The use of wine goes back to times immemorial; the bible, Homer’s epics, Egyptian and Assyrian documents mention it. However, one must wait for the time of the middle Ages before the alchemists discover the active principle, ethanol. At the end of the 17th century A. Van Leeuwenhoek describes years in grape musts and beer worts, but he establishes no relationship between yeasts and fermentation. A century later Lavoisier publishes the first scientific work on fermentation which he considers a purely chemical phenomenon. Only in 1836 does Cagnard-Latour prove the role of yeasts, living organisms which cause biochemical transformations.
In 1866 Pasteur publishes his celebrated work, “Etudes sur le vin,” in which he analyzes the spoilage of wines and prescribes appropriate treatment. This is truly the origin of scientific oeanology, which has continued to progress from that point on.
Taxonomic and systematic studies on the specific microorganisms of musts and wines have multiplied since the beginning of this century. They have led to a good knowledge of the microflora. Since 1950 the development of chemical, chromatographic, and enzymatic techniques has led to a more sophisticated analysis, to the determination of several hundred components of wine, and, consequently, to a better approach to the biochemical phenomena and to a better understanding of the theory of fermentations. The last decades have also seen a significant mechanization of the production of wines. In contrast the transfer of the knowledge of genetics to the field of enology proceeds more slowly.
Traditionally the transformation of must into wine remains a “spontaneous event”. That means, it is induced by indigenous microorganisms which are found on grapes and equipment used in wine making. It is sufficient to crush the grapes in order to start the fermentation. This implies that it is difficult to control the agents of fermentation. Nevertheless, the acid pH of grape must constitute a very selective medium in which only a limited number of microbial species can multiply. These belong to three types of microbes: yeasts (preferentially), lactic acid bacteria and acetic acid bacteria.
The acetic acid bacteria as agents of biochemical change must be inhibited. The yeasts and the lactic acid bacteria, each, participate successively in the two principal stages of the fermentation. In the first stage yeasts transform sugars into alcohol; this is the alcoholic fermentation. The second stage, which occurs sometimes, favors the conversion of malo-lactic fermentation acid to lactic acid by the lactic acid bacteria; this is the malolactic fermentation. The production of certain special wines requires a secondary alcoholic fermentation under anaerobic conditions (sparkling wines) or a secondary growth of yeasts under aerobic conditions on alcohol (sherry).
The composition of the medium, notably the concentration of must sugars, affects the growth of yeasts; the higher concentration of alcohol and the acid pH of the wines affect the growth of lactic acid bacteria. This and the technological operations impose upon the microorganisms a growth cycle and a particular metabolism which constitute the particular characteristic of this biotechnology.
Brandy is obtained by distillation of wines. This procedure was already known in ancient times. Avicienna, an arab physician and philosopher, gave the first description and philosopher, gave the first description of a still in the 10th century. Arnaud De Villeneuve is the first to speak of “alcohol”, and one attributes the discovery of the “eau de vie” to one of his students. Throughout the Middle ages the use of brandy remains strictly medicinal, and its production remains quasi secret. It changes from the art of the alchimist to an industrial art thanks to the work of the physician Boerhave during the 18th century. The “brandwine” (burnt wine) diluted with water became during the 17th century the normal Beverage of Dutch sailors. The tonic can be stored in concentrated form without change, and it is indispensible in the fight against the epidemics of tropical countries. A lack of sales caused by wars leads to the discovery that brandy ameliorates during aging. Today there are several kinds of brandy distinguished by their origin, the quality of the base wine, and the nature of the distillation. The best known are undoubtedly cognac and armagnac.
All prestigious products have been created empirically. They are “the fruit of patient care... . the reputation of the great wine regions having advanced, and greatly thanks to chemical and biological studies.” Therefore one easily attributes the merits of wines to the quality of nature, and sometimes one questions the role of enology.
Ribereau-Gayon and Peynaud supply a precise answer to this question in their “Traite d. “A good wine or a great wine may be obtained without the aid of modern enology. However, the aid of modern enology is in general required to obtain a better quality regularly and with certainty and with the most efficient means.” And, finally, “enology is more than a specialty. It is central interest around which one can create and coordinate a comprehensive program of fundamental research which is authentic and of general interest.”
YEASTS AND THE ALCOHOLIC FERMENTATION
1. Taxonomy, ecology
Numerous studies of wine yeasts have been undertaken in the vine growing regions of the entire world. Their identification is based on Lodder’s “The yeasts” (1970) supplemented by the work of Barnett. It rests basically on a consideration of morphological and physiological characteristics, the metabolism of certain sugars, and the assimilation of diverse nitrogenous compounds.
Wine yeasts belong to the class of Ascomycetes (sporogenous) and Deuteromycetes (asporogenous). Ample documentation shows that the number of species which occur with a frequency surpassing 1% is no greater than about 14. The presence of about 40 other species is fortuitous. In their ecological niches yeasts are often associated with molds, lactic acid bacteria, and acetic acid bacteria. Their isolation and specific counting is done at an acid pH and in the presence of mold inhibitors Identification techniques have been simplified by the use of the “Api system”.
Soil yeasts are spread by wind and insects. Fruit flies and bees play a predominant role in their dissemination. Yeasts are present on vines from the start of ripening. The populations reach a maximum at the time of maturity. Yeasts are not numerous on stems and leaves where they form a pseudomycelium. They colonize all exudates of the openings of stomata and injuries on the surface of the grapes. Growth of cells stops at the point of contact with the cuticular wax. On scanning electron photomicrographs yeast cells can be recognized by their shape, the absence of ornamentations of their surface, and by their bud scars.
condition of the vintage, and climatic conditions, mainly the temperature. The effect of the grape variety is minor certain chemicals applied to fight various parasites of the vines can also effect their distribution.
Each medium is solidified by the addition of an equal volume of 2% agar solution.
On grapes one finds essentially molds, the yeast Aureobasidium pullulans, and yeast species with oxydative metabolism (Rhodotorula) or with little fermentation activity. Among the latter, Hanseniaspora apiculata and its imperfect form, Kloeckera apiculata predominate (99% of the isolated yeasts). Metschnikowia pulcherrima, Pichia membranaefaciens, and Hansenula anomala are less frequent; and other species may occur fortuitously.
All studies confirm the extreme rarity of the occurence of Saccharomyces cerevisiae on grapes. The essential yeast microflora on other supports (beams,vaults,cellar walls) consists of Hanseniaspora uvarum, Hansenula anomala, Metschnikowia pulcherrima, Pichia fermentans, and P.membranaefaciens.
During subsequent manipulations of grapes the yeasts with a strong fermentative capacity develop preferentially and invade the raw material.
In white wine production a selective effect in favour of S.cerevisiae is due to the elimination of the pomace, the clarification of the must (which decreases the total population substantially), as well as to sulfiting of the crushed grapes or the must.
During the first hours of the fermentation one encounters large populations of Hanseniaspora uvarum and Kloeckera apiculata which are rapidly superseded by Saccharomyces yeasts. The species Torulopsis stellata seems to be characteristic of certain regions with”noble rot”(Sauternais). All authors agree on the preponderance of S.cerevisiae several days after the onset of the spontaneous fermentation. S.bayanus often considered the “finishing yeast”exists–often from the start-with S.cerevisiae. It is favoured by the presence of residues from some chemical treatment of the vine.
Under particular circumstances one can induce the growth of Schizosaccharomyces or of Saccharomyces rosei.
In wines, populations of Zygosaccharomyces, saccharomycodes ludwigii, and (as surface growth) species belonging to the genera Pichia and Candida can predominate accidentally. The growth of certain strains of Saccharomyces bayanus as film forming yeasts has been considered for the production of special wines.
2. Industrially important yeasts
The principal yeasts fermenting must and responsible for biochemical changes in wines can be identified on the basis of a limited number of criteria (morphological and physiological). The effect of their occurrence on the essentials of wine quality is known. For the sake of simplicity LODDER has combined in one species some yeasts closely related on the basis of some physiological characteristics, however, distinct on the basis of their interest for enology.
Saccharomyces cerevisiae (syn.: S. cerevisiae var. ellipsoideus, S. ellipsoideus, S.vini). This is the wine yeast par excellence. 80% of the yeasts isolated from fermenting musts in the Gironde belong to these species, as well as 98% of the spices identified at the end of the fermentation. Its shape is globular. The yeast starts fermentations rapidly and has a good resistance to ethanol (8–15 vol%). It disappears rapidly from wines during storage. Certain so called aromatic strains have been selected for their ability to form higher concentrations of esters and higher alcohols. According to Maugenet such yeast can modify the organoleptic properties of wines made from less aromatic vintages.
Saccharomyces bayanus (syn.: S.oviformis, S.beticus, S.cheriensis, S.rouxii). This is the finishing yeast thanks to its capacity to form high concentrations of alcohol, up to 19 vol%. It is found mainly towards the end of the fermentation. The use of S. bayanus has been prescribed for the refermentation of residual sugars in stuck fermentations and for the inoculation of musts from over-ripe grapes. However, its resistance to SO2 (up to 100mg/L) is the reason for almost all of the re-fermentations of sweet, white wines by this yeast. In addition it has the disadvantage of forming relatively important concentrations of acetic acid in the course of the alcoholic fermentation. Recent taxonomic studies by Belin include S.bayanus in the species S.cervisiae. It seems to us that the quite distinct physiological properties of the two species, notably the ability of S. bayanus to grow in films and is inability to degrade galactose, justify the maintenance of two separate species.
Cultures of S. beticus and S. cheriensis are used in the production of sherry. They develop on the surface of the base wine (fortiffed with alcohol) because of their respiratory metabolism of ethanol. They liberate large concentrations of acetaldehyde and esters and degrade acetic acid.
Saccharomyces rosei (syn.: Torulasporarosei). This is a sphercial yeasts of slow growth. It is found on grapes and in musts undergoing spontaneous fermentation particularly in hot climates. Its alcohol producing capacity varies from strain to strain, but is generally quite feeble (6-10 vol%) which explains its absence at the end of the fermentation. The fermentation of acetic acid by S. cerevisiae increases notably at low pH values and at elevated sugar concentrations. In contrast S. rosei produces only small concentrations of acetic acid (0.04g/L) regardless of the composition of the medium. This property can be utilized in the technique of successive fermentations to lower the volatile acidity of wines from grape musts which are too acid. At the end of the growth phase of S. rosei (4th day of the fermentation) one inoculates with a strain of S. cerevisiae which ferments all of the remaining sugar. More recently, use of this yeast has been suggested for the vinification of over-ripe grapes which have been invaded by Botrytis cinerea. The lowering of the concentration of acetic acid in such successive fermentation is the result of two phenomena: (a) S.rosei produces little acetic acid; (b) its growth produces conditions which decrease the rate of the glycero-pyruvic fermentation during the succeeding fermentation by S.cerevisiae or S.bayanus in this medium. Saccharomyces capensis is closely related to S.rosei in its physiological properties.
Saccharomyces uvarum (syn. S.carlsbergensis). Frequently found in beer worts this yeast is also found in musts. Its alcohol producing ability is intermediate. Its growth is readily inhibited by actidione. A certain number of strains of S. uvarum can be found among yeasts capable of liberating small quantities of SO2 during the fermentation.
Saccharomyces chevalieri (syn. S.fruturum, S.lindneri). this yeast approaches S.cerevisiae in its physiological properties, and it is frequently found in fermenting musts. It is distinguished by its inability to ferment maltose.
Saccharomycodes ludwigii. The large cells of this apiculate yeast can attain a length of 25 mm and show bi-polar budding. A single species is known. Rarely seen on grapes this yeast occurs sometimes in sulfited musts. It produces up to 16.8% vol% ethnol and is highly resistant to SO2 (600 mg/L). It is strongly acetogenic and can produce up to 200 mg/L of acetaldehyde during anaerobic fermentations. 70% of the cultures of T. stellata isolated by Domercq in the Gironde come from areas with “noble rot”, and it also occurs frequently in Italian vineyards. It occurs side by side with Hanseniaspora uvarum during the first days of the spontaneous fermentation. It relatively feeble alcohol producing capacity (on the average 10 vol% EtOH) accounts for its disappearance towards the end of the fermentation. This osmophilic yeast also tolerates relatively high temperatures (30-35°C). It ferments fructose faster than glucose.
Hanseniaspora uvarum and its imperfect form Kloeckera apiculata. The small cells are lemon shaped, and may also be recognized by their bippolar budding. They constitute about 95% of the microflora of grapes. Although these yeasts are responsible for the spontaneous fermentation of fresh musts, they are quickly supplanted by Saccharomyces yeasts because of their low alcohol tolerance (3.7-6.4 vol%). They form important concentrations of acetic acid, 1 g/L on the average, and ethyl acetate, 125-374 mg/ L as well as amyl acetate and glycerol. The desirability of these species is controversial. Some authors think that they produce in wine a particular fruity aroma. Others consider them undesirable because of the formation of excessive concentrations of ethyl acetate. A technique of vinification called “la fermentation superquatre” has been suggested for their elimination. Their sensitivity to SO2 explains their small number in sulfited musts, that is, in the majority of cases.
Hansenula. This genus include 25 species. Hansemula anomala is often found on grapes and it has been isolated from musts in many wine growing areas. On a liquid medium this species develops a wrinkled film. The ascospores are hat shaped. Its fermentation capacity is small, and it liberates contaminates poorly maintained cellers and is one of the most feared spoilage yeasts. Its development in wine bottles is characteristic.
An amino acid is generally defined as a compound that possesses amino and carboxyl groups. Some amino acids, however, are iminocarboxylic acids, such as proline, and others are aminophosphonic acids. In nature, there are about 20 amino acids which form a huge variety of complex copolymers, the proteins; 8 of them, the essential amino acids, are required for human growth. All the amino acids are L-a-amino (or imino) carboxylic acids except glycine, which is achiral. In addition to these proteinaceous amino acids, various non-protein amino acids containing D-amino acids and w-amino acids occur naturally in free and bound forms, and play important roles in metabolism. Amino acids are of importance not only as nutrients, but also as seasonings, flavorings, and starting materials for pharmaceuticals, cosmetics and other chemicals.
Amino acids may be prepared by isolation from natural materials, by microbial or enzymatic procedures, or by chemical synthesis. The first two procedures give optically active (usually L-) amino acids, whereas the chemical methods in general produce the racemates, and an additional optical resolution step is necessary to obtain optically active amino acids. Originally amino acids were exclusively obtained by the hydrolysis of plant proteins. L-Glutamate was identified as a taste component of Konbu (kelplike seaweed), used for seasoning in Japan and had been produced up to the 1950s from wheat gluten, soybean and corn proteins, and from sweet turnip molasses by separation of the acid hydrolysate. Other amino acids were also obtained from proteins in a similar way. With the increasing demand for various amino acids, chemical procedures were used for their syntheses. For example, in the Dupont process DL-glutamate is synthesized from acetylene with the intermediate formation of acrylic acid ester and the methyl ester of b-formyl propionic acid:
HC = CH CO2 ROH CH2 = CH—COOR CO2 H2
Strecker Synthesis DL-glutamic acid
In the Ajinomoto process acrylonitrile is converted to b-cyanopropionaldehyde and then to DL-glutamate in a high yield:
CH2 = CH – C = N CO2H2 OHC—CH2—CH2—C=N
Strecker Eynthesis DL-glutamic acid
The enzymatic and chemical methods for the optical resolution of DL-amino acids have been developed simultaneously with the progress made in their chemical synthesis.
The investigation into the accumulation of L-glutamate in bacterial cultures by Kinoshita opened the door to the microbial production of amino acids. The amino acid producing microorganisms were improved genetically to yield amino acids more efficiently. The production of amino acids, e.g., L-lysine, using auxotroph and regulatory mutants has been developed to industrial scales. Numerous studies on the microbial production of amino acids have been carried out, and an increasingly large number of optically active amino acids are now produced using bacteria, with the exception of a few, such as methionine.
On the other hand, the enzymatic procedures for amino acid production have been extensively studied, since Kitahara reported the efficient conversion of fumarate and ammonia into L-aspartate with bacterial aspartase. Several amino acids have been produced industrially with microbial enzymes. The microbial and enzymatic methods have their merits and demerits. In the microbial production (amino acid fermentation), amino acids can be synthesized from simple and cheap raw materials such as glucose, acetate or molasses, and ammonium sulfate or urea, but the production is time consuming and the required amino acids have to be separated from the other amino acids formed and from various impurities including microbial cells. The waste may cause water pollution especially when molasses is used as a carbon source. A few amino acids can be synthesized bacterially from biosynthetic precursors produced chemically; L-tryptophan and L-serine are produced effectively from anthranilate and glycine, respectively.
The enzymatic procedure surpasses the microbial one for the following reasons: it is less time consuming and usually more efficient, and does not produce complex impurities and wastes. However, the enzymatic method is not suitable for the production of amino acids from simple materials. The substrates are either precursors, or other chemicals related to amino acids and are generally more expensive in comparison with the starting materials required in the microbial methods. The enzymes used are neither cheap nor stable. Immobilization of enzymes has been developed to diminish these demerits of the enzymatic methods. The microbial, enzymatic and chemical methods have rapidly advanced in competition with each other to supply us with various amino acids.
MICROBIAL PRODUCTION OF AMINO ACIDS
A variety of microorganisms accumulate amino acids in the culture fluid. However, only bacteria have sufficient productivity to warrant the commercial production of amino acids. Since amino acids are essential components of microbial cells and their biosyntheses are teleologically regulated to maintain an optimal level, they are normally synthesized in limited quantities and subject to negative feedback regulation. The importance of “preferential regulation” in amino acid biosynthesis has also been recognized. Therefore, it is necessary to overcome the regulation to achieve overproduction of amino acids. Microbial amino acid over-production can be achieved using the following procedures: 1. stimulation of the cellular uptake of the starting materials, 2. hindrance of the side reactions, 3. stimulation of formation and activity of the enzymes for the biosynthesis, 4. inhibition or reduction of the enzymes concerned with the degradation of the amino acids produced, and 5 stimulation of the excretion of the product into the extracellular space. The above requirements have been attained by introducing mutation techniques.
A. Production of Amino Acids by Wild Strains
Since the isolation of L-glutamate producing strains by Kinoshita a number of bacterial strains producing more than 30 g/ L of L-glutamate from carbohydrates have been reported. These bacteria have been assigned to various genera and species: Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium thiogenitalis and Micro bacterium ammoniaphilum. In their microbiological characteristics all these bacteria resemble the strain of Corynebacterium glutamicum that Kinoshita isolated first: Gram positive, non-spore forming non-motile, coccal or rod like, and all requiring biotin for growth. Most of them can utilize acetate or ethanol as a carbon source for L-glutamate production, but neither methanol nor n-paraffins.
The nutritional requirement for biotin, and the lack or very low activity of a-ketoglutarate dehydrogenase complex are important factors in the accumulation of L-glutamate. SHIIO et al. observed an overproduction of L-glutamate (2.3 g/L) in an a-ketoglutarate dehydrogenase defective mutant of Escherichia coli. having no requirement for biotin. The parent strain could not accumulate L-glutamate, and therefore, a lack of the enzyme was regarded as the prerequisite for L-glutamate production. The presence of suboptimal amounts of biotin (2.5-5.0 µg/L) causing maximum growth led to efficient L-glutamate production, but in the presence of an excess of biotin (25-30 µg/L), L-glutamate was produced poorly, and other compounds such as lactate and succinate were formed. Oleate was found to be a substitute for biotin, not only for bacterial growth, but also for the accumulation of L-glutamate. An oleate requiring mutant of Brevibacterium thiogenitalis accumulated L-glutamate in a biotin-rich medium with a limited supply of oleate. The utilization of b-lactam antibiotics such as penicilin and cephalosporin C and of surfactants or of C16 - C18 saturated fatty acids also allowed the production of L-glutamate even in a biotin-rich medium, e.g., in media containing cane or beet molasses. Biotin, oleate or C16 - C18 saturated fatty acids cause changes in the fatty acid composition of the cell membrane, particularly in the content of oleate and palmitate, resulting in an alteration of the permeability barrier of the cell membrane to L-glutamate. This phenomenon can be fully explained by the action of acetyl-CoA carboxylase, which contains biotin as its prosthetic group. The enzyme participates in the biosynthesis of oleate and of other fatty acids, and is inhibited by C16 - C18 saturated fatty acids. In contrast, the effect of penicillins on the cell permeability to L-glutamate cannot be ascribed to a decrease in the oleate content. After the addition of penicillin many cells swell or elongate. Therefore, the action of penicillin is thought to cause an incomplete biosynthesis of the cell wall, which results in glutamate excretion. On the other hand, the biotin auxotrophs and the oleate auxotrophs cannot be employed for L-glutamate production from n-paraffins. The degradation pathways of glucose and of n-paraffins are different as shown in Fig. 1. The excretion of L-glutamate in the presence of penicillin by n-paraffins assimilating strains is accompanied by the excretion of phospholipids and of UDP-N-acetyl-glucosamine derivative. Nakao isolated a glycerol auxotroph having a defective L-glycerol-3-phosphate; NADP oxidoreductase, from n-paraffins assimilating Corynebacterium alkanolyticum, in which about 40 g/L of L-glutamate is produced from n-paraffins by addition of 0.01% of glycerol in the absence of penicillin. The extracellular accumulation of L-glutamate by the mutant was found to be dependent on the presence of glycerol, and the maximum production was obtained at low levels of cellular phospholipids. These results suggest that the permeability of L-glutamate through the bacterial cell membrane is controlled not only by the cellular content of unsaturated fatty acids, especially of oleate, but also by the cellular content of phospholipids.
Cabbage & Cucumber Processing
From a historical point of view the fermentations of cabbage (Brassica oleracea) and cucumber (Cucumis sativus) had their inceptions in the Far East, notably China and India. As civilization began to develop and expand into new hemispheres, the art of using lactic acid fermentations became firmly entrenched as an ideal method for preserving fruit and vegetable products.
The use of acidic fermentations by both the home and commercial food producer has been perpetuated because properly fermented products possess distinct and unique flavor characteristics. They are incapable of supporting the growth of microorganisms of public health significance and furthermore, the method permits commodities to be stored for prolonged periods of time without seriously impairing the physical and nutritional qualities of the product.
The spectrum of horticultural commodities that undergoes acidic fermentations is quite extensive (green beans, beets, brussel sprouts, cabbage, carrots, cauliflower, celery, cucumber, olives, onions, peppers, green tomatoes, turnips etc.); however, only a few of these commodities are consumed in quantities of sufficient magnitude, to warrant their production on an extensive industrial scale. For example, The United States only three commodities (cabbage, cucumbers and olives) provide significant contributions to the overall production volume of the fermented food industry.
The transformation of shredded cabbage to sauerkraut is, from a mechanical point of view, a very simple operation; however, from a biochemical and microbiological viewpoint the fermentation is enveloped in an array of complexities.
A schematic diagram depicting the steps involved in producing commercial sauerkraut is shown in Fig. 1, and the functions of the respective operations will be discussed in the ensuing paragraphs.
B. Cabbage Varietals
1. Crop Distribution
Cabbage used for sauerkraut production is generally considered to be a “cold crop”, i.e., its hardiness, proper development and maturity occur under those climatic conditions found within the geographical latitudes of, or equivalent to, those associated with the Northern United States. This area is comprised chiefly of the states of New York, Ohio, Michigan, Wisconsin, Colorado, and Oregon. More than 200000 tons (metric) of fresh cabbage for sauerkraut are harvested annually, and nearly 60% of this yield is produced in New York and Wisconsin.
2. New Hydbrids