora, but in later years (Charley, 1937) the juices wereusually centrifuged or, occasionally, pasteurised before inoculation. TheSwiss, whohave similar sorts of apples, use essentially the samemethods, sanetimes ccntrolling the natural flora with Sllplnlr dioxide and adding the yeast culture several hcurs after su1phiting (Challinor, 1948). In France, the apples used for c1der-makingare totally different in character, being low in acid and relatively rich in t~. ]urther, a rather sweet cider is preferred, and, since artificial. sweetening'is forbidden by law, attempts have been madeto preserve sane' of the natural Slgar of the original juice. Consequently, althcugh such workers as Kayser (1890) and Dienert (1896) isolated yeasts fran ciders, their main object was to find those yeasts incapable of fermenting sucrose or possessing poor powersof fermentation. Attemptsmadeto use such isolates under factory ccnditims proved abortive. If the yeasts were added in large C}!la.ntities, in order to - 2 - overgrow the natural flora of the juice, the fennentation was too rapid and uncontrollable. Attempts to sterilise the fresh juice before yeasting also proved unsatisfactory, since the pasteurising plant then in use gave a persistent "cooked" flavour to the juice (Warcollier, 1928) and, in the absence of acid-resisting metals, often introduced unpleasant flavours from copper-and iran salts. These difficulties might have been overcome,but the exaggerated claims of some of the early exponents of the use of prre yeasts 'Werenot substantiated (Warcollier, 1928). The method thus fell into disuse except for the rapid fennentation of panace extracts destined for distillation as fUel alcohol (Auclair, 1955). Instead, the rate of fermentation was controlled (a) by reducing the nitrogen content of the juice