[This was a ton of work!]
The various yeast species are differentiated from one another by the diastases, and more particularly by the glucidases, which they secrete.
Yeast cells contain several proteolytic diastases, the best known of which is endotryptase. This, probably consisting of a mixture of several diastases, is closer to trypsin than pepsin because it pushes the degradation of albuminoid materials to the amino acid stage (Geret and Hahn). However, it is distinguished from trypsin in that it is favored by a clearly acidic reaction (optimum: 0.2% in HCl). It is hindered by alkalis, sugars, alcohol 10%. It begins to act at 5-6°, has the temperature optimum of 45° and is destroyed by heating for one hour at 60°. Endotryptase is an endocellular distase. However, since it is capable of liquefying gelatin, it must be admitted that under certain conditions it can diffuse through the cell membrane (Will), especially when the yeast is in an abnormal state (dead or diseased cells).
The esterase group comprises, besides the fat enzymes (lipases), a series of other diastases involved in the saponification of esters, phosphoric acid (phosphatases), sulfuric acid (sulfatases), and the like. These different diastases are found in the yeast. They are reversible, that is, susceptible, according to the needs of the body, to cause the hydrolysis or synthesis of fats and esters.
The yeast lipase, which breaks down fats into fatty acids and glycerin, appears to be endocellular: it is involved in the use of the fats found in protoplasm, particularly in the period of sporulation. In some yeasts, however, it is able to diffuse through membranes (Van Tieghem). This would be so in particular for various Torulas (Rogers and Jensen).
According to the nature of the hydrolysed bodies, the glucidases [glucosidase?] are divided into disaccharases or polyases, according to whether they transform disaccharides (sucrose, maltose, lactose and trehalose), trisaccharides (raffinose) or polysaccharides (starch, glycogen, inulin) into hexoses.
Amylase converts starch into maltose and appears to be formed by several distinct diastases. It is found in many species of mold (Aspergillus oryzae, Amylomyces Rouxii, etc.) and bacteria (butyric ferments, Bacillus mesentericus B. subtilis), but rarely in yeasts (Schizosaccharomyces Pombe, Schizosaccharomyces mellacei, Yeast Logos). Inulase, which produces levulose from the inuine, is also found only exceptionally (Schizosaccharomyces Pombe and Mellacei, S. marxianus). On the other hand, yeasts contain an enzyme very close to the amylase, glycogenase, which saccharifies glycogen by first giving maltose, then glucose. This diastase is endocellular and therefore acts only on the glycogen produced by the yeast itself.
Raffinose is split into levulose and melibiose by raffinase (diastase close to sucrase), and melibiose is itself broken down into glucose and galactose by melibiase. Some yeasts can ensure complete decomposition (low brewer and baker’s yeast, S. pastorianus, etc.); others attack raffinose but have no action on melibiose (Schizosaccharomyces Pombe, Schizosaccharomyces mellacei, Yeast Logos).
Sucrase or invertase, which hydrolyzes sucrose to glucose and levulose (invert sugar), is the most common of all diastases. It is found in many molds and most yeasts. Some of these, however, do not contain them (S. Ostosporus, S. apiculatus, S. Rouxii, etc.) and therefore can not ferment the sucrose. In some species, the diastase remains inside the cell, but in many cases it can diffuse to the outside. Sucrase prefers acidic media (optimum pH: about 5). It already acts at 0°, has the optimum temperature 52° and is very quickly destroyed at 70°. The most favorable saccharin concentration is 20%; invert sugar and levulose have a retarding effect on hydrolysis.
Maltase, which converts maltose into 2 glucose molecules, is found in various molds (Asperpillus niger, A. oryzae, Amylomyees, various Mucors) and in many yeasts. It does not exist in S. apiculatus, exiquus, marxianus, or Jorgensenii. It works best in a neutral medium (optimum pH 6.6) and at a temperature of 40°.
Lactase breaks down lactose into glucose and galactose. It is only found in a small number of yeasts (fermented milk yeasts). It is more prevalent in molds.
Trehalase, which gives from trehalose, glucose and levulose, has been found in many yeasts of beer and wine (Kalantar).
Alcoholic zymase splits hexoses into alcohol and carbon dioxide. This diastase is a complex product, probably constituted by a mixture of various enzymes. Harden and Young have shown that it is constituted, at least, by a colloidal part whose fermentative proprieties disappear by heating, the apozymase; and by a non-colloidal heat-resistant part, cozymase. The latter seems to have the structure of a phosphorous organic ether: it would be, according to recent work, a magnesium adenylphosphate.
The zymase is very fragile. Abandoned to itself, the yeast juice rapidly loses its activity, as a result of the destruction of the apozymase by the proteins of the yeast and the hydrolysis of the cozymase by the lipase.
Zymase prefers an alkaline medium. The addition of carbonate and sodium phosphate has a favorable effect on the fermentative power of the diastase. On the other hand, the action of the zymase is stopped by the sublimate at 0.1%, the alcohol at 15%, the formal [formaldehyde?] at 10%.
The diastase is destroyed by heating at 55°; in the dry state and if the desiccation was carried out under vacuum at 40°, it can however withstand up to 140°. The ferment power is maximum around 12-14 ° (Buchner). The optimum temperature appears to be higher, but the protease attack increases as the temperature rises. The best sugar concentration is around 25%.
Autolysis of the yeast.
If the quantity of yeast is less than 40% of the weight of the sugar, the fermentation stops frankly when the sugar is exhausted. But when the weight of yeast is more than 40% of that of sugar, the fermentation continues, the longer the amount of yeast employed is greater. The yeast then lives on its own substance: it is the phenomenon of autophagy or autolysis.
Two phases can be distinguished in autolysis: glycogen fermentation and proteolysis. The glycogenase contained in the cell acts on the glycogene, which is fermented by giving alcohol, carbon dioxide, glycerin, and, according to Salkowski, succinic acid.
At the same time, nitrogenous materials are attacked by various proteases. Among the products of this digestion are nucleic bases, valine, leucine, guanine, lysine, arginine, aspartic acid and choline (Kutscher).
Autolysis is facilitated by the rise in temperature and a slightly acid reaction of the medium (optimum pH = 6). Some salts (acid phosphates, NaCl, acetates, citrates, arsenates also favor the phenomenon while others (neutral phosphates, nitrates, ammoniacal salts) delay or hinder it (Lintner).
Action of Physical and Chemical Agents
In general, when wet, the yeast globules are killed by heating for a few minutes at 50-60°. Some non-resistant species perish at 45-48° while others are only destroyed at 70°. Will has found wild yeasts that can withstand for 30 minutes heating at 70°. The deadly temperature also varies according to the composition of the medium. The heat resistance is lower in acidic medium than in alkaline medium. It is larger in the presence of sugar or starch: thus, in bread, the deadly temperature would be around 68°, according to Wills.
In the spore state, yeasts support temperatures of 5 or 10° higher than those at which the globules perish. Dry yeast resists much better than wet yeast: it can withstand a heating of 5 minutes in a current of air heated to 100°, and even 120° for certain species.
Cold has little effect on yeasts: cells have been cooled to 150 degrees below 0 in liquid air, without altering their vitality, provided that temperature changes are gradual and not abrupt.
The extreme temperatures at which fermentation can take place are close to 0° and 40°. Except for very rare exceptions, all fermentation ceases beyond 40-42°. Cochran and Perkins (1), however, studied yeasts which, heated in a 58° syrup for 30 minutes, continued to ferment the liquid.
(1) Int. Sug. J. XXXV, 26, 1933.
The optimum fermentation temperature is generally between 25 and 35° C. It varies according to the yeast races and the composition of the environment. For distillery yeasts from tropical countries, it reaches 32-35°. It is also possible to acclimate the races of temperate regions, whose thermal optimum is relatively low, to the high temperatures of hot countries (Chaturvedi, Owen).
Madams E. Bachrach and J. Roche have shown that the prolonged action of potassium chloride causes a shift of the thermal optimum to high temperatures. They found that after 10 months of culture on yeast water supplemented with a high dose of KC (10%), the yeast under study had its optimum cell multiplication temperature elevated 3°. The same fact was also observed with the lactic ferment (elevation of 5 to 6° after 3 years). (2)
(2) C. R. CXCIV. 1023, 1932
The optimum temperature zone is, on the whole, very close to that of the deadly temperatures. On the other hand, the toxic action of the components of the must which hinder the functioning of the yeast (alcohol, organic acids, etc.), is found in general considerably increased by the rise in temperature. As the alcoholic fermentation is accompanied by a strong release of heat (20 to 23 calories per 180 grammes of sugar destroyed, according to (Bouffard), which raises the temperature of the liquid, we understand that certain fermentations begun at a temperature too high can stop abruptly in full operation.
It is often necessary, in breweries and wineries, to choose fermentation temperatures much lower than the optimal temperatures, either to hinder the development of certain foreign organisms or to give the fermented liquid particular characteristics from the point of view of taste. For example, in the preparation of the beer by low fermentation, one goes down to 4-10° in the vat room and to 0.5-4° during the stay of the liquid in the cellars.
The light does not seem to have a very marked action. However, according to Lubimenko (1 [this may be a typo?]), it would slow cell multiplication. The fermentative energy as well as the quantity of alcohol formed would be lower for the illuminated yeasts than for those which develop in the absence of light, the difference being all the more pronounced as the temperature is higher. The proportion of acids produced (especially volatile acids) would also be higher and that of glycerine less. According to Owen (3), a short exposure to ultraviolet rays would increase the speed and efficiency of the fermentation of cane molasses.
(3) Ind. Eng. Chem. VI, 480, 1914.
In experiments that have remained famous, Pasteur has shown that the yeast, sown in a sugar solution placed in a flat bowl, burns the sugar with carbon dioxide and water, reproduces abundantly and gives only traces of alcohol. Seeded on the contrary in a solution deprived of air by boiling and kept away from the air, it multiplies much less than in the previous case, but produces only the alcoholic fermentation. Pasteur concludes that: “fermentation is the consequence of life without air, without free oxygen gas … The aerobic being makes the heat it needs by the combustions resulting from the free oxygen gas, the anaerobic being makes the heat it needs by decomposing a so-called fermentable material, which is of the order of the explosive substances likely to release heat by their decomposition”.
The Pastorian theory, after having been disputed for a long time by certain physiologists (Cochin, Brown), has received in recent years a striking confirmation, in particular following the work of Meyerhoff. [I think this is Otto Fritz Meyerhof, but I see his last name spelled differently sometimes.]
There is antagonism between the “plant function” and the “ferment function” of the yeast: the proliferation of the cells is increased, but the intensity of the alcoholic fermentation is reduced in the presence of air. This reduction is, however, quite variable depending on the breeds of ferments: relatively small for yeasts with a low respiratory intensity (bottom fermenting brewer yeasts), it increases sharply when the respiratory power is very great (Torulas), as shown by following figures obtained by Meyerhoff:
CO2 (respiratory coefficient) indicates the number of mmc. of O consumed by mgr. dry yeast and per hour; *** the number of same, CO2, released by the same weight of yeast per hour in a 5% sugar solution, in the presence of air; *** the same amount of CO2, released under the same conditions, in a nitrogen atmosphere.
The best yields are obtained industrially when the yeast is forced to live in anaerobic life, for example by operating in closed tanks under pressure of carbonic acid. A minimum of oxygen is however necessary as exciting cellular: Placed in a culture medium completely devoid of oxygen, the yeasts soon degenerate and die (Cochin, Brown). [cellular functions?]
Yeasts prefer an acidic medium. The optimum pH, corresponding to the maximum development of the cells, varies with the breed and the conditions of culture. It is usually between 4.5 and 5.0. The optimum is close to neutrality, when growing conditions become unfavorable (temperature rise, environmental poverty, addition of antiseptics, etc.). Bacteria are much more sensitive to acidity than yeasts, especially putrefying bacteria, whose optimum pH range is between 6 and 7 and the minimum pH between 4.4 and 5 usually. In industrial technology, this difference in the sensitivity of enzymes is used to favor some of them.
The mineral acids act mainly as a function of the pH. The fermentation is stopped by 0.5% hydrochloric acid, 0.7% sulfuric acid, 0.75% phosphoric acid.
Some organic acids have a much more pronounced action. We shall say a few words of those most often found in the fermentations of cane molasses; formic, acetic, butyric, lactic, citric and oxalic acids.
Johannessohn (1) found that formic acid and its higher homologs, at very low dilution, accelerated the alcoholic fermentation and that the optimum concentration of these acids was directly related to the molecular weight. This concentration would be 5.06 grams per liter, for example formic acid, 6.6 mg for acetic acid, and so on. If it increases, the fermentation is hampered and then stops. The amount of acid which completely stops the functioning of the zymase is, however, not sufficient to kill the yeast.
(1) Biochem. Z. XLVII. 97
According to Henneberg, a dose of 0.08% of formic acid would already weaken the yeast and a dose of 0.2% would stop its development. It is in the presence of this body that it is probably necessary to attribute some of the difficulties that one experiences in fermenting certain molasses, in particular those of the refinery where it has been found up to 0.791% of formic acid. (Zerban).
The different yeasts are unequally sensitive to the action of acetic acid. Meissner (2) found, for example, a 0.25% dose of this acid completely removed the Saaz and Frohberg yeasts [lagers], a dose of 0.375% yeast Logos, while 15 races of wine yeasts were able to finish the fermentation in the presence of 1% of acid. The harmful effects of acetic acid also vary, as Zikes has shown, depending on whether the acid is added to the must or is released during fermentation.
2) Erlangener Dissert, Berlin 1897.
Yeasts are very susceptible to the action of citric and oxalic acids, which can be produced by molds and bacteria during the conservation of molasses. Kayser found that the addition of 0.2 to 0.4% citric acid greatly slowed fermentation. According to Buromsky’s observations, the relative development in the presence of 1% of citric acid would be, for different yeasts: race XII (Berlin) 1.9, Logos 7.2, Frohberg 3.6. Saaz 1.4, S. ellipsoideus 21.3, S. pastorianus 10.3.
Oxalic acid, when its proportion is less than 0.052, acts as a stimulant of the yeast, but at higher doses it is harmful and kills the cells in 2 hours at 0.4-0.5%. With Lebedeff [an old term for dried yeast?], the action is much more sensitive in pure sugar solutions: oxalic acid is already harmful at 0.001% and cells killed at 0.1-0.2%. Although the salts of oxalic acid are much less harmful than the free acid, oxalate shows however toxic at a dose of 0.25%.
Neale and Maerker found that butyric acid retarded the development of yeast at the 0.05% concentration and stopped it completely at 1%. Müller, however, observed that a 0.5% dose of butyric acid slowed down only slightly the fermentation, but much more strongly affected cell growth. This one would already be hampered by the low concentration of 0.005%, according to Juslin. Butyric acid, at relatively low doses produced during the storage of molasses which have been exposed to moisture, may therefore be the cause of lazy fermentations of certain musts.
Yeasts show, on the other hand, a high tolerance towards lactic acid, which is one of the most commonly used antiseptics in distillery of starchy materials. Hayduck, for example, has observed that this body has a retarding action on yeasts only if it reaches a concentration of 1.35%. At 0.5%, it stimulates cellular development. This is so when the temperature remains relatively low. But if it exceeds 40° during fermentation, lactic acid can be very harmful and greatly reduce the yield of alcohol, this being partly due to the increase in the toxicity of the acid with the rise of temperature, but especially to the preponderance taken by lactic acid bacteria.
In Jamaica, Ashby has studied the action of acetic, lactic and butyric acids at various concentrations on budding yeast (Saccharomyces) and on fission yeast (Schizosaccharomyces) seeded in a cane juice mash and vinasse, having a density of 15° Brix and an initial acidity of 0.24% SO4H2 [sulfuric acid]. He obtained the following results:
The antiseptic power of alcohols increases rapidly as the number of carbon atoms increases. Thus, Regnard observed that a solution of 2 gr. of glucose in 250 cc. of water no longer fermented in the presence of:
The various yeasts are very unevenly resistant to high alcohol concentrations. As soon as it reaches 6 to 8°, ethyl alcohol becomes antiseptic for some of them, while other breeds can produce 15 to 16% alcohol without being inconvenienced. Went and Geerligs have observed, for example, that Saccharomyces Vordermannii, found in Java distilleries, can rapidly ferment solutions containing 18-19% glucose. Inui has described, under the name of Saccharomyces Awamori [from a Japanese wine], a yeast whose activity begins to be hindered only by 13% alcohol and is completely stopped only from 20% alcohol. According to Gray (1). tolerance to alcohol would not be specific to the genus or species, but would depend solely on the yeast race.
(1) J. Bacteriology XLII, 561, 1941.
The action of alcohol is dependent on the temperature. Thus, Muller-Thurgau (1) noted, for the particular yeast he studied, that the fermentation was stopped by the amounts of alcohol below (by weight):
(1) In F. Falar – Technical Mycology. London, 1898-1910.
Under the name of antiseptics, substances which hinder the development of ferments are designated. Depending on the dose used, they can paralyze certain vital phenomena (reproduction, fermentation, etc.) or cause complete cell destruction. At low doses, they interfere with the multiplication of yeasts and increase the ferment capacity.
The mode of action of antiseptics is very variable. They can act as oxidants (K permanganate, hypochlorites), as hydrolyzers (strong acids), by coagulating protoplasmic colloids (phenols, creosote) or by forming with them combinations of absorption (corrosive sublimate, heavy metals), etc.
The antiseptic action depends not only on the nature and origin of the microbes or the yeast, but also on many other factors: temperature, reaction, chemical composition of the medium, and so on. It is increased by the rise of the temperature, the acidity of the must and its poverty in nutritive matters. Biernacki indicates, for various products, the concentrations accelerating and preventing the fermentation:
Yeasts may be accustomed to withstanding increasing doses of anti septic. They thus acquire a new physiological state, which can persist even after a series of cultures and be manifested by a variation in the composition of the products formed. In the case of acclimation to fluorides, for example, the yeast is enriched more and more in mineral substances: the lime that appears inside the cell insolubilizes fluorine, in the form of lime salt. There is a reduction in the amount of glycerine and succinic acid formed.
The most commonly used antiseptics in the fermentation industries are sulfur dioxide (vinification), hydrofluoric acid and fluorides (distillery), dilute sulfuric acid, lime milk, bisulphites, formalin (cleaning of the vat room).
Hayduck first reported in 1909 the existence in yeast of an endotoxin capable of killing it, when extracted from the cells and introduced into the culture medium.
Ferbach (1) confirmed this existence and observed that the toxic substance seems to play with regard to yeast, and also with respect to bacteria, the role of an antiseptic. The endotoxin of yeast shares with some known toxins the property of passing through porcelain filters and of being destroyed at a temperature of 100°. But it is clearly distinguished by its volatility: it is easily entrained with water vapor, when distilling toxin macerations under reduced pressure, so as not to exceed 40° (Fernbach).
(1) C. R. CXLIX, 437, 1909.
Under certain conditions, the toxins produced by the yeasts could diffuse into the culture liquid and prevent a new development of the ferment in this medium (vaccinated or immunized solution). Boulard (2) has indicated a method for obtaining this result with fermented beverages.
(2) C. R. CLXXXIII, 1422 1926
“With a liquid, such as a wine must, for example, containing 250 grammes of sugar per liter, a first fermentation is determined by the usual method, with one or more yeasts, and the fermentation is well declared, and that 20 or 30 grammes of sugar have been transformed into alcohol, the liquid is heated for about an hour, at a temperature a few degrees higher than the deadly temperature of the yeasts which are in the wine. As a general rule, it suffices to reach 45° C. The liquid is then cooled, then brought back to the optimum temperature of fermentation, it is once again sown with the same yeasts, then when the fermentation is again clearly declared, it is heated a second time as it has been previously. In general, it suffices for 3 operations of this type to stop all fermentation and to render the liquid unfermentable even after the addition of a significant amount of yeast and while the dose of unfermented sugars is still greater than 150 gr. per liter.”
This phenomenon appears moreover very general, and it would be possible, by this method, to vaccinate any liquids before their complete transformation and to prevent their subsequent invasion by a certain ferment (Boulard).
Longevity of the yeasts.
Yeasts are likely to remain in the same environment for a long time without perishing. Duclaux, examining old Pasteur cultures after 11 to 17 years, observed that out of 26 yeasts, only 6 could not revive. Klöcker found that living cells were found in sucrose and beer must solutions after 20 and 30 years. Finally, Gayon and Dubourg examined wines made in 1810, 1818, 1819, 1832, 1836 and 1846 and found that they still contained yeasts capable of provoking alcoholic fermentation.
The faculty of conservation also depends on several factors: race and origin of the yeast, presence or absence of light, culture medium, etc. According to Will, wild yeasts have a more prolonged vitality than those of culture, some of which can die very quickly. Sunlight seems pretty damaging to conservation. It would be yeasts living aerobically on the surface of liquids that would have the greatest vitality, according to Henneberg.
Culture, isolation and examination of yeasts Culture methods.
The classical methods of bacteriology are used for culturing yeasts. The necessary equipment is: test tubes, Roux tubes, Petri dishes, Erlenmeyer flasks, Chamberland autoclave, etc. and, for physiological research, the balloons of Pasteur [glass balloons], Chamberland, Feudenreich, the flasks of Hansen, etc.
However, unlike bacteria, yeasts require a slightly acidic reaction. Culture media should be placed in thin layers, so as to achieve the maximum aeration, if we want to obtain the multiplication of cells; and, on the contrary, in deep vials, if one wishes to provoke the alcoholic fermentation. The most commonly used liquid media are:
This solution has been used by Pasteur in most of his studies on alcoholic fermentation.
Any fermentable sugar may be added to this solution, which has been used by Laurent in his work on the hydrocarbon nutrition of yeasts.
Pairauit advises, for the study of rum yeasts, the following solution:
Filter if necessary and sterilize for 20 minutes in an autoclave at 120°. The formula of the special nutrient mixture is: Am phosphate: 100, K sulfate: 60. Mg sulfate: 10, Ca acid phosphate: 30.
Culture media are also often used: beer must and malt extracts; fruit juice; decoctions of carrots, potatoes, etc .; yeast water. The latter is prepared by boiling 100 gr. fresh yeast in 1 liter of distilled water; it is filtered and sterilized. To obtain a high proliferation of cells, it is important to add sugar.
As solid media, slices of potato, turnip, carrot, etc., or beer, fruit juice, with 8% gelatin or 1.5% of agar can be used.
The study of yeast sporulation requires a special technique. First of all, the cells must be young and well-nourished, which is obtained by growing the yeast, for about 48 hours, in a nutrient medium (beer must, for example) with repeated transfers. Then, the cultures are subjected to a period of starvation, by seeding on block of plaster or in a not very nutritive medium.
In the Engel-Hansen method, a conical or cylindrical plaster block is prepared by mixing plaster of Paris with 3 parts of water. The block is placed in a glass box, at the bottom of which we put a little distilled water or beer beer, up to about half of the height of the block. The box is covered with a lid, without it preventing the free flow of air. To reduce the risk of infection by bacteria, it is advantageous to replace the glass box with a Hansen bottle. After the apparatus has been sterilized, by heating at 115° C. for 1/2 hour, the yeast is deposited on the plaster using a sterilized platinum wire [inoculating loop], and the oven is heated to room temperature, more favorable for sporulation (25-30°). After about 100 hours, most cells formed ascopores.
In the Gorodkowa method, simpler than the previous one and which gave excellent results to Guilliermond, a gelatin medium is seeded with young and vigorous yeast, having the following composition:
Yeast develops vigorously after plating, but budding stops soon, due to the small amount of sugar available. After 2 to 3 days, sporulation is complete [spores may be though of as the seeds of yeast?].
Many other media have been used to obtain spore formation; yeast water, gelatin or nutrient agar, carrot slices, etc. Carrot culture is particularly indicated for cytological studies (Guilliermond). Most yeasts, especially those of the genus Schizosaccharomyces, produce ascospores in this medium after 6 to 8 days and sometimes in less time.
Purification and isolation.
The principle of the methods currently applied to purify and isolate yeasts is to dilute the liquid containing the microorganisms, so that there is more than one cell in a given volume of the culture medium. [There is a beautiful demonstration of this in Lallemand’s Alcohol Textbook (6th edition) from the head distiller at Jack Daniel’s isolating lactic acid bacteria.]
Hansen’s method. – First, dilute the yeast mixture with distilled water. A drop of the liquid is taken and the number of globules found in the microscope is determined by means of a grid glass. Then dilute so that there is only one globule in each second drop. During these manipulations, it is important to strongly agitate the contents of the flasks, to have a complete separation and a uniform distribution of the cells in the dilution water. A droplet of liquid is then sown in a series of flasks containing sterile must and the flasks are left on their own until the colonies have developed on the bottom of the containers.
Balloons showing a single yeast spot contain a pure culture, coming from a single cell.
A second method, devised by Koch and perfected by Hansen, uses solid media, gelatin or agar. The yeast mixture is first diluted in distilled water and, after counting the globules, a drop is sown in a suitable volume of gelatinized must at 30° C. The mixture is stirred well to separate the microorganisms and spread a drop of the mixture on a grid slat [streaked]. This is placed in an ordinary wet chamber or, better still, a Bottcher wet chamber (also called Van Tieghem and Lemonnier cell), which can be examined under a microscope at any time. It is easy, therefore, to follow the development of the colonies by regular microscopic observations. When the observer perceives a well insulated germ, he marks it by the pointer or by inscribing the square dummy number of the grid slat. It remains only to carry the colony in a liquid medium, after sufficient development. [I think these are now called perfusion chambers.]
A disadvantage of culture in a solid medium is that the food reaches the cells only by diffusion and so slowly that they can die before their complete development.
Lindner method. — Lindner, after having diluted the yeast (in a beer must, for example), so that the liquid contains only one globule per drop, successively touches a certain number of points of a sterile Petri dish with the liquid, which was introduced into a flamed pipette with a fine opening. Wherever there will be a single cell, a single spot will be formed. It is then easy, using platinum wire, to transport the colonies in a nutrient medium in order to multiply them.
Another method, known as the Lindner droplet method, consists in depositing, by means of a sterilized metal pen, small droplets of the dilution of yeasts, in more or less tight lines, on a slide which we return to the humid chamber to allow microscopic examination. [These now may be called perfusion chambers and have inlets, outlets, and temperature regulation for live cell observation.]
Lindner’s method is simpler than Hansen’s, but less reliable. The microscopic examination aiding, it gives however very good results.
Cell counting.— The yeast globules which are suspended in water contained in a very small container of known dimensions are counted directly by means of a microscope. [hemocytomer and now their are lots of affordable automated versions with image recognition]
The apparatus generally employed is constituted by a slide on which is fixed, with putty, a coverslip of 0.2 mm. thick, in the middle of which we made a circular opening. It is closed at its upper part by a slat applying exactly on the bowl. The object is equipped with a micrometric network, Each square of the network is 0.05 mm. side and forms the basis of a prism with a volume of 0.0005 mmc. It is also possible to use a hemocytometer constructed for counting blood cells.
After having diluted and agitated the wort strongly, so as to obtain a homogeneous distribution of the yeasts, a drop of liquid is deposited in the bowl of the apparatus, which is covered with its slat, avoiding imprisoning an bubble. air. A few minutes are waited for the cells in suspension to settle and the count of the globules (at a magnification of 300) in a series of squares placed next to one another. A second preparation is then carried out, on which the same procedure is followed and continued until the average number of cells contained in 5 squares, for example, no longer varies significantly.
Yeast development. — It is easy to follow the development of yeasts under a microscope. After having diluted the culture medium, so that a drop of it contains only a small number of cells, a droplet of liquid is placed on a coverslip and placed in a humid Bottcher chamber. The yeast cells can thus be stored for a period of 8 days without danger of contamination and follow the phenomena of budding, sporulation, spore germination, etc. [perfusion chamber]
To observe the development of ferments at different temperatures, small ovens were built (Ranvier, Vidal, etc.), which fit on the platter of the miscroscope and which are maintained at the desired temperature by a regulator. But, most often, we can do without these devices, putting the cells in an ordinary oven during the interval of operations. [employing some of these ideas may be useful because Pombe yeast can operate at higher temperatures than other yeast which may be key to their dominance.]
Examination of spore germination presents some difficulties, as there are still some cells in the preparations which do not sporulate and which develop before ascospores. This disadvantage can be overcome by operating as follows. A small portion of the yeast culture, made on solid medium (gelatin, carrot, etc.), is extended by means of a spatula on a sterile slide, and this is placed in an oven at 55-60° for 12 hours. Vegetative cells are killed and only ascopores survive. The mixture is then wetted with a little water, and a drop is placed in the humid chamber. It is also possible, as advocated by Hansen, to treat yeast with absolute alcohol or 50% alcohol; vegetative cells are killed in one minute, while ascopores resist for a long time. [It is still unclear at this point why you want to isolate the spores. If they are like a seed, are they the healthiest thing to preserve and/or regenerate the population?]
Microscopic reactions. — In general, living yeasts do not take the usual dyes. However, treated with neutral red (in aqueous solution at 1:10,000), the metachromatic corpuscles are slightly tinted, while the nucleus and protoplasm remain colorless. Dead yeasts easily stain with 0.5% methylene blue and gentian violet.
To study the nucleus, fixation is first carried out by means of the Bouin picroformol solution or the Perenyi solution, and then stained with Heidenhain’s ferric haematoxylin. It is also possible, after fixation in the Bouin solution, to use the Delafield hematoxylin method.
The yeast membrane is stained, after fixation, with Ehrlich methylene blue or Hanstein aniline.
Tincture of iodine (Lugol’s solution) stains glycogen in red-brown, and osmic acid (Flemming solution) in yellow or blackish-brown fat globules. To distinguish the latter from oil droplets, which are often found in the cells of the film yeast, it is treated successively with alcohol and concentrated sulfuric acid: the oily droplets then take on a greenish-gray hue, which finally becomes blackish-brown.
Physiological properties. — To determine the resistance to acidity, one can use the very simple method of Duclaux. It consists in inoculating, with the yeast, tubes of sweet wort acidulated with 1, 2, 3% of tartaric acid. The time elapsed between seeding and the appearance of a disorder, due to the development of the yeast, is noted.
The optimum temperature of fermentation is determined by seeding the yeasts in a must rich in sugar, to be sure that it will remain unfermented sugar, and by bringing to the oven at various temperatures, kept constant (23°, 30°, 35°, etc.). After about ten days, the remaining sugar is measured. A similar procedure is used to determine the ability of the yeast to ferment the must rich in sugar. A must containing 30% glucose is inoculated and the remaining sugar is measured after fermentation.
The ability to ferment various sugars is a very important trait for the distinction of yeasts. This property can easily be appreciated by means of Lindner’s method. A drop of aqueous solution of yeast is placed in a regular moist chamber with the help of the platinum wire, and a small quantity of sugar to be studied, which has been pulverized beforehand, is added. Cover with the coverslip, whose sides are closed with a little Vaseline, and take to the incubator. By examining the microscope preparation the next day, the appearance or absence of carbon dioxide bubbles indicates whether the sugar is attacked by the yeast. To make sure that it is CO2 bubbles, we can drop a few drops of caustic scale on the pore-object: the CO2 bubble contracts and disappears. It is essential to make the seeding with an imperceptible trace of yeast otherwise, the glycogen contained in the cells could give rise to a release of carbonic acid.
It is also possible to seed the yeast to be studied in invert sugar solutions of sucrose, etc., and to follow, with the aid of the polarimeter, the variations of the polarimetric rotation of the liquid. We are thus aware of the more or less rapid disappearance of the C6 sugars from the speed at which the C12 sugars are exchanged, and so on.
The determination of the ferment and activity of yeast, which are of special importance from the industrial point of view, is carried out by different methods. The dosage of the alcohol or sugar can be carried out during the fermentation process, but this has the disadvantage of requiring a lot of time, if many determinations are necessary.
It is more convenient to measure, volumetrically or gravimetrically, the quantity of carbonic acid released. Volumetric dosing requires the use of a special apparatus to collect all the gas produced during the fermentation: we can use a regular nitrogen, which is filled with mercury, to avoid the absorption of gas which is would produce with water or other liquids. More simply, we can content ourselves with making successive weighings of the fermentation tank, equipped with a special closure allowing the free release of carbon dioxide and retaining the entrained water vapor, the Meissl valve or the Alwood valve, for example, which force the gas to bubble into sulfuric acid before escaping into the atmosphere. [very clever!]
In the commercial test of yeasts, we dilute 5 gr. pressed yeast (or 50 g of liquid yeast) with 400 cc. of 10% sugar solution in distilled water. It is introduced into a fermentation flask, closed with a fermentation closure and the flask is weighed. This is then placed in a water bath or oven rigorously set at 30° C. After 24 hours, we weigh again and the weight loss in gr. of carbonic acid represents ferment power. [This will come in handy! I learned a ton about this and its math while studying the Champagne method.]
The activity, or impulsive power, of the yeast is determined as follows, by the method of Meissl. A small quantity of yeast (1 gr.) Is weighed and diluted with 50 cc. of sugar solution, to which nutrient salts (1) have been added, in a small fermentation flask. We weigh everything. The apparatus is placed for 6 hours in an oven at 30 ° C. and, after passing a stream of air to expel carbonic acid which is still in the flask, weighed again. Weight loss, calculated in gr. for 100 gr. of yeasts, is the number that measures impulsive power. Meissl calls normal yeast the one which, under these conditions, equals 1.75 gr. of CO2, and gives it the value of 100. [Again very clever, and these days I think we may de-gas ultrasonically]
(1) The sweet solution consists of 400 gr. refined sugar, 25 gr. monoammonium phosphate and 25 gr. mono-potassium phosphate. Dissolve 5 gr. mixing in 50 cc. well water.
Hansen’s work has shown that yeasts can be stored for a long time in 10% pure sucrose solutions. Most species can survive for periods ranging from 15 to 17 years. Holm recommends the following solution:
A Hansen vial is usually used to preserve the yeast.
Another method, advocated by Will, is to dry the yeast, so that it contains only 15 to 20% water, and to mix it with pulverized silica, plaster of Paris and coal. The mixture is dried at 400 and placed in hermetically sealed containers. By this method, it was possible to keep for 9 years some yeasts. During this period, the yeasts form ascopores (Hansen).
It is prudent to regenerate the yeasts kept in the laboratory from time to time (every month for example), by reseeding in new tubes of sterile sweet medium. [We see a great modern description of this in the Alcohol Textbook 6th edition regarding Jack Daniels.]
Identification and classification.
The polymorphism and variability of the physiological properties of yeasts make characterization of species identification difficult. Thanks to Hansen’s work, we can, however, succeed in differentiating these with a very satisfactory precision. Hansen used as distinguishing features: the shape and dimensions of cells at different temperatures and in different environments; the shape of the ascospores and their mode of germination; the optimal and extreme temperatures of budding, sporulation and haze formation: the appearance of the veil and cultures on solid media (agar, gelatin); the biochemical properties, and more particularly the action on the different sugars. Lindner added: the appearance of “giant colonies”, obtained by inoculating a large plate of gelatin in its middle.
The most important characters are the temperatures at which veils and ascospores form. Also, when a yeast does not produce a veil or give spores, its identification becomes much more delicate, if not impossible. [We probably need to learn more about veil in this context versus other film yeast yeast phenomena.]
We give below the classification of Hansen, modified by Guilliermond.
Yeasts are divided into two families: Saccharomycetaceae, or true yeasts, which form ascospores – and non-Saccharomycetaceae, or non-yeasts, which do not give spores.
They are subdivided into 5 groups:
1st group. – Yeasts with cylindrical cells, rectangular or oval, multiplying by transversal partitioning [fission]. Ascus with 4 or 8 ascopores, generally resulting from isogamic conjugation. Yeast vegetating on must of beer, form a deposit. This group contains only one genus: Schizosaccharomyces. [Ascus: a sac, typically cylindrical in shape, in which the spores of ascomycete fungi develop.]
2. group. – Yeasts multiplying by budding. Ascus from a conjugation (sometimes rudimentary). This group includes the following types:
Zygossaccharomyces: ascus resulting from heterogamic copulation; ascospores with a thick, smooth membrane;
Debaromyces: ascus resulting from a coupling usually heterogeneous; globular ascospores, with a verrucous membrane;
Nadsonia: ascus derived by budding from a cell formed by heterogamic conjugation; verrucous membrane ascospores more or less thick;
Schwanniomyces: traces of conjugation; ascospores with verrucous membrane, formed of two unequal parts separated by a salient ring;
Torulaspora: rounded cells, resembling those of torulas, with a large globule of oleaginous in the center; traces of conjugation in the formation of asci.
3. group. – Yeasts multiplying by budding, forming in the sugary solutions first a deposit, then a more or less mucous veil, without occlusions of air; smooth, round or oval ascospores with 1 or 2 membranes, germinating by budding. Usually produce alcohol.
This group includes the following types:
Saccharomycodes: cells dividing by an intermediate process between bourgeonnement [budding] and scissiparity [fission], ascospores with a single membrane, germinating in the form of a tube;
Saccharomycopsis: ascospores with two membranes, germinating by budding;
Saccharomyces: round, ovoid, ellipsoid or oblong cells, ascospores with a single membrane, germinating by budding, sometimes with rudimentary mycelium formation;
Hansenia: apiculate cells, that is to say, provided at one of their extremities or both of a small point, which makes them resemble a lemon; hemispherical ascospores hat-shaped, with prominent rim.
The genus Saccharomyces, the most important from the point of view of fermentation industries, is subdivided into six subgroups:
a) Yeast fermenting dextrose, maltose and sucrose, but not lactose: S. cerevisiae, carlsbergensis, pastorianus, intermedius validus ellypsoideus, turbidans, willianus, Vordermanii, sake, etc.
b) Fermenting yeasts of dextrose and sucrose, but not maltose and lactose: S. marxianus, exiguus, mandshuricus, Zopfii, coreanus, etc.
c) Yeasts fermenting dextrose and maltose, but not sucrose or lactose: S. Rouxii Soja, Lindnori, Mangini, Chevalieri, etc.
(d) Yeasts fermenting dextrose but not maltose, sucrose or lactose: S. mali Duclauxi, unisporus, etc.
e) Yeasts fermenting lactose: S. lactis, fragilis, etc.
(f) Yeasts which do not produce alcohol and whose fermentation characteristics are little known: S. conglomeratus, theobromae, etc.
4. group. – Yeasts with budding, forming from the beginning, on sweet medium, a dry and opaque veil, with occlusions of air. Characteristic ascospores, provided with a kind of membrane and often with a projecting rim. Most of the species in this group do not give alcohol, but produce aromatic esters. The group includes the genres: [I wonder if Suaveolens is in here as a non ethanol producer? At this point in history, I think it was still classified as a mold.]
Pichia: often cylindrical cells; hemispherical, irregular or angular ascospores; mycelium rudimentary, fairly developed;
Willia: Ascospores in the shape of a lemon or hat, with a rim or a protruding ring.
5th group. — budding yeasts whose affinities are poorly known. Fusiform ascospores. This group includes the following types:
Monospora: ascus with a single needle-like ascospore, germinating laterally by budding;
Nematospora: ascus with several fusiform ascospores, terminated by an “eyelash” [un cil].
This family, which groups all yeasts not forming ascospores and whose place in the classification is uncertain, includes the following genera:
Torula: Generally spherical cells, often with a large globule of oil in the center. Species of this genus very often form a veil (or in some cases a ring), but only after fermentation. The veils are always viscous and without air bubbles. A number of torulas contain red or pink red pigments, more rarely black or brown.
Pseudosaccharomyces: Apiculate-shaped cells.
Mycoderma: Cells most often elongated, cylindrical, tending to remain united in thin chains. They form from the beginning of the culture on must of beer as folded veils, filled with bubbles of air. Some species contain a red or pink pigment. Mycoderms normally vegetate on contact with the air and without giving alcohol.
Cryptococcus: Yeasts resembling Torulas, parasites of humans and animals.
They include species belonging to the most diverse groups. The most interesting from the point of view of the fermentation industries are the genera Endomyces, Monilia, Oidium.
Endomyces. – Mushrooms forming, on the basis of beer, from the beginning, a thick, fluffy veil consisting of a typical mycelium, disarticulating into arthrospores. The cells from the budding of the articles of the mycelium give asci containing 4 ascospores.
Oidium. – Mushrooms with the same characteristics as the previous ones, but never producing asci.
Monilia. – Vegetable mushrooms on beer, in the form of a mycodermitic veil, more rarely a ring, first composed by yeasts, then a typical mycelium, giving rise by lateral or terminal budding to yeasts and never producing asci. Sometimes the mycelium disarticulates into arthrospores.
The most important yeasts from the point of view of fermentation industries belong to the genera Saccharomyces, which includes most crop breeds, and Schizosaccharomyces, which includes some interesting yeasts from hot countries. The following are the main species involved in the fermentation of sugar cane products.
Principal rhummerie yeasts
Schizosaccharomyces Pombe Lindner.
Cells generally rectangular, rounded at the ends, measuring 7 x 4.5 mus [μ] on average. They multiply by partitioning, the transversal partition dividing the cell into unequal parts. Under certain conditions (absence of air), the cells lengthen a lot and can have many transverse partitions, without separation of the elements. Sometimes lateral branches are formed. The cells do not contain glycogen.
Sporulation occurs easily on slices of carrots (after a few hours), in old gelatin cultures and in musts after fermentation. Ascus shaped dumbbells. Ascopores 4 in number, arising in pairs in each bulge of the ascus, measuring about 4 mus of diameter and presenting on the surface of their membrane an amyoid substance which is colored blue by the iodine.
Yeast does not form a veil on must of beer; but it produces a ring after a month. On gelatin, it gives a compact layer of fine flutes, with liquefaction of the medium. Beijerinck has so far reported the existence of a sporogenous variety, forming white colonies on gelatin and an asporogenous variety, producing brown colonies on the same medium.
Sch. Pombe is a high attenuation surface yeast [top fermenting?], causing a vigorous fermentation. Minimum temperature 25° C; optimum temperature 30 – 35°. It fermented glucose, levulose, sucrose, maltose, raffinose, inulin and dextrin. [77°F, 86°F-95°F]
It was discovered by Saare and Zeidler, in a millet beer made by the natives of tropical Africa, the dollo or pombé. It has sometimes been used successfully for the production of pure yeast rum (Arroyo).
Schizosaccharomyces mellacei Jorgensen.
A species very close to the preceding one, of which it is distinguished by its somewhat larger cells (1) and by the property it possesses of fermenting mannose, on which the yeast Pombé has no action. It is a yeast with high attenuation limit, giving good yields in alcohol and supporting a relatively high acidity. However, it is less active and ferment sugars more slowly than most Saccharomyces.
(1) Guilliermond observed that, grown on slices of carrot, the cells of Sch. Pump measured about 7 m long and 4.5 m wide and those of Sch. mellacci 9.5 x 5 mus.
Isolated from molasses and vinasses sent in 1893 from Jamaica to the Jorgensen laboratory in Copenhagen, Sch. Mellacei was later studied by Greg, Allan and Ashby. [Search through the blog and you will find all of their papers.]
Greg pointed out the interest shown by this yeast, which gave him at the same time excellent alcohol yield, a very aromatic rum. But the duration of the fermentation, from 3-6 days in the case of budding yeasts, was increased to 12 days (must at 21° Brix).
Allan has found that in Jamaica it is almost the only yeast found in distilleries producing grand arôme rum. In the other rhummeries, we find, in roughly equal proportions, the breeds with budding and those with scissiparity. The former usually predominate at the beginning of the rhummière campaign, often being supplanted after a while by the seconds, when the acidity of the musts increases.
Ashby isolated several races of Schizosaccharomyces. Most of them are top fermenting yeasts, joined in chains of 4 cells or more, and forming on the surface of the musts a brownish white hat. Some, however, are low-growing, showing isolated or 2-folded cells (2). The latter ensure a faster fermentation (6-8 days instead of 8-10 days, in laboratory tests of the author). The attenuation limit is about the same in both cases, but the low yeasts give a proportion of alcohol a little stronger than the high yeasts, which produce more esters and dry matter. [Ashby showed there were both top and bottom fermenting varieties. The top fermenting were superior for aroma.]
(2) These low yeasts could under certain conditions become high yeasts. Ashby has, indeed. observed that one of the yeasts isolated by him originally produced a low fermentation, but that after being kept for 2 months in a fermented and rejuvenated cane juice, it behaved like high yeasts. [I don’t not remember ever seeing this note from Ashby which leads me to believe there may be other papers to find.]
With respect to alcohol resistance, Ashby found that the scissiparity yeasts examined by him could produce 12-14% alcohol, but only after a prolonged period of time (20-24 days). During the first 7-9 days, during which 9-10% alcohol is formed, the fermentation is regular and relatively fast, after which it slows down considerably. The doses of alcohol that hinder and stop the activity of top fermenting yeasts are 8.5 and 12.5% respectively, while for bottom fermenting yeasts they reach 9.5 and 14% by volume. In practice, a concentration of 16% of sugars is the maximum limit for fermentation to be completed after a reasonable lapse of time (10-12 days).
Schizosaccharomyces are much more tolerant of acidity than Saccharomyces. While they see their activity stopped by 1% acetic acid or 0.15% butyric acid, the first are barely hindered.
On the other hand, when the medium is not very acid, the budding yeasts are more active and quickly take possession of ferment. Also, in vesou musts [fresh juice], whose acidity is generally less than 0.5%, only budding forms are found. In contrast, yeasts with scissiparity prevail in the mash of acidic molasses and are practically the only ones present when the acidity exceeds 1%.
According to Cousins, the Schizosaccharomyces would be conveyed by the air, because we see them appear suddenly in the distillery musts, where it is not possible to meet them at the beginning of the campaign rhummière. [I never seen this note from Cousins. What I think was learned later is that they had a far lower frequency of occurrence than saccharomyces yeast.]
N. Deerr also isolated from a molasses of Peru a Schizosaccharomyces, with elongated cells, sometimes club-shaped, measuring 7.5-12 mus X 3.5-4.5 mus, ascospores often irregular, 4 in number of ascus. This yeast had some differences with the Sch. Pombe and Sch. mellacei.
Pairault observed that scissiparian yeasts dominated at the beginning of the century in the molasses rums of Martinique and Guadeloupe. “This yeast,” he writes, “is found exclusively in our West Indies molasses cuvées, especially when the temperature is exceptionally high.”
According to Kayser, the Schizosaccharomyces of the French West Indies differ from Sch. Pombe and Sch. mellacei because they do not have the property of fermenting dextrin. The same author has pointed out, moreover, the existence of several races (or species), differentiating among themselves by the shape and the dimensions of the cells, the appearance of the giant colonies, etc. It describes in particular the following four forms:
Yeast IV (Martinique): Schizosaccharomyces branched and elongated 9.1 to 20 mus of length over 2.1 to 3.6 wide. Giant colony small, slightly discoid, slightly elevated center, a little craterform.
VI (Martinique): unbranched Schizosaccharomyces, very long and uniform; 11.7 to 18.2 muses long on 1.8 to 3.6 wide. Colony slightly scalloped and domed, yellowish in color, shiny appearance.
IX (Guadeloupe): Schizosaccharomyces with rectangular globules; 8.5 to 14 mus long and 2.2 to 2.7 wide. Colony with slightly elevated center, with scalloped, yellowish lobes.
XII (Guadeloupe): Schizosacchraromyces short, branched, 7.5 to 14.5 mus long on 2.7 to 4.5 wide. Colony similar to that of VI.
Kayser has, moreover, reported an interesting property of yeasts with scissiparity: that of giving amounts of higher alcohols substantially less important than budding yeasts. [I think this is translated correctly, but the rule of thumb is known backwards. I wish I had a citation here.]
Schizosaccharomyces asporus Eijkmann. [Eijkmann was the nobel laureate that discovered vitamin B and worked in Batavia.]
This yeast with scissiparity was encountered by Eijkmann in arrack distilleries in Batavia. It is different from Sch. Pombe by the fact that it does not produce endospores. Beijerinck regards it as an asporogenous variety of the latter species. According to Groenwege, it is the main agent involved in the fermentation of Batavia arrack It supports very high sugar concentrations, the density of musts in the arrack distilleries being about 1.130 (27 ° Brix) Note that Ashby , in Jamaica, was also fermented by the Sch. mellacei mash of molasses and vinasse, having as density 30 ° Brix and containing 23.3% of sugar at the beginning.
Schizosaccharomyces formosensis Nakazawa.
Ellipsoidal or irregular cells, measuring 9.2-16.8 mus of 4.8 mus; ellipsoid ascospores without glycogen, but with membrane impregnated with amyloid. The yeast forms on a beer must a veil at 25-27° and a ring at 25-37° [I think that is brix, not temp]. It fermented glucose, levulose, sucrose, maltose, galactose, mannose, raffinose, inulin and dextrin. Optimum temperature of propagation: 32° C. [89.6F]
This yeast was isolated from sugary products in Formosa by Nakazawa, who found in the same habitat two other species with scissiparity; Sch. sautawensis Nakazawa and Sch. Nokkoensis, whose characters are not very different from those of Sch. formosensis.
Zygosacharomyces major Takahashi et Yukowa.
Spherical cells of 3.7-7.5 mus of diameter, sometimes oval, containing glycogen. Spores transparent, round or ovate, 3-4.5 mus of diameter in general, 1 to 4 in number of asci.
Yeast, which belongs to the group of low yeasts, forms on a “koji” extract a ring after 3 days. It fermented glucose, levulose, mannose, sucrose, maltose, but not galactose, lactose, or raffinose.
The Zygosaccharomyces major, isolated by Takahaski and Yukawa from the maturing “Shoju” (Japanese soybean condiment), was met by Hall, James and Nelson in Barbados cane molasses, along with Zygosaccharomyces. Nussbaumeri Loghead and Heron.
Saccharomyces cerevisiae Hansen.
Round or oval cells. Limited development temperatures in beer must: minimum 13°, maximum 40°. Ascospores 1 to 4 in asci (sometimes 5), very cold and with a distinct membrane; diameter ranging from 2.5 to 6 mus. At 37° 5 and at 9°, the ascospores are not formed: they appear at 36-37° after about 24 hours; at 30° (optimum) after 20 hours, and at 11-12° after 10 days. Limiting temperatures for haze formation: 5° and 38°. The veil appears at 33-34° after 9-18 days: at 6-7° after 2-3 months; and at 20-22° (optimum) after 7-10 days. At 20-34° the cells of the veil have an elongated shape and a bizarre appearance; in the old veils, we meet all kinds of cells, some very long, having the appearance of a mycelium. Yeast ferment glucose levulose, sucrose, maltose, but not lactose.
S. cerevisiae, found by Hansen in the breweries of London and Edinburgh, is very common in breweries and distilleries. Many yeasts, high or low, of which the systematic position is poorly known, are attached to this species. Particularly noteworthy are the Frohberg type and the Saaz type, the first with low attenuation and the second with average attenuation. These yeasts include various widely used breeds, especially those of the Frohberg group, in industrial fermentations.
Saccharomyces Vordermannii Went and Prisen-Geerligs.
Round or piriform cells, measuring 6-7 x 5-5 mus, the youngest remaining long enough welded to the old ones. In old agar cultures there are also elongated, filiform cells surrounded by ordinary cells. Ascospores numbering four in each cell (2-3, according to N. Deerr.). Yeast does not form a veil on the surface of sugary liquids, but only a ring in contact with the walls of the container.
It fermented glucose, levulose, sucrose, maltose and raffinose, but not lactose or dextrin. Fermentation, which is very fast, ceases in the presence of 9 to 10% alcohol.
S. Vordermanii was discovered by Went and Geerligs in the ragi, a leaven used for the fermentation of Batavia arrack. Some authors attach it to S. cerevisiae, the characters that distinguish the two species being of little importance.
S. Peck and N. Deerr studied the isolated ferments of cane molasses samples from various rum producing countries (Cuba, Demerara, Java, Mauritius, Natal, Peru, Trinidad). They met only budding yeasts, except in the molasses of Peru, where there was exclusively a yeast with scissiparity [fission]. The differences they noted between the agencies did not seem sufficiently clear to them to be considered specific. These authors look accordingly at the budding yeast isolated by them as varieties of Saccharomyces Vordermanni. From the physiological point of view, they develop slowly from 20 to 25° and very quickly to 32°: they ferment glucose, levulose, sucrose and maltose.
Pairault, who has examined rum yeasts from the French West Indies, considers that they, with the exception of Schizosaccharomyces, are related to the yeast of beer (S. cerevisiae).
“They are cordially round,” he writes, “sometimes oval, those of the vesou are generally smaller and rounder than those taken in tanks of molasses: the first are from 3 to 7 mus in length, the others from 7 to 9. Rum yeasts are fond of high temperatures, their activity is very low below 23°, their preferred temperature seems to be 35°, but some still stand valiantly at temperatures of 41-42 and even 43°. Many rum yeasts do not ferment maltose, out of 40 tried, 18 were in this case. In the same rhummeries, I have a few days apart met yeasts that fermented maltose and others that did not. The others appear to be about as active, according to the tests made. Rum yeasts are generally held at the bottom of the vats, where they congregate in compact masses [flocculate]. Some yeasts are surface yeasts and form beautiful yellow skins above the vats, which are usually removed. These high yeasts are usually Schizosaccharomyces.”
Kayser has noted the presence, in the molasses received from the French colonies (Martinique, Guadeloupe, Reunion), with both scissiparity and budding, yeasts; low, high, and film. Surface Saccharomyces were generally not very active and had low fermentative power. Most of the yeasts examined had a heating time of 10 minutes at 55° and even one of them 2 minutes at 60°. The vast majority fermented levulose, glucose, sucrose, maltose, mannose, raffinose, galactose and inulin, but none attacked dextrin. Some high yeasts did not secrete sucrase and left sucrose intact. The alcoholic strength, quantity and quality of the acids (fixed or volatile) produced, the optimum reaction of the medium, the odor released during the fermentation, the appearance of the giant colonies, etc., varied from yeast to the other.
It is therefore probable that, besides the yeasts of the cerevisiae group, there are other Saccharomyces in the rhummerie musts, differing in particular in their action on the various sugars and the cultivation characteristics (formation of giant colonies). The identification and classification of these yeasts require further study.
Yeast of great industrial importance, but whose morphological characters have been little studied. It has elongated cells, resembling those of Saccharomyces pastorianus Hansen. Isolated by Van Laer and Denamur from the yeasts employed at the Logos et Cie brewery in Rio de Janeiro, it seems to have originated from a spontaneous fermentation of cane juice. It is a low fermentation, slow yeast, producing little alcohol, but having a very high attenuation limit. It fermented glucose, levulose, sucrose, maltose, galactose, mannose, inulin and dextrin.
Saccharomyces ellipsoideus Hansen.
Elliptical or round cells, developing on must of beer between 0.5 and 40-41°. Ascus generally ellipsoidal and small, containing 1 to 4 spores measuring 2 to 5 mus. At 4 °and 32°5, the ascospores are not formed; they appear after 26 hours at 30.5-31°5; from 21 h to 25° (optimum) and 41 days to 10°5. The veil is not formed at 5 ° or 38 °; it is fully developed at 33-340 (optimum) after 8-12 days, and at 6-7 ° after 2-3 months. The cells of the veil are often sausage-shaped. Yeast ferment glucose, sucrose and maltose. [Some of the degree signs in here get wacky and it may be worth consulting the original page. I can’t quite tell if he is talking about temperature, brix, or attenuation, and how he is denoting half measures.]
The ellipsoideus were discovered by Hansen on the surface of the grapes. They play the leading role in the fermentation of wine, and many breeds are attached to it. Wine yeasts, especially those of Champagne, are quite often used in the manufacture of rum by pure culture; they are used especially in Cuba and sometimes in Martinique.
Saccharomyces Zopfii Artari.
Spherical or ellipsoidal cells, 3-6 mus of diameter. Maximum sprouting temperatures 33-44°, optimum 28-29°. Spherical ascospores, 1.5-3 mus of diameter, numbering 1-4 per cell (2 in general): maximum temperature of sporulation 32°. The vegetative cells can withstand a temperature of 66-67°, and even, according to Owen, 90° for 10 minutes. The yeast ferment glucose, levulose and sucrose, but have no action on maltose or lactose.
Isolated by Astari during the production of sugar in Saxony, Zopfii was found by Owen in the sugars of Cuba, where it is one of the main agents of deterioration of this product, as well as in “blaskstrap” molasses. Schweizer and Fischlin also found it in the musts of cherries and found that it produced high amounts of volatile acids and esters. The eau-de-vie obtained was of inferior quality and had a very pronounced fusel odor.
Like other Saccharomyces involved in the fermentation of cane molasses, the following species have also been reported:
Saccharomyces secundus Groenewege. This yeast, which is attached to the cerevisiae group, was found by Groenewege (1) in a Batavia distillery. It would play a relatively important role in fermentation.
(1) Arch. Suikerind. Ned, Indie XXIV, Med. N° 16, 1916.
Saccharomyces javanensis Groenewege. Same origin as the previous yeast; very secondary importance.
Saccharomyces formosensis Nakazawa (1). Isolated by Nakazawa, in Formosa, from a must of fermented cane molasses.
(1) J. Agr. Chem. Soc. Japan IX, 285, 1933.
Saccharomyces robustus Nakazawa and preciosus Nakazawa (2). Isolated from a must of cane juice fermented in Manila, these yeasts, which have a high ferment capacity, attack glucose, levulose, sucrose, galactose, mannose, raffinose and trehalose, but not maltose, inulin and dextrin. Optimum temperature: 33 and 33°5; Optimum pH 4.7-6.4 for the first species and 5.3-6.1 for the second species.
(2) J. Agr. Chem. Soc. Japan XII, 356, 1936.
Pichia californica Siefert.
Cells generally oval, measuring 4-8 mus long and 3-5 mus wide, enclosing a very refractive corpuscle. They form a delicate veil, which falls to the bottom of the container when stirred. Limiting temperatures of budding, in a wine at 8°: 7-12° and 33°; optima 28-30°. In the must, the maximum temperature is 39°. Sporulation limit temperatures, 5-6° and 39-40°; optimum close to 34°. Spherical and refractive ascospores, measuring 2-3 mus of diameter, forming only in a 12% alcoholic solution.
Isolated by Siefert from a red wine from California, this yeast was found by Saito (3) in the fermentation of cane molasses musts at the island of Bonin (Japan) and would be the main agent of this fermentation.
(3) Z. Spiritusind. XXX, 565, 1908.
Willia (Torula) Van Overeem.
Isolated by De Kruyff from the “ragi” and the “tapé” of Java, this yeast plays an important role in the manufacture of Arrack from Batavia.
These yeasts constitute a rather heterogeneous group, characterized by the generally spherical shape of the cells and the absence of sporulation. They often ferment sugars, especially glucose and levulose. Many forms have been described by Hansen, Will, Pearce and Barker. Wehmer, etc …
Very common in molasses, Torulas are frequently involved in the spontaneous fermentation of sweet musts. Gifted with a great oxidizing power, they produce a lot of esters, whose quantity increases with the nitrogen richness and aeration. They are aerophilic, prefer the amide nitrogen and have an optimum pH of 4.5, according to Kayser (1). [I suspect all they produce is ethyl acetate.]
(1) C. R. Acad. Agr. XI, 449, 1925.
Ashby has isolated in Jamaica a species that spontaneously ferment molasses, diluted or undiluted (90° Brix). It does not form a veil, but only a ring. Incapable of inverting sucrose, it only weakly fermented the cane juice; more, in molasses musts, it gives about 8% alcohol. It determines a very slow fermentation and produces considerable quantities of esters: up to 18% of the alcohol formed, after 24 days. [probably all ethyl acetate]
Browne has described, under the name of Torula communis, a species he found in the raw sugars of Cuba, which fermented invert sugar, but not sucrose. It can develop in the most concentrated sugar solutions and plays an important role in the deterioration of raw sugars, especially between the age of 9 and 15 days after manufacture.
Monilia spp. – Odium spp.
The genus Monilia includes mushrooms growing on must of beer, in the form of a mycodermic veil or, rarely, a simple ring.
Browne has encountered two species: Monilia nigra and M. fusca Browne, with black pigment, in raw sugar (which they cause deterioration) and cane molasses.
On sucrose agar, M. nigra‘s colonies first appear as star-like spots. They consist of hyphae arranged radially and giving rise to trans parent cells. When the colonies have reached a certain size (1-15 mm), the hyphae break up into clusters of black conidia. Yeast cells, elliptical in shape, can give rise to new hyphae or multiply by budding like yeasts. M. fusca differs from M. nigra in the greenish-brown coloring of conidia, the shorter length of hyphae, and the less pronounced tendency to give yeast-cells.
These two fungi grow well in raw sugar solutions, except the most concentrated ones, with light gas production and a fruity smell. They determine the inversion of sucrose.
Went and Prinsen-Gleerligs (2), in Java, isolated a yeast from the “ragi”, which they called Monilia javanica and which, according to these authors, would be used in the fermentation of Batavia’s arrack. It appears in the form of rounded or piriform cells, sometimes irregular, forming, after 1-2 days, a veil on the surface of sugary liquids. On agar, we obtain mycelial filaments, giving rise to yeast-shaped cells. M javanica fermented glucose, levulose, sucrose, maltose and raffinose but not lactose or dextrin. It produces about 5% alcohol after about ten days. The resulting alcohol has a rather unpleasant taste.
(2) Verhand. Königl Akad. Weteneh, Amsterdam IV. N° 2. 1895
Peck and Deerr found in a cane molasses from Natal, a Monilia neighbor of the M. javanica. It presented the interesting property of giving during the fermentation a fruity smell reminiscent of that of the best Jamaican rum. In pure culture on molasses must, it produced 7.5 esters per 100 of alcohol formed. The esters consisted mainly of ethyl acetate and butyrate. [that sounds promising!]
Various other species of Monilia have been observed to cause the sugars to boil, giving alcohol. To mention in particular Monilia candida Bonorden and M. vini Osterwalder. Their fermentative power is usually low.
Finally, Arroyo, in Puerto Rico, used in the fermentation of cane juice (see Chapter VI) an Oidium, the O. suaveolens, found in the sap of a shade tree used in coffee plantations. This mushroom forms on the surface of musts a thin film. It has little action on sugars, but by attacking the protein materials of the medium, it gives rise to significant amounts of organic acids and esters. The dominant smell of the bouquet is that of ripe apple.
PREPARATION OF RHUMMERIE MUSTS
The must, or “grappe” (1), is obtained by mixing, in certain proportions, the sweet raw materials (juice, syrup, molasses or scums) with vinasse and water so as to obtain a determined density. The acidity is usually adjusted by the addition of a small amount of sulfuric acid. Sometimes an antiseptic and nutrient salts are added to facilitate the development of the yeasts. The proportions of the various ingredients vary within wide limits, depending on the type of rum that one wants to obtain.
(1) The term “grappe” [cluster/bunch] is still widely used in the French West Indies
from the beginnings of colonization.
Pretreatment of raw materials
The sweet raw materials used in the brewery have certain defects, which may need to be corrected in order to obtain a good fermentation and a better quality eau-de-vie.
Cane juice is very rich in microorganisms, some likely to cause lateral fermentations leading to the production of aromatic principles unpleasant to taste. Molasses may contain, in addition to harmful ferments, excess mineral matter or organic acids which not only hinder the development of alcoholic yeasts, but also adversely affect the bouquet of the rum. Also, various authors recommend to pre-sterilize juices and molasses, and, in the case of the latter, to eliminate unwanted constituents. In the preparation of yeasts for pure yeast fermentation, it is obviously essential to sterilize, at least partially, the must.
Treatment of molasses.
Beet molasses, rich in organic acids harmful to yeast, were formerly treated by the process of de-nitration (2). To the molasses, diluted at 25°-28° Baumé, was added an amount of sulfuric acid (2 kgs., Acid at 60° B. p. 100 of molasses on average) sufficient to decompose the organic salts, and it was boiled for a quarter of an hour in a steam-heated tank equipped with an air bubbler to facilitate the removal of volatile acids. [wow, that is gnarly!]
(2) Today most plants have removed de-nitration, to use yeasts acclimated to the volatile acids of molasses or, more often to carry out fermentations in the presence of ansiseptics (fluorides).
Except in the exceptional case of products having undergone during their conservation a butyric or putrid fermentation, the molasses of cane, generally little charged with volatile fatty acids, do not require to be subjected to such an energetic treatment. Most often, they are used as such in the preparation of musts.
Barbet process. Barbet, however, recommends sterilization by means of apparatus similar to that used for denitration. He devised a continuous device, functioning as follows:
The molasses is diluted to 28-30° Baumé in 2 wooden or copper tanks, serving alternately, and added, if necessary, a small amount of sulfuric acid to remove fatty acids. It then passes inside the tubes of a tubular heat recuperator, heated by hot molasses from sterilization: a regulator makes it possible to feed the recuperator uniformly. At the outlet of the latter, the molasses enters, almost boiling, in a sterilizer, constituted by a closed copper tank, made of a steam-bubbling heating system, a perforated tube allowing the injection of air in the liquid and a gas vent. If the vapors that emerge have a bad odor they are expelled into the atmosphere. In the opposite case, they are condensed in a tubular cooler and added to the must or, if one operates by the method of “repasse”, to “brouillis”.
On leaving the sterilizer, the molasses flows into a dilution tank, to be brought to the fermentation density, by mixing with hot water coming from the condensers or with hot vinasse coming out of the distillation column. A special device allows to adjust the proportions of these ingredients. The dilution tank is equipped with a steam bubbler, which allows the wort to be warmed again after dilution and thus completes sterilization (1). The duration of the stay of the must in the tank is 20 minutes. Finally, after serving to heat the fresh molasses in the heat recuperator, the diluted molasses is brought to the temperature required for fermentation in a container, arranged so that no contamination of the must can during cooling.
(1) The temperature can be increased to 100° in the sterilizer and then in the dilution tank. However, with molasses feeds, which have an acidity generally greater than 2 gr per liter, practically sufficient sterilization is obtained by heating at 80° C.
In the case of small distilleries, the above device is simplified and reduced to a single tank, in which the molasses is diluted to 30° B., brought to the boil and finally added water or vinasse to obtain the suitable density.
Arroyo process — Arroyo proposes to perform, in addition to a sterilization at 80° C., a heating (2) and an acidification of the molasses.
(2) The purification with lime was already recommended formerly. Unfortunately the sulphate of lime tends to quickly incrust devices (trays, condensers, tubulars, etc.).
In an open cylindrical tank, equipped with a heating coil and a mechanical stirrer, the molasses is introduced, which is mixed with a certain quantity of milk of lime, determined in advance, so as to obtain a raising the pH of the raw material by 0.5 [titratable or pH?]. After addition of the milk of lime, the agitator is set in motion and hot water is introduced until the density of the mixture is 50-55 Brix. The temperature is then raised to 70-80° C., and maintained at this level for half an hour.
While continuing a vigorous stirring of the mass, the liquid is passed, at the end of this period of time, in a centrifugal separator (Alfa Laval centrifuge for example), to remove from it solids which have been precipitated or separated during treatment (3). The clear liquid is returned to a second tank similar to the first, but the coil is fed with cold water instead of steam. As soon as the coil is covered with the liquid, cooling is begun and the agitator is set in motion. Once the temperature has fallen to 35-40° C., the nutrient salts, if any, are added then the amount of sulfuric acid needed to obtain a pH between 5.0 and 5.6. The liquid passes a second time, for clarification, to the centrifuge, and is sent to “composition”.
(3) One can also use for this purpose, either a pressure filter or the decantation, But the most expeditious process is that of centrifugation. [Arroyo later demonstrated decanting.]
This pretreatment of the molasses not only destroys the microbial flora, allowing only heat-resistant spores to survive, but also determines chemical modifications of the medium, which facilitate the progress of the fermentation and favor the production by the yeast of aromatic principles improving the aromatics of the rum (mainly rum oil).
Lime neutralises the fatty acids present in the molasses, thus preventing their volatilization during the subsequent heating. These acids are then released, after the addition of sulfuric acid, and intervene in the constitution of the bouquet of the brandy. It continues, under the combined action of pH and heat variations, abundant precipitation of organic impurities (gums, etc.) and minerals which increases the saccharine richness and decreases the viscosity of the liquid.
Here are some analyzes, due to Arroyo, which show the modifications brought to the material by the treatment (figures related to the primitive molasses):
The treatment eliminated 30 to 40% of the mineral substances and gums, with the consequent increase of the sugar content of 3 to 5% and a decrease of the Brix density of 4 to 7%.
The benefits of molasses purification are not limited to fermentation. The wines sent to the distillation being clear, one avoids the clogging of the columns, reduces the expenses of vapor and improves the output of the apparatuses. The bottom of the tank is composed almost exclusively of yeast. The recovery of the latter, in the form of fodder yeast or food, is greatly facilitated. Finally, the pre-treatment increases the rate of potassium salts in the vinasse and makes it more economical their recovery by concentration and incineration. [Not sure about this last potash point. My understanding is that it is a challenge to dispose of regardless.]
Arroyo advises treating cane juice as molasses, by defecation with lime. The vesou is brought to 80° C, added lime milk to have a pH of 7, then sulfuric acid to reduce the ionic concentration to pH 5.8. It is then clarified by filtration or centrifugation. [I’ve always wondered if there was a solar solution to achieving this.]
This treatment regulates and accelerates the fermentation of the vesou, destroying the many microorganisms that are in the liquid, while improving the bouquet of rum. In particular, it increases the production by certain breeds of yeast of rum oil. Liming, if not done with great caution, may, however, have deleterious effects, an excess of lime liberating organic bases and bodies of the group of alkaloids, which can pass into the distillate and communicate to the rum an unpleasant taste.
Arroyo gives the following results, obtained using the same raw cane juice (1), (2) heated to 80°, (3) heated and defecated with lime as indicated above. During fermentation and distillation, the conditions were kept as similar as possible for the 3 samples.
As early as 1895, Greg drew attention to the role that liming played in the production of the aroma of rum, which he attributed to a particular essential oil. During experiments in Jamaica, he noted:
(1) that un-limed cane juice and rums derived therefrom did not contain rum oil;
(2) that the vesous defecated with lime and having fermented under the action of certain yeasts (No. 18 in particular) contained rum oil;
(3) that industrially produced rums, made from a mixture of cane juice, molasses and scums of defecation [skimmings], also contained, in very variable proportions, the same essential oil.
The author accordingly admitted that this aromatic matter resulted from the action of certain yeasts, made possible by the lime juice. Excessive liming releasing organic bases from the pyridine group also spoiled the quality of the rum. These conclusions have been confirmed in recent years by Arroyo, who has specified, depending on the pH, the optimal amounts of lime to use. [Circular 106]
In the French West Indies, where vesou rum has been produced for more than half a century, distillers have also become aware of the influence of defecation on the quality of eau-de-vie. They have known for a long time that heating the vesou gives a bouquet to the rum that is more full-bodied and more persistent, aging more quickly. Liming, followed by a concentration of the liquid, accentuates these qualities, at the same time that it makes disappear the special aroma, called “taste vesouté”, of the raw juice.
However, in Martinique and Guadeloupe, it is the manufacture of rum of raw vesou that prevailed over that of rum of cooked vesou and rum of syrup. The first product is, in fact, easier to obtain by spontaneous fermentation, and its bouquet appeals more to Creole consumers. However, from an export point of view, the other two types have indisputable advantages.
In Jamaica, on the other hand, the cane juice used in the composition of musts is very often defecated with lime. This operation is considered essential for the production of quality rum.
Musts of vesou and syrup.
The operation which consists in mixing the raw materials entering the constitution of the must is known, in the French West Indies, under the name of “composition”. [Arroyo used the term batición sort of like cake batter.]
Vesou musts are often composed by simply diluting the cane juice with water, so as to have a density varying between 1.035 and 1.065 and a sugar content of 8 to 14%. In Martinique, the density is normally between 1.035 and 1.045 (at the temperature of the observation), while in Madagascar it reaches 1.060-1.065.
When it is desired to obtain a more full-bodied product, more particularly suitable for export, the water is partially replaced by vinasse. In this case, the composition usually varies between the following limits:
Pairault indicated at the beginning of the century as the average composition of musts de vesou in the French West Indies:
The musts of syrup are sometimes prepared simply by diluting the syrup with water, so as to have a density of 1.043 – 1.050. More often, however, a high proportion of vinasse (60 to 70%) is added, the rums of syrup of drums being generally intended for the export and having to be consequently more full-bodied. Hereinafter the composition adopted for the musts of a well-known brand of rum from Martinique: [I got drum from their use of “batterie” which I think implies storage.]
The fermentation of the must above, abandoned for spontaneous seeding, is rather slow (5-6 days); the rum obtained has a coefficient of impurities close to 400. [that really looks like it translates to abandoned!]
In Haiti, where the main raw material used in the production of rum is the drum syrup, the proportions would be on average, according to Pairault:
The fermentation lasts about eight days, sometimes less, often more, [pombe yeasts…]
According to E. Baker (in Litteris [correspondence]), the must is currently obtained by mixing 35-38° Baumé syrup with water in the ratio of 1 to 4 or 6, so as to obtain a liquid of density varying between 8° and 10° Baumé. In general, vinasse is added to the composition in a proportion of 1/5 to 1/3. Some distillers, very rarely indeed, use Am sulphate and sulfuric acid, at the respective doses of 0.5 and 1 p. 1,000. The fermentation lasts from 3 to 8 days, more often from 6 to 8 days. This variability is explained by the following reasons: no seeding is done at the time of fermentation; the feet of the vats [pieds de cuve] are only exceptionally used; the temperature of the fermentation rooms varies considerably from one season to another; finally the density of the musts is not regularly followed. [They may likely be riding the line between a Pombe ferment or not.]
The manufacture of rums of syrup was known in the Antilles as early as the 18th century. Ducœurjoly writes about it:
“We express the cane juice, crushing them in the mill, in the usual way: one cooks one third of this juice, or vesou, until the consistency of syrup; we take the other two thirds, which is boiled for about an hour, and until it has rejected all the coarse skimmings that come to the surface of the chauctère. This latter liquor is used instead and in the same way as the scums, and the first to hold place of syrup … ” [SOS this may not be translated the best, especially the last line.]
As another way of preparing grappes with cane juice, the same author reports: “The vesou is cooked, in the good foaming, until the consistency of light syrup, the froths that have been drawn serve instead of the fact that boilers are extracted, when sugar is made. In the composition of the grappes, this syrup and foam are twice as much as if it were sugar syrup and ordinary scums. The rum that is distilled is very good, and it is called, in Barbados, where he manufactures a lot, spirit of rum”.
The composition of the molasses musts presents great variations, according as one wants to have a more or less aromatic product.
Often, it is enough to dilute molasses with water. This is how they operate in particular in English Guiana, where they add to the final molasses the amount of water needed to have a density of 1.060-1.063. The proportion is 15-16 volumes of molasses per 100 volumes of water and the total sugar content of about 9%.
In the United States, molasses musts “blackstrap” for the manufacture of alcohol have a density a little higher: 1.065-1.075 in general. In some cases, however, it is 1.080. The normal dilution rate is 1 volume of molasses per 5 volumes of water.
In the French colonies (Martinique, Guadeloupe, Reunion), musts are also sometimes composed with molasses and water only. More frequently, however, a certain amount of vinasse is employed. This is 10 to 30% generally in Reunion and La Guadeloupe.
In Martinique, it amounts to 50-80%. In some factories, it even reaches 90%. It is then no longer used with water, except in very small quantities to bring back to the initial degree the density of the vinasse, which is concentrated as the production season progresses. The proportion of molasses (by volume) is usually 10%; it rarely reaches 15%. As for the density of the must, 1.040-1.050 most often, it rises to 1.075-1.080, when one wants to get a full-bodied rum. The sugar content is usually between 6 and 8%. Exceptionally for the manufacture of grand arôme rum, the must, composed only with molasses and vinasse is very acid (15 gr per liter) and high density (1.090), reaches up to 1.115: fermentation is in this very slow (9-10 days). In the old industrial distilleries of Saint-Pierre, the most used proportions were:
For the manufacture of certain types of rum, mixed musts of vesou and molasses are prepared. According to Suzuki, we would obtain the best results from the point of view of fermentation and alcohol yield, by using a mixture of 60% of vesou at 15° Brix and 40% of molasses at 20° Brix, added with 50 gr. Ammonium sulphate per hectolitre of must.
Molasses and scum musts. [skimmings]
In the beginnings of the rhum industry, the musts were generally composed of a mixture of molasses, defecation foam and vinasse.
Ducœurjoly (1802) indicates, for the composition of the grappes, various formulas, according to the nature and the quality of the raw materials available. He advises, for example, for the beginning of the season, when one does not yet have syrup (molasses), and per tank of 300 gallons:
When you start having some molasses, Ducœurjoly reports the following combinations:
For the time of the harvest:
To continue to make rum after the end of the sugar manufacture, when we no longer have scums:
Finally for grappes without scums or dunder [vidanges]:
[“24 h. after and when you have brewed and skimmed well, add syrup”
Soleau (1) wrote in 1835 about the making of rum in English Guyana:
(1) Ann Marit. Col. 1835, t. 2, 40.
“In general, there are few molasses distilled, the sale being more advantageous and not giving a rum as perfect as that which is obtained with the scums. The proportions of the different parts that make up the grappe vary with the amount of molasses that we have, or that we want to distill. Here are the proportions of a grappe: it contains 30 gallons of scum, 30 of water, 30 of dunder, 10 of molasses. It is calculated that 6 parts of skimmings correspond smoothly to a part of molasses. Now, if we vary the respective quantities of skimmings and molasses, we manage to have, by the reduction and the number we give to the foam, 15% of douceur [translates as gift payment, so extra?] and to complete the 100 parts in quantity by the water and the dunder”.
Porter indicates as proportions frequently used in the beginnings of the last century in the West Indies:
The amount of sugar was about 15%.
In Jamaica, preference was given to:
The average density was 1.050 and the sugar content was 12%. “In some distilleries, Porter writes, the sugar content is 14-15%, which does not seem to be exceeded, because the formation of too much alcohol prevents the fermentation from ending and the sugar to be completely transformed “.
Wray recommends a similar mixture consisting of 10% molasses, 20% skimmings, 50% vinasse and 20% water.
At present, it is more than in Jamaica that we continue to use the foam. In this country, the proportions of the ingredients used vary from one distillery to another, according to the type of rum that one wants to obtain (common clean, medium rum or german rum). Usually the musts are formed by 10% molasses (70 to 85° Brix), 20 to 40% skimmings and 10 to 20% water.
The density varies from 16 to 25° Brix, musts for grand arôme rum being generally thicker than the others. The sugar content varies between 8 and 15%; in some rare cases, it may exceed 16%. The acidity is relatively very high: from 10-15 gr. per liter (as sulfuric acid) on average, it sometimes rises to 20 gr. and rarely drops below 8 gr. This acidity, due to the use of scums and acidic vinasse, is constituted, in the proportion of 70 to 90%, by fixed organic acids.
The fermentation time, from 4-6 days for light rums, reaches 2-3 weeks in the case of grand arôme rums.
In distilleries not annexed to sugar factories, the skimmings are replaced by cane juice, which is limed and often left to itself for a few days in a juice tank before being used.
For the manufacture of german rum, a certain quantity of aromatic liquids, the acid (acid) and l’arôme (flavor) obtained by subjecting the cane juice to a special treatment, are added to the must (see Chapter II). Arroyo gives the following example of grand arôme rum composition:
Practice of composition.
In the past, must composition was often done in several stages. “In the Leeward Islands,” Porter writes, “vinasse and water are sometimes mixed together in equal proportions. When these ingredients are well mixed and the temperature is appropriately regulated, fermentation is sufficiently advanced after 24 hours to allow the addition of the molasses, which is introduced in the proportion of 3 gallons per 100 gallons of liquid. After 1 – 2 days, when everything is well fermented, pour the same amount of molasses again.” The author advises, however, to use molasses in one go, after the beginning of the fermentation, the successive additions tending to stop the fermentation and to lengthen the duration.
Wray indicates the following process for composing a 1,000-gallon tank. “First of all, 200 gallons of well-clarified foam are squeezed in, 50 gallons of molasses and 100 gallons of clear vinasse are mixed together, then the fermentation is allowed to settle in. This is done very quickly. 50 more gallons of molasses, 200 gallons of water are added, and the mixture is left standing for an hour for the fermentation to take place, at which point 400 gallons of vinasse are poured into the tank and mix thoroughly with the mass”.
Even today, in certain regions where the manufacturing processes have remained primitive, the must is prepared in stages. Thus Prinsen-Geerligs reports the following operation mode observed by him in a distillery on the peninsula of Malacca [Malaysia]. The molasses is diluted with water to a density of 15° Baumé and poured into large vats which are half filled. After 3 days, when the fermentation is almost complete, the tanks are filled to the top and distilled 3 days later. “If the tanks had been filled from the beginning,” writes the author, “the rise in temperature and the formation of acetic acid would have been such that the yield of rum would have diminished a great deal.”
De Sornay, in Mauritius, indicates a similar procedure, “Some distillers, instead of diluting the molasses with water at one time, dilute first at 16 or 17° Baumé, seeding [ensemencent? add yeasts?] and it is only when the ferment is well developed (7 or 8 hours later), that they continue the addition of cold water to arrive at 10° Baumé”.
In Haiti, one also operates on the composition several times [very interesting phrasing!]. The quantities of syrup and water required are poured into the vats and, after fermentation has begun, the vinasse is added. Some put to start the first part of the syrup and the water, then the second half of the syrup, then the vinasse (Pairault).
On the other hand, in the rum producing countries where the manufacturing methods have been modernized, the composition of the musts is generally done in one go. It should be pointed out, however, that Arroyo has recently recommended a method of fermentation with a thick must in which the addition of molasses is done in several stages.
Depending on the importance of the installation, the composition [batición!] is carried out either in the fermentation tanks themselves, or in a special vat (which should preferably be copper and cylindrical) or a pit dug in the ground. The first way of doing this has the disadvantage, in the case where one practices the seeding, to oblige to add the yeast to the must, whereas it is better to pour it on the leaven. [This is a big Arroyo idea, must should be added to yeast, not yeast to must and he describes the reason in Circular 106.]
In the French West Indies, if small agricultural distilleries usually mix in the fermentation tanks, the industrial rhummeries most often use for this purpose a masonry pit, embedded in the ground and coated with a cement layer. The capacity of this one varies between 5,000 and 10,000 liters.
The mixing can be carried out as follows. First we get molasses, whose volume is measured by passing through a special tray under load, or using a graduated rule placed in the pit. The vinasse, which comes from the boiler of the distillation apparatus, is then directly poured without being cooled or, more often, after passing through a tubular cooler which lowers the temperature to 30 °. Finally, complete with water. The volumes of vinasse and water are generally measured by means of the graduated rule placed in the composition pit. In other cases, the vinasse is first poured, which is brought to the desired density by addition of water, then the molasses and finally the water.
Mixing is carried out in small installations by means of a board of rectangular or rounded shape pierced with holes, placed at the end of a long handle. In larger plants a mechanical stirrer or an air injector is used. The latter is not advisable when working with sterilized musts, because, unless previously filtered or disinfected by passage through an antiseptic liquid, the injected air introduces in the liquid foreign micro organisms. As the maintenance of the filters is a rather delicate operation, it is preferable to use a mechanical stirrer to perform the stirring.
Most often, we calculate the proportion of materials to be used to obtain a given density, as follows:
The difference between the final density and the densities of the two component materials is differentiated; the remains, placed inversely, indicate the proportions to be adopted. Or, for example, to mix a molasses with 1.400 with water, so as to have a wort at 1.050 density. The calculation is as follows:
The proportion will be 50 l. molasses for 350 l. of water. This method of calculation gives only approximate results, but which nevertheless suffice in the conditions of the practice, where one does not try to obtain a very rigorous density.
The vats and the composition pit must be kept perfectly clean. It will be good, at the end of the day, to wash them with hot water or with exhaust steam, then with an antiseptic solution. The next morning, wash again with warm water. Carefully avoid leaving bottoms of must there.
Saccharine richness and must density.
In order for the alcoholic fermentation to take place under good conditions, the sugar concentration of the must must be maintained within certain limits.
A too low density favors the development of certain parasitic ferments and hinders that of the yeast. It is not advisable to go down, although it is sometimes done in a vesou distillery, below 1.040 (at 15°C), a density which corresponds to a sugar content of 9-10% in the case cane juice or syrup and 7-8% in the ordinary molasses musts.
On the other hand, it is economically advantageous to work with as concentrated liquids as possible. This makes it possible to reduce the necessary vat room capacity (and consequently the costs of first establishment), the fuel expenses for the operation of the distillation column and the pumps, the volume of the vinasse obtained.
Factors determining the concentration.
The factors that intervene to limit the concentration are: the duration of the fermentation, the action of the constituents of non-sugar, the tolerance of the different races of yeast with regard to the products of the cellular metabolism, finally the temperatures reached during fermentation.
From a certain saccharin concentration, close to 15%, the transformation of the sugar slows down rapidly, because of the inhibitory action exerted by the alcohol on the yeast. In the manufacture of industrial alcohol it is not indicated, for reasons of cost, to extend the duration of the fermentation beyond 48 hours. The concentration limit of the musts in sugar is, consequently, located at about 15%.
It is not the same in rhummerie, where the cost price does not need to be as tight. The prolongation of the fermentation is even advantageous in the case of the grand arôme rums, because it is accompanied by a more important formation of aromatic secondary products (esters, acids). A short fermentation is indicated, however, for obtaining light rums. The type of eau-de-vie that one wishes to obtain will therefore be the main factor determining the saccharine richness and the density of musts.
The nature and proportion of non-sugar elements are also important. Cane juices, especially if they have been deprived by the defecation of a certain amount of nitrogenous and phosphatic materials, as well as syrups, are usually relatively poor in nutrients. This defect increasing by the dilution, it will be necessary to allow the normal nutrition of the ferments, to raise the saccharine concentration around the maximum tolerated by the yeast for the complete transformation of the sugar into alcohol in a normal lapse of time.
In the case of molasses, on the contrary, especially when they have been used up, the high proportion of certain mineral substances may considerably hinder or even stop the fermentation, if the dilution ratio of the raw material is close to the maximum concentration of sugar. Vinasse intervenes in the same direction, the inhibitory action of the salts being in this case increased by that of certain organic acids. The depressive effect of the constituents of the non-sugar is especially pronounced towards the end of the fermentation, when there remain in the musts only small quantities of sugars. The presence in the wines of residual sugars is often much less the infermentable character of the latter than an unfavorable balance between non-sugar and sugars, the exhaustion of the nutrient medium and the production of toxins by the yeast.
Also, the saccharine richness of molasses musts, especially when the water is partly or wholly replaced by vinasse, is usually lower than that of vesou musts or syrup: 9 – 12% instead of 10 – 14%. Sometimes, it falls to 7-8%, although the density of the must is very high (1.100 and more for the must of grand arôme rum in Martinique).
In the case of rhummerie ferments, concurrently intervening with alcohol, to disturb the yeast, the other products of cell metabolism: esters, higher alcohols and especially acids. The antiseptic power of these, practically negligible in the production of industrial alcohol, is all the more accentuated that it aims to obtain a more robust eau-de-vie, that is to say, richer in aromatic principles.
The behavior of different races of yeast in musts with high saccharin concentration is also very variable. Here are some results obtained by Arroyo, in Puerto Rico, by fermenting with pure yeast musts with various sugar contents:
The above results were obtained at the Laboratory under optimal conditions of fermentation (temperature, acidity, etc.). In industrial manufacturing, where optimal conditions are rarely achieved, sugar concentrations must necessarily be lower.
It is generally accepted that the sugar content of musts for light or medium rums must be between 9 and 11%. In some cases, however, the rate is substantially increased, and up to 16%. This is particularly the case in the manufacture of the German Rum of Jamaica, or yeasts with scissiparity [fission, Pombe], particularly resistant to alcohol and acids. But then, the duration of the fermentation is abnormally prolonged and the yield of alcohol often mediocre (1).
(1) However, by the process of recovery of yeasts, the wealth of alcohol can be pushed to 12-13% and even 14%, following the observations made in France in 1944, in the work of skimmings of sugar (Mariller ).
As we have previously reported, the rise in temperature increases the antiseptic action of alcohol. While many yeasts can be acclimated to high alcohol concentrations, few are able to withstand the combined effects of high concentrations and high temperatures (Owen). In the tropical distillery where, as a result of the temperature of the cooling water, it is generally not possible to go below 30 ° and where the temperature is sometimes raised to 38-40 °, it would nevertheless be possible in many cases, to obtain much better fermentations, by acclimatizing ordinary yeasts to these unfavorable conditions (Owen, Chaturvedi). This acclimation is usually accompanied by a slowdown in the speed of fermentation.
The state of development of the yeast intervenes finally in the resistance to the temperature-alcohol complex. When the musts are pitched with an insufficiently ripe yeast footing, containing a large proportion of young cells, it is not rare that the fermentation stops prematurely, before complete exhaustion of the sugars. [I think the logic of this may be backwards. I think you want young and vigorous cells with strong cell walls.]
Increase in the concentration of musts.
In addition to the selection and acclimation of yeasts, various processes have been recommended to allow the fermentation of must with high saccharin concentration. These processes have been studied for the production of industrial alcohol. Tests should be done to specify the action they can have on the formation of the principles of the bouquet and on their value in the rhummerie.
Delbruck observed that the addition of rye grains to concentrated sugar solutions improved fermentation. He attributed this effect to the mechanical action exerted by the grains, which facilitates the elimination of the carbon dioxide produced. [I think this is accurately translated as rye: “grains de seigle”. I’ve heard of this done with bagacillo which is pulverized bagasse to restart a stuck fermentation, but I’ve never encountered an explanation of the mechanics.]
Owen found that charcoal and cane bagasse enjoyed the same property. The use of activated carbon at a dose of 200 gr. about 1,000 liters, allows to raise the initial density of the must to 30 ° Brix and more, while the maximum concentration generally accepted for the manufacture of alcohol industry is only 20 ° Brix. In his research, Owen observed that not only was the alcoholic yield of musts at 35 ° Brix with added coal as high as that of ordinary musts at 18-200 Brix, but that the acceleration of fermentation was such that the time required for the exhaustion of sweeteners was about the same in both cases. [I wonder if these days ultrasonic treatment could de-gas the beer.]
Bagasse also has a very clear accelerating action on the fermentation of molasses musts, especially when they have a high density. This action is however less pronounced than in the case of coal. It is even larger if the amount of bagasse is higher: however, beyond 25 gr. per liter, there would be no appreciable increase of the stimulating action.
The yeast recovery fermentation method (see Supra) makes it possible to significantly raise the density of musts. In the Hildebrandt and Erb process, the alcohol yield is higher when the sugar content is higher, as shown, for example, by the following figures given by Owen (1):
(1) Sugar XXXVI, NO 3, 26, 1942.
Similar results were obtained by Owen, by introducing into the musts autolyzed yeasts from the treatment of bottoms. [This may be a way to increase yeast nutrition?]
Finally, Arroyo, by purifying the molasses so as to eliminate substances (gums, excess mineral matter) and undesirable microorganisms, was able to obtain excellent fermentations with musts containing 18 to 22% sugar and giving wines at 10%. – 11% alcohol. During the tests carried out, the duration of the fermentation usually varied between 24 and 48 hours and the alcohol yield between 90 and 100% of the Pasteur coefficient.
In the Arroyo thick-must process (2), the molasses is diluted with water, so as to have a density of 55-63° Brix, and then added with concentrated sulfuric acid, in a quantity sufficient to lower the pH by 0.5, and, if applicable, nutrients (Am sulfate, lime superphosphate). The thick must thus obtained is carried in a tubular heater at a temperature of 80° C. During this time, the mineral or organic impurities precipitate, the microorganisms are destroyed by autolysis and the sucrose undergoes some inversion. The clarified liquid is then decanted and sent to the fermentaton, while the deposit is taken in a special tray with two or three times its volume of water, to recover the sugars contained therein. After re-settling, clear supernatant water is used to prepare the normal must and the residue is discarded.
(2) U S. Patent 420 898, 28 Nov. 1942. By thick must, one hears a very concentrated mash. In France, this expression is reserved for liquids containing suspended solids (musts of grains, potatoes, etc …)
The clarified thick must from the settling tank is cooled in a temperature exchanger at 40° or below, before being sent to the fermentation tanks, where it is diluted with water.
The quantity of water necessary to carry out the desired dilution is first introduced into the tank, which is closed and equipped with a mechanical stirrer, then, progressively and continuously stirring, 50% of the thick must, so as to obtain a normal must having a density of 18 to 24° Brix. We adjust the acidity, material that the pH is about 5.0 (variable depending on the yeast race used), and finally we add a starter or leaven, equivalent to 10 or 15% of the total volume of the must. After 12 hours, when the Brix dropped from 55 to 63%, 30% thick must is again introduced with gentle stirring and, after another six hours, the remaining 20%. The density after mixing must be a little lower than the initial density: if it is 22° Brix for example, the density following the first molasses intake will be 20° Brix and following the second intake of 18° Brix approx.
During the fermentation, the temperature is maintained between 28 and 30° C, by continuously passing the wort in an external refrigerant, and the pH to 5.0 -5.2, by addition of lime or, better, of 10% ammonia water. To reduce the amount of foam that forms at the time of the additions of thick must, a small quantity of anti-foam product (“Turkey red oil” for example, with 1 liter of oil per 16,000 l. must). The fermentation is usually completed 15 to 30 hours after the last introduction of thick must.
This process has important advantages. The must is freed, by the preliminary purification, not only of the foreign ferments, but also of a large part of the mineral and organic impurities present in the molasses and which counteract the fermentation. The efficiency of the fermentation is substantially increased, the fouling of the columns reduces. As the density of musts is higher and the alcoholic richness of the wines raised, the capacity of the distillery is increased by 35 to 50%, the expenditure of manpower and fuel diminished.
We reproduce some data relating to fermentations carried out by this process in a distillery of Puerto Rico (1).
(1) Sugar XXXVIII No 2, 18, 1943
The acidity of must deeply influences the process of alcoholic fermentation. The concentration of H ions acts both on the development of the ferments and on the nature of the products formed. Insufficient acidity promotes the multiplication of bacteria, and causes yeast formation of a relatively large amount of organic acids at the expense of alcohol production. An excess of acidity paralyzes the yeast. The proper acidification of musts is therefore one of the most important points in the production of industrial alcohol and spirits.
This acidification is performed in rhummerie by the addition of sulfuric acid or acidic vinasse. In distillery of starchy materials, it is sometimes acidified, but more and more rarely, by subjecting the must to a preliminary fermentation by lactic acid bacteria. When sulfuric acid is added to a solution of molasses, it is first applied to the alkalis, which are neutralized. Then he releases the volatile organic acids. When the dose of acid used is quite high, the fixed organic acids are in turn displaced and the excess of sulfuric acid remains, if necessary, in the free state. The complex nitrogenous materials are more or less degraded, according to the dose of acid, and transformed into more assimilable substances for the yeast. Finally, sulfuric acid begins the reversal of sucrose into glucose and levulose. This hydrolyzing action is however quite weak in the conditions of the practice: it takes some importance from 1 gr. ac. sulfuric acid per liter.
Practice of acidification in the rhummerie.
The addition of sulfuric acid to the must of molasses and vesou is a practice that has been in use for quite some time.
Pairault reports that at the end of the last century a large number of distillers in the French West Indies and English Guiana added small quantities of sulfuric acid to their composition in order to obtain purer fermentations. At present, the use of sulfuric acid is widespread, in the manufacture of ordinary rums. On the other hand, when one wants to have a grand arôme rum, one carefully avoids using it, the degree of acidity necessary for the yeast being obtained by the addition of vinasses or scums.
It has been proposed, on various occasions, to substitute for SO H various other mineral or organic acids: hydrochloric acid (Mohlant), hydrofluoric acid (Effront), phosphoric acid (Collette and Boidin), lactic acid, etc. The use of these has not developed. However, hydrochloric acid is sometimes used in sugar beet molasses distillery, to avoid encrustation of the columns and evaporators of vinasse (formation of soluble lime chloride).
The quantities of sulfuric acid added to the must are quite variable according to the country, the type of rum that one wants to obtain, the quantity and the acidity of the vinasse entering the composition, etc…
In English Guiana, 1 l. is usually used of acid for 1,000 l. of must. It is about the same in the United States. In the Magne process, which is frequently applied in the latter country, fermentation tanks receive 0.80 to 1 l. of acid per 1,000 l. must, intermediate tanks 1.20 l. for 1,000 l. and leavening appliances an even higher dose.
Martinique is used in the manufacture of ordinary molasses rum 0.1 to 0.4 l. and in the rum of vesou 0.2 to 0.5 l. SO H, for 1,000 l. of must. The acidity, given that provided by the vinasse, is 1.4-1.6 gr. per liter for musts of cane juice and 3-4 gr. for those molasses. Exceptionally, it reaches up to 15 gr. in the case of grand arôme rum.
In Guadeloupe, a small amount of sulfuric acid is also usually added to the must of molasses, so as to have an acidity of 2 – 3 gr. per liter, while in Madagascar and Reunion, acid is rarely used. In the latter two countries, the initial acidity of musts, often consisting of a simple mixture of molasses and water, is low: 1.5 to 1.9 gr. about per liter.
In Jamaica, sulfuric acid is never used. As the composition of high proportions of skimmings, vinasse and fermented juice (acid, flavor) are very acidic, the acidity of musts is dependently high: 10 to 20 gr. per liter.
As regards the influence of vinasse on the composition of the distillate, we can notably mention the observations of Kayser, who has noticed a significant increase in the rate of volatile acids. This author obtained, among others, the following results, with two musts with 12% molasses by volume. The first prepared with water, the second replacing half of the water by an equal volume of vinasse:
Adjusting the acidity.
Most often, in rhummery, the addition of sulfuric acid is empirically made without taking into account compositional variations of the raw materials and the special requirements of the ferments employed. It is only in recent years that the conditions for rational acidification of the nutrients have been specified by means of pH control.
At the beginning of the century, Pairault warned Rhummeries against the use of an excessive quantity of sulfuric acid, which paralyzes the action of sucrase at relatively low doses. He indicated as the maximum quantity, that it is most often good to reduce, that of half a liter of SO4H2, for 1,000 l. of must.
Humboldt (1) recommends adding only 1 l. of acid for 10,000 l. of cane molasses, and Arnstein (2) a sufficient dose to obtain an acidity corresponding to 1.5-2 cc. of normal soda per 100 CC. Next, Effront and Prescott, the best practice in cane molasses distillery would be to achieve an acidity of 1 to 2.5 gr. per liter, whereas, according to Williams, molasses must should have a free sulfuric acidity of 0.1%.
(1) Louisiana Plant. LXVIII, 206, 1922.
(2) Louisiana Plant. LXVIII, 126, 1922.
Freeland insisted that the amount of sulfuric acid used must be a function of the quality of the molasses and the type of fermentation. A mediocre molasses requires a higher proportion of acid than another of good quality, easily fermenting. In pure fermentation, much less acid is required than in spontaneous fermentation. “It is generally admitted,” he says, “that the appropriate acidity of the must is between 0.15 and 0.20%, evaluated as sulfuric acid, but this dose is often exceeded and may be as high as 3%, if the molasses contains a lot of free acids. Usually 0.75 to 1 liter of sulfuric acid is added per 1,000 l. of cane molasses.
Owen and Bond found that the dose of sulfuric acid corresponding to the maximum alcoholic yield varied according to the yeast used and the composition of the molasses. They obtained the following results, during tests carried out with musts (at 16-17° Brix) of blackstrap molasses:
These results show that the optimum dose of sulfuric acid depends on the race of yeast. It is, with the yeast N ° 83 for example, of 1.50 cc. for Louisiana molasses and 1.25 cc. for that of Cuba. In the case of Magné yeast, the maximum alcohol yield is given by an acidification of 1.25 cc. for both types of molasses. The tolerance of the various breeds with regard to the variations of the acidity is greater or less: the difference between the maximum and minimum alcoholic yields corresponding to the various acidity levels is highest for the yeast N° 74 and the lower for Magné yeast. The amounts of acid required to obtain the maximum alcohol yield varied more in the case of Louisiana molasses (1.0 to 1.5 cc, depending on the yeast races) than in the case of the Cuban molasses (1.25 cc). This seems to be due to variations in the “buffer power” of molasses. [Magné was a system for growing pure yeasts that we should probably learn more about.]
Finally, the figures above show that the addition of insufficient amounts of acid results in a reduction in the alcohol yield. This fact, quite often observed, is difficult to explain. It is probably due to the action of organic acids liberated by sulfuric acid. Owen and Bond conclude from the foregoing that it is important to specify experimentally by laboratory tests, for each race of yeast and for each type of molasses, the acid needs of the must.
The dosage of the alcohol produced in the presence of variable amounts of sulfuric acid is a rather delicate method, the difference between the yield of the optimum solution and that of the solutions containing inadequate amounts of acid, being generally low. Another more practical method is to measure the acidity at the beginning and at the end of the fermentation. [Δ acidity!]
It has indeed been observed for a long time that the higher the acidity during the fermentation, the lower the alcohol yield. Fernbach (1) in particular has shown that if the acidity produced by pure yeasts can vary with the races, it is always stronger when the liquid is primitively less acidic. It has long been accepted in beet distilleries that the increase in acidity should not exceed 0.2 gr., in good fermentations (Boullanger). [He may be referring to pH crash in unbuffered ferments.]
(1) C. R. CLVI. 77, 1913.
Hildebrandt, in the special case of cane molasses, has found that the maximum alcohol yield is obtained when the pH of the must is not modified during fermentation. Here are some of the results obtained by this author:
In the above tests, the optimum pH is close to 5, but it is likely to vary, depending on the yeast races, between 4 and 6. The true criterion of optimum acidity is that the final pH does not tend to be higher or lower than the initial pH. The easiest and safest way to regulate the acidification of the musts will therefore consist in achieving the constancy of the pH during the fermentation. [Either a wider buffer or periodic adjustment. pH was a very new concept back then which opened a lot of doors.]
This is so when one wants to get the maximum alcohol yield. If it is proposed to produce an alcohol of mouth, whose bouquet is a function of the secondary products formed during the fermentation, it is not the same.
When the pH is relatively low (4.5-5.0), the fermentation is fast and the rum obtained light. If it is high (5.5-6.0), a larger quantity of fatty acids and esters is produced. The eau-de-vie is more full-bodied, more mellow and finer. But fermentation is longer and tricky to conduct. In particular, it is important for the must to be sterilized beforehand in order to eliminate bacteria whose development interferes with that of yeast in low acidity environments.
According to Arroyo, the optimum pH for rum production would be between 5.5 and 5.8, and the titratable acidity between 1.5 and 2 cc, of N / 10 soda per 100 cc, must. However, if the must has not been previously sterilized (at least partially), it may be necessary to go down to 5.0 and even 4.5, to avoid contamination by bacteria and wild yeasts. In the production of very full-bodied rums, where scissiparous [fission] yeasts are used, which adapt to high acidity, the pH will be even lower. The distiller will have to determine experimentally what is the optimum pH for the particular breeds of ferment he uses and the special conditions of its manufacture (saccharine richness of must (1), fermentation temperature, type of rum, etc…).
(1) The development of yeasts is closely dependent on the alcohol – temperature – acidity complex. If the temperature and the saccharine richness are high. It is necessary to have a relatively high pH, otherwise the action of the yeast will be paralyzed. In the case of high temperature and acidity, it is important to lower the sugar content of the must.
Arroyo also studied the fermentation at pH maintained constant by neutralization of acids during fermentation. By this process, he obtained rums that were more aromatic, more mellow and quicker to ripen. He attributes these results to the fact that the lower fatty acids (formic acid, acetic acid), which have a pungent taste, are eliminated in the form of salts, whereas the higher fatty acids are less easily neutralized by the added alkalis. and tend to form valuable esters chemically during fermentation and distillation; on the other hand that the formation of rum oil is increased when the pH is high.
Here are some results obtained by the author above, adding liquid ammonia to the must during fermentation (fermentation temperature 27-29 ° C.).
Arroyo advises to operate industrially as follows:
The previously sterilized must is brought to pH 5.8-6.0, then 1 gr. of sterile lime carbonate per liter of must, avoiding stirring the liquid so as not to cause a change in pH; the carbonate is deposited at the bottom of the tank and has almost no influence on the pH, if one does not carry out agitation. The pH of the yeast footing is also adjusted to about the same value as that of the mash. [I feel like I missed Arroyo advising this.]
According to the author, this method would give a rum of high quality, very difficult to imitate. It is unfortunately difficult to apply because it requires work in aseptic environment, to protect the yeast against the occurrence of bacteria.
Nutrients and catalysts.
The musts of cane juice often have a deficiency in certain elements necessary for feeding the yeast, and in particular nitrogen. Molasses, on the other hand, may contain an excess of mineral matter, which hinders the development of yeast, and the harmful action of which may be diminished by the addition of antagonistic salts. Finally, the use of small quantities of substances acting as stimulants or catalysts is likely to accelerate the fermentation and increase the yield of alcohol.
Although so far only the use of ammonia salts and, to a lesser extent, phosphates has been of importance in rhumming, numerous tests have shown, however, that the use of various other substances acting on yeast nutrition.
Sulfate of ammonia. – Sulphate of ammonia was already commonly used in the French West Indies at the end of the last century, usually at a dose of 400-500 gr. for 1,000 l. of must. At present, we add, in Martinique, 100 to 300 gr. of this salt per 1,000 l. In a vesou distillery, the most common proportion is 300 gr. per 1,000 l., while in a molasses rhummerie, the sulphate of Am. is sometimes removed, especially when you want to get a full-bodied rum. It is about the same in Guadeloupe, where the dose of 200 gr is often used. of sulphate per 1,000 l. molasses must. In Reunion and Madagascar, musts are almost always composed of Am sulphate.
In English Guiana 1 kg of Am sulphate is added per 1000 liters of must. The dose is lowered in the United States, for the work of blackstrap molasses (50-55% of sugars), 100 gr. per 1,000 l. must at 16-18 ° Brix density. In the Magné process, only 250 gr are introduced. Am sulphate. for 1,000 l. in the yeast propogation apparatus, 350 gr. p. 1.000 in the intermediate tank and nothing in the fermentation tanks. On the other hand, in the work of Cuba’s invert syrups, much larger quantities of up to 3 kg per 1,000 lbs. to achieve maximum alcohol yield.
In Jamaica, the sulphate of Am. is never used.
The addition of nutrient salt, by causing a more vigorous development of the yeast, makes the fermentations faster and reduces the dangers of infection by foreign ferments. However, it is an optimum dose, according to Pringsheim, between 0.004 and 0.008% assimilable nitrogen, above and below which the fermentation is slowed down and the yield decreased.
The proportion of molasses nitrogen is generally sufficiently high for the must to contain an amount of this element at least equal to the above optimum. It seems, therefore, that the use of ammoniacal salts in the fermentation of cane molasses may be dispensed with. Nöel Deerr, for example, observed that in the case of the Hawaii molasses examined by him, which contained 0.24 1.06% nitrogen, corresponding to 0.05-0.2% nitrogen in the mash, Am sulate, had no influence either on the duration of the fermentation or on the yield of alcohol.
Arroyo, however, considers that if the total nitrogen content falls below 1% in molasses, it becomes necessary to add ammonium sulphate (0.2 to 0.5% of the molasses by weight) to these to obtain a good fermentation in the rhummerie. From 1% of total nitrogen, the addition of this nutrient is no longer motivated. According to the same author, Puerto Rican molasses would have, with a few exceptions, a generalized deficiency of nitrogen. The optimum nitrogen concentration for musts at 18-21 Brix, containing 11.5 to 13.5% of total sugars, would be 75 to 100 mgr. N per 100 cc, must.
Cane juices, especially if they have been defecated beforehand, as well as battery syrups, generally have a deficiency in nitrogenous matter. Owen, for example, has shown that to give the maximum yield of alcohol, invert syrup mashes at 24° Brix density, required a contribution of 3 gr. Am sulphate per liter.
Kozo Suzuki, in Formosa, has found that the optimum amount of Am. sulphate, for vesou musts containing 8% sugar, is 0.5 gr. per liter, if this nutrient salt is used alone. But when using bipotassium phosphate at the same time, the best results, as regards both the yield of alcohol and the rapidity of the fermentation, are given by a dose of 1 gr. of Am. sulphate and 1 gr. of phosphate per liter.
According to Iwata, the most appropriate proportions for cane juice from the first and second mills are 1 gr. of each of the above salts per liter. For the juice of the third mill, a dose of 0.5 gr. of Am. sulphate and 0.1 gr. K phosphate is sufficient. As for the juices, which are richer in nitrogen and mineral salts provided by insufficiently ripe canes, damaged by parasites, or by “white tips”, they do not need to be supplemented with nutrients. More than half the amount of nourishing salts is required for the overweight canes than for normal canes. [Fascinating!]
The determination of the nitrogen in the raw material will provide interesting indications on the needs of the must in Am sulphate. However, comparative laboratory tests will be necessary to specify the optimum dose, which may vary with the yeast race.
The use of ammoniacal salts has some disadvantages, when one wants to obtain a full-bodied eau-de-vie. It decreases the amount of higher alcohols produced. Most of these come from the transformation of the amino acids of the must by yeast, which tends, when it has a more easily assimilated nitrogen source, not to attack the amino-acids. [very key concept]
Various nitrogenous materials have been proposed from time to time in place of Am sulphate. Although their use is not extended, it is interesting to say a few words, because of the advantages that they can present in certain cases.
Ammonia and various ammoniacal salts. — The use of ammonia has been advocated by Owen, for the fermentation of invert syrups. If the high doses required by these last come only from Am sulfate, the fermentation begins satisfactorily, but, as a result of the accumulation of SO4 ions, it soon becomes languid and stops before the sugars are completely exhausted. To counter this inconvenience, the author proposes to replace half of the sulphate of Am. by a corresponding dose of ammonia, which is added to the must when the fermentation is about in its middle. [another key trouble shooting insight]
In this case, the use of Am. phosphate or, what ever is cheaper, albuminoid nitrogen, in the form of dried blood or distillery bottom for example, can also be advantageous. [I’ve never heard of this blood usage before.]
The use of ammonia, in the form of a 10% solution of NH3, was also recommended by Arroyo for the preparation of starter. Ammonia accelerates the multiplication of yeast cells and is more effective than Am sulphate in lowering the pH, as shown by the tests below. The doses used were 4 gr. pure Am-sulphate (all at once) and 1 cc. pure ammonia (several times) per liter of must.
By using ammonia in solution in the fermentation of the main must instead of Am sulphate, the above mentioned author has also observed a reduction in the duration of the fermentation and an increase in the alcohol yield, at the same time as a decrease in the level of higher alcohols and an increase in that of other products. In the tests below, the fermentation was carried out at a temperature of 27-29 ° C.
Various other ammoniacal salts: carbonate, phosphate, Am. Hydrochloride, have been experimented with. Under ordinary conditions, they do not offer any clear advantages over sulphate, and, as they are more expensive, they are not to be recommended. It should be noted, however, that Azzi (1) in Brazil obtained the maximum yields in the fermentation of cane molasses by the use of Am phosphate at a dose of 150-200 g. per hl.
(1) Bol de Agricult. São Paulo XXXVI, 330, 1935.
Urea. — Lindner and Wüst, then Bokorny, observed that urea was an excellent source of nitrogen for yeast, which develops remarkably in a urine solution with added sugar. According to Zeller (2), the addition of urine to the musts would determine an increase in the activity of the fermentation, up to 200%. This would be due in part to the ammoniacal salts present, but especially to a stimulating substance soluble in alcohol, precipitable by insulin and whose action would be comparable to that of vitamin B of Gigon or biocatalyst Z Euler. The optimum dose of urine is 1-2%, and the urine of the night is more active than that of the day.
2) Biochem. Z. CLXXII, 142, 1926.
Peptonized and autolyzed yeast. — The degradation products of the albuminoidal substances (albumins and peptones, amino acids) are easily assimilated by the yeast. On several occasions, it has been recommended to add the bottom of the vat, previously treated so as to carry out the peptonization or autolysis of the yeasts.
Barbet obtained an improvement in the fermentation of beet molasses, by the use of peptonized bottoms by the addition of sulfuric acid under pressure.
The muddy yeast residue is boiled in a Krüger, for example, and distilled until the alcohol is exhausted. A dose of sulfuric acid corresponding to that required to ensure the acidification of the normal must is then added; it is closed and brought to a pressure of 2 kg. The yeast cells are gradually dissolved. The acid mixture is then cooled and added to the must. It takes about 500 gr. peptonized yeast per 100 kg. molasses worked. [This is quite interesting and may be useful.]
In the Bauer process, peptonization is carried out by the bacteria. The yeast is left to itself for 3 or 4 weeks in small vats, which receive daily the amount of yeast corresponding to consumption. [This sounds very much like muck and now we are seeing it relates to peptonization.]
Autolysis can also be carried out by keeping the yeast previously diluted in water in a vacuum at a temperature of 50 ° for a few days.
If you start from a brewing yeast or bakery, you can make a dough with it by diluting with distilled water. An equal weight of a saturated solution of white sugar is added.
Owen was able to obtain, by adding autolysed bottoms to cane molasses musts, appreciable surpluses, as shown by the figures in the table below:
Owen found that yield increases were most pronounced in the case of high-density, high-sugar musts.
Kayser has shown that in the presence of autolyzed yeast, there is greater production of impurities, especially higher alcohols. Here are some results obtained by fermenting a 12% molasses must with 25% vinasse with 2 rum yeasts, in the absence and in the presence of 2% autolysed yeast:
In another test with autolyzed rum yeast (2%), the same author obtained:
The proteolytic products resulting from autolysis act not only on the quantity, but also on the nature of the esters obtained. The alcohol provided by the autolysed yeast ball of the previous test had a very good taste and was more perfumed than that of the control ball. The composition of the esters was as follows:
Finally, the fermentation of the balls that had received the autolyzed yeast started more quickly and ended earlier. Kayser therefore recommends the addition of autolyzed yeast in the case of fermentations made with certain pure yeasts, which are not very productive of higher alcohols, and also to activate fermentations. [Fascinating ideas here. I think I am correctly translation ballons to ball.]
It should be pointed out that the sludge from the fermentation tanks and the lees of the distillation apparatus, mainly made up of yeast cells, appear to play an important role in the preparation of the grand arôme rum. These materials, which are collected in the “muck hole”, constitute a source of nitrogenous materials, whose transformation, by the yeasts and the bacteria, would contribute powerfully to the production of the bouquet (Allan). Similarly, in the manufacture of the grand arôme rum of Martinique, the yeast deposits, instead of being evacuated with each new fermentation, are kept in the vats: it is only when their level exceeds a certain height that they eliminate some of it. [A very unique insight about Martinique!]
Phosphates play an important role in alcoholic fermentation. Various authors have shown that they stimulate in a very marked way the fermentative activity of the yeast (Delbruck, Elion, Young). Elion (1) in particular has found that monopotassium phosphate and neutral phosphate of ammonia determine an increase in the release of CO2, varying, in a given time, from 28 to 63%, depending on the race of yeast.
(1) Zent Bakt. Parast. XIV. 1893/.
However, although molasses musts are generally low in phosphoric acid, the addition of phosphates has rarely yielded industrially profitable results.
Peck and Deerr, for example, were unable to achieve fermentation acceleration or increased alcohol yield by adding lime phosphate to the Hawaii molasses feed, which contained less than 0.03% P2O5.
On the other hand, Owen and Chen found that Cuba’s invert syrups, to give the maximum alcohol yield, required a relatively high intake of phosphoric acid: 1 gr. of potassium phosphate per liter of wort at 20° Brix.
In the industrial fermentation of the final molasses of Puerto Rico, 250 to 500 gr. of lime phosphate per 1,000 l.
According to Arroyo, the optimal dose of phosphoric acid in molasses fermentation will vary between 0.2 and 0.25% of P2O5. Phosphoric acid deficiency would be much less common in Puerto Rico molasses than nitrogen. An excess of P2O5, which suffers from an insufficiency of N, seriously hampers, according to the observations of the same author, the good progress of the fermentation which could even be completely stopped in extreme cases. In such cases, the addition of nitrogen, in an amount sufficient to bring the P2O5 / N ratio to about 1/5. would restore normal fermentation immediately. The optimum phosphoric acid concentration for musts at 18-20 ° Brix, containing 11.5 to 13.5% total sugars, would be 15 to 20 mgr. P2O5 per 100 cc. of must.
The experiments carried out by Kozo Suzuki and Iwata in Formosa on cane juice showed that the best results were obtained by the addition of 1 gr. K phosphate per liter, with normal juices. The vesou provided by immature canes or strongly attacked by insects does not require phosphoric acid.
Salts of magnesia, manganese, etc.
Various authors have pointed out the favorable action exercised on the yield of alcohol and on the rapidity of the fermentation by certain metallic salts. It is, moreover, most often difficult to specify whether these intervene by supplying the yeast with a nutritive substance which is lacking in the must, or if they act as stimulants of the zymatic function. This stimulation can itself be of a biocatalytic nature, or result from the neutralizing action exerted with respect to certain toxic principles produced by the yeast.
The use of sulphate of magnesia is often advised in the fermentation of invert syrups and molasses of high saccharine richness. Owen and Chen obtained a slight increase in alcohol yield and a significant increase in the speed of fermentation, adding to the must of invert syrup from Cuba (at 20° Brix) sulphate of Mg, at the dose of 1.5 gr. per liter. It is possible that, in certain cases, the sugar raw materials, and particularly the defecated cane juice, have a deficiency in magnesia, which makes it useful to add this element to musts.
Kayser observed that magnesium salts also act on secondary products of fermentation, notably by increasing the level of higher alcohols and decreasing the level of esters. This author obtained, in a fermentation test with and without phosphate of magnesia, the following results (in grams per hl) of alcohol at 100°):
Guanzon and Lopez found that Mg sulfate, employed at the concentration of 0.005 to 0.01 mol. (0.60 to 1.20 grams per liter), increased the alcohol yield of cane molasses (1) in proportions ranging from 2.46 to 8.88%. With the sulphate of Ca, used under the same conditions (0.68 to 1.36 grams per liter), the yield in alcohol was increased from 8.27 to 11.12%. The useful effect of Ca sulphate was decreased by the simultaneous use of Mg sulphate. These results are due to the antagonistic action exerted by the Ca-ions vis-à-vis the K ions and by the MS ions vis-à-vis the Na ions. In the tests which they carried out, the above authors indeed observed a reduction of increase of yield, when the sulphate of Ca was employed in the same way with the sulphate of K, and the sulphate of Mg with the Na sulphate. This last salt, used alone, gave lower yields than the controls. As the cane molasses contain often very high proportions of K or Na salts, the addition of Ca or Mg sulphate to the must would counterbalance the harmful influence exerted on the nutrition of the yeast by a physiologically poorly balanced solution. Ca and Mg, which have a pronounced antagonism between them, must never be used simultaneously.
(1) The molasses, diluted at 18 ° Brix, contained per liter: Ca 0.5825 gr. ; Mg 0.1464 gr .; K 0.4316 and Na 3.9590 gr.
Lasnitzki and Szorenyi (1) found that potash salts (KCL) greatly increased the rate of fermentation (by about 150%). Even in the case of cane molasses, which are, however, very rich in K, the addition of potassium salts could in certain cases cause increases in yield. Guanzon and Lopez were able to obtain an increase of 1.01 to 3.69% in the balloons treated with K sulphate at the concentration of 0.005 – 0.01 mol. They attribute this to the antagonistic action exerted by K on metal ions other than Na, which are normally found in molasses. [SOS I’m not sure about “ballons” here. Does it refer to their small scale testing glass?]
(1) Biochem. J. XXIX, 580, 1934.
According to Sanzo and Pirrone (2), if seawater is added to glucose solutions, a marked acceleration of the alcoholic fermentation is observed (20 to 24% with 13% of water). This action would be more pronounced when the water was freshly collected. At a high dose (above 20%), sea water causes a marked delay in fermentation. Brill and Thurlow (3) have observed in the Philipine Islands, where molasses is often diluted with sea-water for the preparation of musts, that the alcohol yield diminishes proportionally with the quantity of sea-water, when it exceeds a certain rate. The yield of alcohol (% theoretical yield) of 71.6 in the case of molasses diluted with distilled water added with 3 equivalent of NaCl (0.48 gr per l.), fell to 63.4, when using 1/4 of seawater, and at 55.5, if only the latter was used.
(2) Atti R. Acad. Linell 161, XIII, 140, 1931
(3) Philippine J. Sc. A XII, 267, 1917.
Mitra (4) in California, found that Na, K, Ca and Mg chlorides, if they reach a certain concentration are more or less toxic to yeasts. KCL presented itself as the least toxic and NaCl as the most toxic.
(4) Univ. of California. Public. in Agr. Sc. III, 63, 1917.
Kayser and Marchand (5) observed that the addition of manganese sulphate to a concentrated sweet must (1 gr and 1.5 gr of sulphate per liter to 24% of sugar) had the effect of pushing the fermentation much further and giving an increase of alcohol sometimes reaching 3%. The amount of glycerine and volatile acids is also increased, while the fixed acids decrease. The lactate, the acetate and the nitrate of Mn behave substantially like sulphate, but with the succinate and the phosphate one observes, for a greater disappearance of sugar, lower contents of alcohol than in the control. The addition of yeasts to manganese salts would, according to Kayser and Marchand, lead to more complete fermentations. Manganese phosphate would increase the level of impurities, particularly that of higher alcohols and Kayser esters.
(5) Ann. Inst. Agr. 121 VI, 355, 1907.
More recently, Hildebrandt and Boyce, experimenting with cane molasses, found that by the use of small amounts of manganese sulphate, copper sulphate and sodium cyanide, an increase in alcohol yield varying from 1 to just over 2%. The stimulating action was more pronounced and more regular, when instead of adding the metal salt to the normal must, it was added to the yeast starter, at a dose of 1 per 1000 to 10,000 in the case of the sulphate of Mn. for 1000 to 3,000 in that of Cu sulphate and 1 in 10,000 in that of Na cyanide. The operation must be renewed with each new yeast starter, the action of salts not being felt beyond the second generation. This way of making it possible to reduce to a small number the quantity of necessary salts, makes them relatively inexpensive for use in industrial fermentations.