CHEMICAL AND CYTOLOGICAL CHANGES
DURING THE AUTOLYSIS OF YEASTS
A thesis submitted to The University of New South Wales as fulfilment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
by
Tatang Hernawan
Sarjana Farmasi, Apoteker (I T B, Indonesia) Master of Applied Science (UN SW, Australia)
Department of Food Science and Technology The University of New South Wales Kensington, N. S. W. Australia
February 1992 D E C L A R A T I O N
The candidate, Tatang Hernawan, hereby declares that this thesis is his own work and that, to the best of his knowledge and belief, it contains no material previously published or written by another person nor material which to substantial extent has been accepted for the award of any degree or diploma of a university or other institute of higher learning, except where due acknowledgement is made in the text of the thesis.
T a t a n g H e r n a w a n A C K N O W L E D G E M E N T S
I wish to express my sincere gratitude to Associate
Professor G. H. Fleet, Department of Food Science and
Technology, University of New South Wales, Australia, as my supervisor for his guidance, advice and encouragement during this project and thesis production.
I would like to acknowledge the assistance of laboratory staff of the Department of Food Science and
Technology, Mr P. Mark, of the Electron Microscopy Unit, and Ir E. Poerwanto, a fellow postgraduate student,
University of New South Wales.
I would like to thank fell ow students in the
Department of Food Science and Technology for a friendly situation throughout this study and Ms H. N. Ivan for helping me during studies in Australia.
Sincere thanks are also given to my father, Mr
H. Sadeli, during his life, and my mother, Mrs H. Oyoh
Sadeli for their strong encouragement and support.
To my children, Rogydesa and Bethasari, and my wife, Ora Ny. Nila Merli ta, I would like to thank them for their support during this study.
I would like to thank the Commonwealth Government of Australia for granting me a Colombo Plan
Award, and the Department of Food Science and Technology,
University of New South Wales, and the Department of
iii Pharmacy, Bandung Institute of Technology, Indonesia, for facilitating my PhD studies.
iv T A B L E 0 F C O N T E N T S
Page
ACKNOWLEDGEMENTS ...... iii
TABLE OF CONTENTS ...... v
ABSTRACT ...... ix
1. INTRODUCTION ...... 1
2. LITERATURE SURVEY ...... 6
2.1 Autolysis - General Description ...... 6
2.2 Subcellular Organisation Of Yeast ...... 7
2.2.1 Cell wall...... 7
2.2.2 Cytoplasmic membrane ...... 11
2. 2. 3 Organelles ...... 12
2.3 Cytological Changes During Autolysis Of Yeast 16
2.4. Biochemical Changes During Autolysis ...... 19
2.4.1 Carbohydrates ...... 19
2.4.2 Proteins ...... 22
2. 4. J Nucleic acids ...... 34
2.4.4 Lipids ...... 38
2.4.5 Vitamins ...... 50
2.5 Commercial Significance Of Yeast Autolysis . 51
2.5.1 Leavening power of compressed baker's yeast ...... 52
2.5.2 Autolysed yeast extracts 52
2.5.J Industrial fermentations 53
3. MATERIALS AND METHODS ...... 57
3 .1 Yeast Strains And Culture ...... 57
3. 2 Autolysis Of Yeast Cells ...... 59
V 3.3 Cell Viability ...... 59
3.4 Cell Dry Weight; Soluble Autolysate ...... 59
3.5 Total Carbohydrate, Reducing Sugar. And Glucose...... 60
3. 6 Organic Acids ...... 61
3.7 Protein 62
3.8 Amino Acids 62
3.9 Ribonucleic Acid 64
3.10 Deoxyribonucleic Acid ...... 66
3.11 Lipids ...... 68
3.12 Free Fatty Acids ...... 70
3 .13 Glycerol ...... 71
3.14 Cytological Observations ...... 72
4. RESULTS ...... 75
4.1 Comparative Survey Of Autolysis In Several Species And Strains Of Yeasts ...... 7 5
4.1.1 Introduction ...... 75
4.1.2 Results ...... 75
Cell viability 75
Solubilization of cell biomass ...... 76
Reproducibility of data on dry weight analysis ...... 78
Carbohydrates 78
Organic acids 78
Protein 84
Amino Acids 84
Ribonucleic acid 88
Deoxyribonucleic acid ...... 92
Lipid 94
vi Free Fatty Acids ...... 104
4.2 Chemical Changes During The Autolysis Of Saccharomyces cerevisiae 2180a, Kloeckera apiculata 202 And Candida stellata 8008 ..... 104
4.2.2 Introduction ...... 104
4.2.3 Results ...... 104
Cell viability 104
Solubilization of cell biomass ...... 106
Carbohydrates 106
Organic acids 111
Protein 114
Amino acids 114
Ribonucleic acid (RNA) 119
Deoxyribonucleic acid (DNA) 119
Lipids ...... 124
Glycerol ...... 130
4.3 Effect Of Cell Concentration On The Kinetics Of Yeast Autolysis ...... 130
4.3.1 Introduction 130
4.3.2 Results ...... 134
Effect of cell concentration on the kinetics of cell death during autolysis. 134
Effect of cell concentration on the kinetics of cell weight changes and cell solubilization during autolysis ...... 134
4.4 Observations Of Yeast Autolysis With The Electron Microscope ...... 136
4. 4 .1 Introduction ...... 136
4.4.2 Results ...... 139
Scanning electron micrographs of auto- lysing yeasts ...... 139
Transmission electron micrographs of thin sections of autolvsing yeasts ..... 139
5. D I S C U S S I O N ...... 149
vii 5.1 Cell viability 150 5.2 Solubilization of cell biomass ...... 152
5.3 Carbohydrates 154
5.4 Organic acids 156 5.5 Proteins and Amino Acids ...... 159 5. 6 Nucleic acids ...... 164 Ribonucleic acid 164
=D~e~o~x-y~r~i=b~o~n~u=c-=l=e=i~c---aaa~c~i____ d ...... 166
5.7 Lipids ...... 168
5.8 Cytological changes ...... 173
6. CON CL US ION ...... 176
B I B L I O G R A P H Y ...... 182
viii ABSTRACT
The chemical and cytological changes that occurred during the
autolysis of three yeasts, Saccharomyces cerevisiae, Kloeckera
apiculata, and Candida stellata, were examined. The yeasts were
grown in yeast extract glucose broth for 48 h, after which the
cells were harvested and autolysed by incubating in 0.1 M
phosphate buffer, pH 4.5, at 45"C for up to 10 days. Three
strains of each species were examined.
Autolysis was characterised by rapid loss in cell
viability and solubilization of cell biomass. After 10 days,
25-35 % of the cell dry weight was solubilized. The soluble
autolysate consisted of carbohydrate (3-7 %), organic acids (3-6
%), protein (12-13 %), free amino acids (8-12 %), nucleic acids
(3-5 %) , and lipids (1-2 %) , Carbohydrate in autolysates was
predominantly polysaccharide with only traces of glucose or
reducing sugars. The main organic acids in autolysates were
propionic, succinic, oxalic, acetic, formic, and malic, with
some variation in their individual concentrations depending on yeast species. Sixteen different free amino acids were found in
autolysates, the main ones being phenylalanine, glutamic acid,
leucine, alanine, and arginine. The concentrations of
individual amino acids varied with yeast strain. Cellular RNA was 85-90 % degraded during autolysis with most of the degradation products appearing in the autolysates. Cellular DNA was only partially degraded and recovered in the autolysates.
Approximately 40 % of cell lipid was lost during autolysis and
recovered in autolysates. Both phospholipid and neutral lipid
classes were degraded, with neutral lipids but not phospholipids being found in autolysates. The composition of cellular lipids
as well as degradation of the different lipid classes varied
with the yeast species. Autolysates contained 0.2-0.6 %
glycerol.
Scanning electron microscope and transmission electron
microscope observations showed the cell shape and cell wall to
remain intact during autolysis, although a decrease (13-14 %) in wall thickness was noted. After 5 days of autolysis, there was
a retraction of the cell membrane from the cell wall, and some mernbraneous vesicularization. Such cytological changes were
less evident in K. apiculata.
There were minor variations in autolytic behaviour between strains of one species. The autolytic character of K.
apiculata showed significant differences from the other two
species.
X 1
1. INTRODUCTION
Autolysis or self degradation is a natural event that occurs after yeast cells have completed their normal growth cycle and have entered tt.e death phase. In general terms, it is characterized by a loss of membraneous function and organization, degradation and solubilization of cell macromolecules by th~ action of endogenous enzymes and the release of intracellular constituents and br_eakdown products into the extracellular environment.
The phenomenon of autolysis in baker's yeast was first observed by Salkowski at the end of the 19th century (see
Farrer (1956) for historical review), and since that time it has attracted the sporadic interests of scientists. Of potential commercial interest, were the findings that yeast autolysates were rich in amino acids, proteins, vitamins, and ctegradation products of nucleic acids, and that the autolytic process could be significantly accelerated or triggered by incubating yeast cells at high temperature, by addition of salt, or by treatment with various organic solvents, such as, toluene, chloroform, or ethanoJ.
The commercial implications of yeast autolysis are varied and have been recognized for a long time.
Indeed, they have attracted more attention than endeavours to understand the scientific basis of the process. Yeast autolysates, which are rich in nutrients 2
and strong in flavour profile, quickly found application
as ingLedients in the food processing industries (Peppler
1982, Dziezak 1987ab). Autolysis was also seen as a
process for preparing proteins, polysaccharides and
pigments from various yeasts for use in the food and feed
industries. Several enzymes, such as invertase and ~- galactosidase, also used in food processing, were prepared from yeast autolysates (Peppler 1982). On the negative side, it was quickly realized that autolysis was
the process that decreased the leavening power of baker's yeast (Saccharomyces cerevisiae) during storage and that uncontrolled autolysis of brewer's and wine yeasts would lead to of £-flavours and bacterial spoilage problems in beer and wines. Properly controlled, however, yeast autolysis could be used to benefit in wine and beer fermentations (Arnold 1981b, Masschelein 1986). These various commercial implications highlight the need to better understand and control the autolytic process.
The chemical and biochemical changes that occur during yeast autolysis have been studied by numerous researchers throughout the past 75 years, and have been reviewed by several authors (Joslyn 1955, Farrer 1956,
Hough an~ Maddox 1970, Arnold 1981b). Nearly all the studies have been u~dertaken with one yeast species, namely, Saccharomyces cerevisiae because of its commercial significance, and information about the autolytic behaviour of other yeasts is very limited. 3
The most evident and best studied change that
occurs during the autolysis of S. cerevisiae is the breakdown of intracellular protein and accumulation of
amino acids, peptides, and proteins in the extracellular
autolysate. There is little doubt that proteolytic
enzymes are responsible for these changes. The breakdown of cell nucleic acids, principally RNA, is also well demonstrated with the degradation products (bases, nucleotides, etc.) appearing in the autolysates. The fate of DNA, which represents only a small percentage of the total nucleic acid, during autolysis is not clear. While some authors have reported its degradation (Hough and 1"~c. Maddox 1970) others have not found this to be1 case (Ohta et al. 1971, Trevelyan 1976, 1977). The fate of cell polysaccharides, principally those of the cell wall, during autolysis is not resolved. Many cytological studies have shown that the cell wall remains largely intact and entire at the end of autolysis. Nevertheless, it is believed that some changes must occur in its porosity because intracellular macromolecules can be found in autolysates. There is accumulating evidence that the degradation of lipids plays a key role in the regulation of autolysis. Loss of membrane structure, organisation and function is considered to be a
triggering event of autolysis. Such changes are needed to overcome the barriers of compartmentalisation and permit macromolecular substrates to come in contact with their
specific hydrolytic enzymes. Altered permeability of the
cytoplasmic membrane or plasma membrane to allow movement 4
of autolytic products out of the cell, also suggests
changes in lipid composition. However, despite the
significance of lipids in the autolytic process their
fate during autolysis has not been well characterized
{Arnold 1981b).
Al though important progress has been made in describing the chemical changes that occur during yeast
autolysis, the research to date has been, to a large
extent, uncoordinated. Most studies have focussed on the
autolytic degradation of proteins, a few have examined
the degradation of nucleic acids and even fewer have considered the autolytic fates of lipids or carbohydrates. In some studies, autolysis was induced by high temperature incubation of the cells, while in others organic solvents or salt was used to induce the reaction.
The autolytic fate of the major macromolecules, as measured in the one experiment under the one set of conditions has not been determined, so that it is not possible to correlate the kinetics of degradation for the different cellular components. Such information would be important in determining the sequence of events that occurs during autolysis and, therefore, in developing a better biochemical understanding of the overall reaction and its control. As noted already, nearly all studies on yeast autolysis have been conducted with S. cerevisiae, and virtually nothing is known about the autolytic behaviour of other yeast species. 5
As part of a larger researc!'l program to. gain fundamental biochemical knowledge about yeast autolysis, this thesis has the following aims:
( i) to examine cell degradation
(solubilization) during autolysis;
(ii) to determine under controlled, comparative conditions the degradation of proteins, carbohydrates, nucleic acids (DNA and RNA) and lipids during autolysis, and associated cytological changes;
(iii) to compare the autolytic behaviour of Saccharomyces cerevisiae, Kloeckera apiculata, and Candida stellata.
The last two species have been chosen because of their importance in wine fermentations and the potential contribution of their autolytic reaction to wine quality { Fleet 1990a) . As noted already, virtually nothing has been reported about the autolytic behaviour of yeasts other than S. cerevisiae. 6
2. LITERATURE S U R V E Y
2.1 Autolysis - General Description
Autolysis is a term that is used to describe the breakdown of cell constituents as a result of the activity of endogenous enzymes. It occurs naturally in yeasts when they have completed their growth cycle and have entered the death phase. In general terms, this process of "self-destruction", "self-digestion", or self solubilization" is characterized by an 1/']crt!ue "" cell permeability, degradation of the cell macromolecules and the release of intracellular constituents and breakdown products into the extracellular environment. Apart from cell death and ageing, several external factors are known to induce or trigger yeast autolysis. These include incubation of the cells at 45-50 °c, exposure to organic sol vents or high salt concentration, disruption of the cell wall by lytic enzymes and mechanical disruption of the cells.
Autolysis was first observed in yeasts by
Salkowski in 1889 who noted the accumulation of sugar and amino acids in the supernatant of stored baker's yeast.
Since that time, numerous authors have noted the
autolysis of yeast cells and have correlated this property with the release of proteins, amino acids, nucleic acids, nucleic acid bases, vitamins and other
substances into the extracellular environment. In addition, the role of enzymes, especially proteases, in 7
this process has been firmly documented. The early
studies on yeast autolysis as well as interesting
accounts of the historical development of knowledge on
the subject have been reviewed by Schryver et al. (1927),
Joslyn (1955), Joslyn and Vosti (1955) and Farrer (1956).
More recent accounts of yeast autolysis are those of
Hough and Maddox (1970), Arnold (1981b), and Babayan and
Bezrukov (1985).
2.2 Subcellular Organisation Of Yeast
Autolysis involves breakdown of yeast organelles. This section provides general background information on the subcellular structure and composition of yeasts. Unless indicated otherwise, the information presented relates only to S. cerevisiae as few other yeast species have been studied in this context. The reader is ref erred to the review of Robinow and Johnson ( 1991) arid chapters within the book "The Yeasts. Yeast Organelles" ( Rose and
Harrison 1991) for more detailed information on this subject.
2.2.1 Cell Wall
Cell wall composition and structure have been reviewed by
Fleet ( 1991) . The wall represents 15-25 % of the dry weight of the cell and consists mostly (approximately 80-
90 %) of polysaccharides and about 10 % of protein.
Electron microscope studies show the wall as an outer 8
envelope about 100-400 nm thick, that consists of three
layers of different electron density (Lloyd and Cartledge
1991, Robinow and Johnson 1991). Although somewhat
flexible, the wall provides rigidity and shape to the
cell, and maintains the integrity of the cell protoplast
against the osmotic differential of the external environment. Apart from this basic purpose, the wall also has a number of other important properties. It serves as a site for the specific reception of some macromolecules, such as killer toxin, mating factors, and antibiotics. It has a certain charge and porosity, and it has a limited range of enzyme activities.
The polysaccharides of the wall are comprised of glucan (30-60 %) , mannan (25-50 %) and chitin (1-2 %) .
Three different glucan molecules have been reported to occur within the wall. The main glucan is an alkali insoluble - acid-insoluble polysaccharide that represents about 35 % of the wall and is responsible for cell shape and rigidity. It is a slightly branched molecule, consisting of glucose residues connected by (1,3)-~ linkages. Branching (about 3 %) occurs through the (1,6)
~-linkage. An alkali-soluble glucan, represents about 20
% of the wall and also contains a preponderance of (1,3)
~-linkages and a small percentage ( 3-4 %) of branching through (1,6)-~-linkages. It differs from the alkali insoluble glucan in that it also contains a percentage
(8-12 %) of glucose residues that are connected by (1,6)
~-linkages. The function of this glucan is not clear, but 9
it could serve as a ·matrix that permeates the wall. It may be covalently linked to the mannoprotein of the wall, serving as an anchor for that principal wall component.
The third glucan component represents about 5 % of the wall. It is highly branched and is predominantly linked by (1,6)-~-bonds. Its function is not known, but in some strains it may act as a receptor site for killer toxins
(Fleet 1991).
The bulk of the wall mannan is covalently linked to protein and is more correctly ref erred to as mannoprotein. It is mainly located on the outer surface of the wall where it serves as a receptor site and contains the antigenic determinants of the yeast cell.
Some enzyme activities are associated with the mannoprotein. Mannoprotein consists of about 90 % mannose and 10 % protein. Depending on the strains, small but varying amounts of phosphorus (0.1-1 %) are also present.
Mannose is mainly connected to the protein by a double N acetylglucosamine unit through asparagine. A small amount
(about 10 %) of mannose, in the form of oligosaccharides, is connected to protein by O-glycosyl bonds through the hydroxyl groups of serine and threonine. The mannan polysaccharide linked through asparagine, consists of about 250-260 mannose residues of which about 70 units are linked by a-(1,6)-bonds, forming a backbone to which oligosaccharide side chains of mannobiose, mannotriose and mannotetraose are attached. The mannose residues of the side chains are linked by a-(1,2)- and a-(1,3)-bonds, 10
and some of the residues may also contain phosphorus
(Fleet 1991).
Chi tin in S. cerevisiae comprises about 1-2 % of the wall and is mostly located in the region of the bud scar. Only a small portion (about 10 %) may be located outside the budding area. It is a linear polysaccharide consisting of residues of N- acetylglucosamine linked by 13-(1,4) bonds. These chains may be associated through hydrogen bonding to give very insoluble complexes. With current cell wall extraction techniques, chitin is always contaminated with glucan and mannan, and there is some evidence that chitin may be covalently linked to these other wall components (Fleet
1991).
Protein represents 5-15 % of the wall, but this content varies with the species and conditions of growth and depends on the purity of the wall preparation as well as the degradative activities of proteases during wall preparation. Wall protein is a complex of molecules that serve several functions including (i) cross-linking of wall polymers because part of the protein is covalently linked to mannan and possibly other macromolecules such as glucan (ii) enzymatic, as some enzymes that cleave nutritional substrates (e.g. invertase) , and as others which participate in the turnover of wall polymers during cell morphogenesis, and (iii) some protein molecules serve as receptor sites and agglutination factors in cell-mating interactions (Fleet 1991). Of the cell wall 11
located enzymes, the (1,3)-~-glucanases and proteases
could be active· during autolysis (Charpentier and
Freyssinet 1989)
Lipids represent 2-14 % of the wall in S.
cerevisiae. This content may be higher or lower with
other species. However, there is some doubt as to whether
lipid is a true wall component, as it is difficult to
prepare walls without contamination with cytoplasmic
membrane. Also, no biological function has yet been
proposed for any wall lipid. Only glycerides
predominantly composed of unsaturated palmi toleyl and
oleyl residues have been reported to be the major lipids
in S. cerevisiae walls, while phospholipids r1nd sterols
are absent (Arnold 1981a, Fleet 1991).
2.~.2 Cytoplasmic membrane
The cytoplasmic membrane which is sometimes called the plasmalemma or plasma membrane or cell membrane is a barrier separating the aqueous interior and exterior of
the cell. Its function is { i) to control the uptake of nutrients and other external substances into the cell, and the movement of cell constituents to the external environrne11t, (ii) to facilitate cell wall synthesis, and
(iii) to form an expandable cover and protective barrier
for the protoplast (Matile et al. 1969, S:.iornalainen and
Nurminen 1976, Kokova-Kratochvilova 1990, Henschke and
Rose 1991). 12
The cytoplasmic membrane comprises 10-20 % dry weight of the cell (Kokova-Kratochvilova 1990) and consists predominantly of proteins and smaller portions of lipid (24 %) and carbohydrate (17 %) (Arnold 1981a).
This lipid consists mainly of neutral lipid (92 %) and a small amount of phospholipid (8 %) • The dominant phospholipids are phosphatidylethanolamine, phosphatidyl choline, phosphatidylinositol, and phosphatidylserine.
Phosphatic acid was also reported, but only in small amounts (Arnold 1981a, Henschke and Rose 1991). The neutral lipid consists mostly of triacylglycerol ( 36 %) followed by sterol esters (28 %) that contain mainly the esters of zymosterol, ergosterol and dehydroergos terol.
Free sterols contribute 13 % of the neutral lipid. The main free sterol is ergosterol (Arnold 1981a). The most prevalent fatty acids within the lipids are palmitic and stearic acids (saturated acids) and palmitoleic and oleic acids (unsaturated acids). Free fatty acids and others are present in only small (< 23 %) amounts (Arnold
1981a).
2.2.3 Organelles
Nucleus
Aspects of the yeast nucleus have been reviewed by
Williamson (1991). Each cell contains a nucleus that is roughly spherical in shape and 2 µm in diameter. The nucleus is separated from the cytoplasm by a porous 13
nuclear membrane which is a double membrane composed of two unit membranes separated by a perinuclear space. The nucleus contains the chromosomal DNA which, in the haploid state, composes about 14 Mega base pairs (Mbp) .
Haploid strains of S. cerevisiae possess 16 chromosomes that range in size from 200 kilo base pairs (kbp) to over
2 Mbp. The DNA of the nucleus is complexed with basic proteins (protamines and histones) to form chromatin.
Some RNA as well as enzymes are also found in the nucleus (Williamson 1991).
Vacuoles
Vacuoles are highly conspicuous within the yeast cell on observation by light and electron microscopy. They form part of the internal membraneous network within the cell.
Each cell usually contains one large vacuole, mostly spherical in shape, and several smaller ones. Their diameter ranges from 0. 3-3 µm and they are bound by a single unit membrane (Kockova-Kratochvilova 1990) . The functions of the vacuoles within the cell are emerging to be very complex and significant and are discussed in detail by Schwencke (1991). There is little doubt that the vacuoles are organelles of metabolite storage and are organelles in which a diverse range of hydrolytic enzymes including proteases, lipases, and nucleases are located.
Consequently, they appear to serve a major function in the recycling of cell macromolecules. However, they also 14
seem to be involved in a range of other activities including transport and secretory phenomena.
Mitochondria
Mitochondria occur as membraneous organelles that are distributed throughout the cytoplasm. They are usually
0.3-1 µm wide and 1 µm long and occur in varying numbers.
Diploid cells of S. cerevisiae, for example, contain upto
29 mitochondria. They are comprised of an outer smooth membrane and an inner membrane which has a granular appearance and is folded to form lamellae or cristae that protrude into the inner space of the organelle. Both membranes are essentially lipoproteins and are the sites for location of the enzymes of the citric acid cycle and of cytochromes, whose prime function is the generation of energy (ATP) for the cell (Kockova-Kratochvilova 1990).
Endoplasmic reticulum
The endoplasmic reticulum is a system of double membranes found within the cell cytoplasm. Some of these membranes are attached with dictyosomes or are connected with the outer nuclear membrane or plasma membrane. The outer surface of endoplasmic reticulum is more granular, while the inner surf ace is smooth. The endoplasmic reticulum is the main site for the synthesis of peptides and proteins (Kockova-Kratochvilova 1990). 15
Ribosomes
Ribosomes are globular organelles of 20-30 nm in size, and are associated with the outer mitochondrial membrane, the endoplasmic reticulum, and the outer nuclear membrane but not with the plasma membrane or the vacuolar membrane. The ribosomes are characterized by the Svedberg sedimentation constant of 70S or 80S. The cytoplasmic ribosomes are 80S and are often found in groups of polysomes that are linked with messenger RNA (mRNA) strands. However, ribosomes themselves contain ribosomal
RNA (rRNA) and are associated with protein units. The main function of the ribosomes is protein biosynthesis
(Kockova-Kratochvilova 1990, Lee 1991).
Golgi body
The Golgi apparatus is an organelle which is commonly found in normal growing cells, and occurs as flattened cisternae with vesicles sloughing off at the opposite poles of the cisternae. This organelle appears to function in cell wall synthesis (Kockova-Kratochvilova
1990) .
Microbodies
Microbodies are organelles, measuring O. 2-1. 5 µm, that are frequently associated with strands of endoplasmic reticulum, and contain a fine granular proteinaceous matrix and also may contain crystalline repertoires. 16
Depending on their function, microbodies may be called as peroxisomes when they act in oxidative metabolism and contain oxidative enzymes catalyzing reactions that produce hydrogen peroxide. Also, they are called glyoxisomes when they contain reserve enzymes of the glyoxylate cycle, namely, lyase and malate synthase
(Veenhuis and Harder 1991).
Cytoplasm
Cytoplasm is naturally bounded by the cytoplasmic membrane at the outer space and the nuclear envelope at the inner space. It contains organelles such as, mitochondria, vacuoles, and the ribosomes, and cytosol which contains the soluble enzymes for glycolysis and other metabolic pathways (Robinow and Johnson 1991).
2.3 Cytological Changes During Autolysis Of Yeasts
The first cytological observations of yeasts < s. cerevisiae) during autolysis were recorded using the light microscope. Joslyn (1955) reported that the response of cells of S. cerevisiae to the Gram stain changed during autolysis, indicating general cytological alteration within the cell. The cells, normally Gram positive, tended to become Gram negative on autolysis, but such observations were not always consistent. However, increasing granulation of the cytoplasmic material along with globule or vesicle formation was 17 consistently observed as the cells underwent autolysis.
Such changes have been confirmed by electron microscopic observations of thin sections of autolysed wine yeasts
(Avakyants 1982, Piton et al. 1988). Avakyants (1982) observed plasmolysis along with detachment of the plasma membrane from the cell wall, followed by plasma membrane breakdown. Piton et al. (1988) examined the autolytic behaviour of cells of S. cerevisiae during the ageing of champagne wine. After six weeks of storage, the cytoplasm of the cells had undergone major transformation that correlated with the development of polysaccharide granules and lipidic vesicles. From the third month onward, all yeast cells were plasmolyzed. The cell membrane had detached from the wall and enclosed the membranous, vesiculated remnants of the cytoplasm, which now appeared very degraded.
As reviewed by Vosti and Joslyn (1954a) and
Farrer (1956), early studies suggested that the cell wall was dissolved during autolysis. However, it is now firmly concluded that this is not the case. Light microscopy observations of extensively autolysed cells of
S. cerevisiae, clearly show the cell wall to remain intact and to retain the basic shape of the cell (Schryver et al. 1927, Joslyn and Vosti 1955, Farrer
1956). However, as pointed out by Farrer (1956) and also noted by Hough and Maddox ( 1970) , such observations do not preclude some basic alteration within the wall structure. In fact, most authors agree that the porosity 18 of -the wall must increase during autolysis in order to allow the diffusion of cell constituents (especially large molecules) to the external environment (Joslyn
1955, Arnold 1981b, Babayan et al. 1981).
The retention of wall structural integrity during autolysis of S. cerevisiae has been confirmed by observation with the electron microscope (Babayan et al. 1981, Charpentier et al. 1986, Piton et al. 1988). Even after 15 years of champagne storage, the walls of the associated yeast sediment remained intact (Piton et al. 1988). However, the endostructure and composition of the wall have been reported to change. Al though Babayan et al. (1981) reported a thickening of the wall during autolysis, this observation has not been supported by the studies of Charpentier et al. (1986) and Piton et al. (1988). Charpentier et al. (1986) noted that the cell wall of S. cerevisiae grown in a synthetic medium decreased by 50 % during autolysis for 14 days, but for the same yeasts grown in wine medium, the wall thickness did not change during autolysis. For cells grown in both media, however, wall composition changed during autolysis. This was characterised by decreases in both the protein and polysaccharide contents, and an increase in the ratio of mannan with respect to glucan. Piton et al. (1988) reported a decrease in thickness of the walls of yeasts during ageing ( autolysis) in champagne. Some layers of the wall were lost on prolonged ageing, in particular the mannoprotein layer. Charpentier et al. 19
(1986) proposed a role of (1,3)-~-glucanases in the alteration of wall structure and composition during autolysis, possibly by degrading some wall glucan, leading to increased porosity of the wa] 1. Loss of the wall mannoprotein, also caused by glucanase action, could increase its porosity {Zlotnick et al. 1984, Fleet 1991).
It is not known if other yeast species follow the same cytological alterations as S. cerevisiae during autolysis. In particular, it is not known if their cell walls remain intact or are disrupted during autolysis.
2.4 Biochemical Changes During Autolysis
2.4.1 Carbohydrates
Carbohydrates comprise upto 40 % of the dry weight of S. cerevisiae {Manners 1971). This carbohydrate is represented mostly by that which forms the cell wall
{principally glucan and mannan, Section 2. 2 .1) and that which forms the storage polymers. The storage polymers are mainly glycogen and trehalose and their content within the cell can vary significantly (15-30 % and 0.4-
15 %, respectively) depending upon the culture environment. The metabolism of storage carbohydrate within yeasts has been reviewed by Manners ( 1971) and
Panek (1991). Several authors have noted that carbohydrates are solubilised during autolysis, but the amounts released were very low compared with nitrogenous components {Hough and Maddox 1970, Babayan et al. 1981). 20
Changes in storage carbohydrates during autolysis.
The early studies on yeast autolysis mentioned the breakdown of cell carbohydrates, and it was suggested that the proteolytic phase of autolysis was preceded by a stage of autofermentation of glycogen reserves within the cell. This behaviour was used to explain the short latent period of several hours ( 8-12 hours) that was observed before the commencement of proteolysis (Vosti and Joslyn
1954, Farrer 1956). There appears to be no further study of the fate of storage carbohydrates during autolysis.
Neither Manners (1971) nor Panek (1991) mentioned the metabolism of either glycogen or trehalose during autolysis in their reviews of yeast storage carbohydrates. Consequently, the behaviour of these storage substances during yeast autolysis remains an open question.
Changes in cell wall polysaccharides
As discussed in Section 2.3, the cell wall of S. cerevisiae retains its structural integrity and shape during autolysis. Nevertheless, electron micrographic studies (Babayan et al. 1981, Avakyants 1982, Charpentier et al. 1986, Piton et al. 1988) suggest a certain degree of alteration to its structure and composition during autolysis. Also, there is general agreement that the porosity of the wall is increased during autolysis, further suggesting some degree of change or degradation. 21
During the autolysis of brewer's yeast, Hough and Maddox (1970) briefly noted that the carbohydrate released during autolysis was in the form of glycoprotein and suggested that· it might originate from degradation of the cell wall. Charpentier and coworkers (Charpentier et al. 1986, Piton et al. 1988) provided the first solid evidence that the chemical composition of the walls changed during autolysis. During autolysis of both S. cerevisiae and S. bayanus, the wall polysaccharidic layers decreased by 20-40 %, the external parts of young cell walls became much thinner, and some wall compounds disappeared. Polysaccharides containing vie-glycol groups and mannoproteins were eliminated. The decrease in wall amino acids was associated with a decrease in wall glucan of cells grown in wine medium. However, the glucans of the cells grown in a synthetic medium were less hydrolysed. Glycoproteins (mannoproteins) were recovered in the autolysates of intact cells.
Charpentier and coworkers (Charpentier et al.
1986, Piton et al. 1988) concluded that (1,3)-(3- glucanases are active during autolysis and degrade part of the cell wall glucan to release mannoprotein components. These observations are consistent with earlier studies by Fleet and coworkers (Fleet and Phaff
1974, Hien and Fleet 1983, Fleet 1991), and Sanz et al.
( 198 5) who found that isolated cell walls of S. cerevisiae as well as other yeasts, underwent autolysis on incubation and that (1,3)-(3-glucanases were active 22
during this process. Arnold (19,2) had also proposed a role of ( 1, 3) -!3-glucanases in the process of yeast autolysis. Subsequent to these observations, many authors have now demonstrated that certain (1,3)-~-glucanases hydrolyse glucan components (possibly the alkali soluble glucan) within the wall to release mannoprotein, without disruption to overall wall integrity and shape (Fleet
1991) . Presumably, such changes also alter the porosity of the wall.
2.4.2 Proteins
Degradation
Proteins represent 40-60 % of the yeast cell dry weight
(Kinsella 1986, Kockova-Kratochvilova 1990). About 10-15
% of this protein is associated with the cell wall (Fleet
1991), the remainder being of intracellular origin.
According to Kinsella (1986), about 50-60 % of the total protein forms a complex with nucleic acid through hydrogen bonding and electrostatic forces. During autolysis cell protein is degraded by proteolytic enzymes to produce polypeptides, peptides and amino acids. The degradation products are commonly estimated as total nitrogen, soluble nitrogen, protein nitrogen, free amino acids and amino nitrogen which are released into the external medium. Measurements of these products are often used as parameters to determine the degree of autolysis, the protease activity and the enzymatic process by which 23
the yeast cells undergo autolysis. ·According to the reviews of Joslyn (1955) and Farrer (1956), as much as
90 % of the cell protein can be solubilized and released into the autolysate during autolysis.
In an early study, Schryver et al. {1927) reported a decrease in the protein content of cells of brewer's yeast that were kept in a loosely-covered dish for a fortnight. This decrease corresponded with the recovery of nitrogenous constituents from washings of the cells. Approximately 25 % of the cell protein had been solubilized after two days. By six days, 36 % of the protein had been solubilized and by 12 days (end of experiment), this value had increased by nearly 55 %. He associated these changes with protein degradation brought about by autolysis.
Vosti and Joslyn (1954) studied autolysis of s. cerevisiae by incubating cell suspensions of commercial baker's yeast in phosphate buffer, pH 5.0, at 53° ± 1° C for 72 h. They followed the kinetics of autolysis by measuring the concentrations of solubilized total nitrogen and of amino acid nitrogen. The percent of cell nitrogen converted to soluble nitrogen (that recovered in soluble autolysate) after 12 hours was 15 %, and after 72 hours this value was 45-50 % • The corresponding values for amino acid nitrogen were 10 % (12 hours) and about 20
% (72 hours). These kinetics of nitrogen solubilization were affected by the type of buffer, buffer concentration, buffer pH, and temperature. Autolysis 24
conducted in citrate buffer gave similar ~mounts of soluble and amino nitrogen to that conducted in phosphate buffer, while that conducted in arsenate buffer gave about 50 % more solubilization of the cell nitrogen.
Moreover, a greater proportion of the total nitrogen occurred as amino nitrogen during autolysis in arsenate buffer. Nitrogen solubilization was about 100 % higher , for autolysis in 0.1 M citrate buffer than in 0.5 M citrate buffer at pH 3-...1-3.2. However this difference was not observed at pH ~-4. 3. The optimum pH for autolysis in O. 5 M citrate buffer was 4. 0 as measured by both total nitrogen and amino acid nitrogen released. The optimum temperature for autolysis was 45 °c, with almost no autolysis occurring at 35 °c. After autolysis in 0.5 M citrate buffer, pH 4.4, at 45 °c for 72 hours, approximately 7 5 % of the total cell nitrogen had been solubilized and about 30 % of this occurred as amino nitrogen (Vosti and Joslyn 1954a). The kinetics of autolysis was further examined by Vosti and Joslyn
(1954b) using several pure culture yeasts. The susceptibility to autolysis varied with the yeast species. Saccharomyces carlsbergensis 421, for example, was most susceptible to autolysis, and released some 80 % of its cell nitrogen after autolysis for 24 hours at 45
0 c. Also, the optimum temperature and optimum pH for autolysis varied with the species. Growth of cultures under aerated conditions compared with non-aerated conditions increased their susceptibility to autolysis. 25
Such variations are illustrated in Table 1 which collate some of the data reported by Vosti and Joslyn (1954b).
Hough and Maddox ( 1970) studied the autolysis of brewer's yeast (Saccharomyces carlsbergensis) after suspension in distilled water at pH 6.5 and incubation at 45 °c for 14 h. Sampling was done at intervals of one hour. After 4 hours, less than 5 % of the cell protein had been solubilised by autolysis. Maximum solubili zation of protein (18 %) had occurred by about 8 hours.
The protein in the autolysate decreased after this time and correlated with an increase in the concentration of amino acids in the autolysate, thereby suggesting proteo lysis of the protein to amino acids.
Trevelyan (1976, 1977) studied the influence of several variables on the kinetics of protein decrease in cells of commercial baker's yeast during autolysis.
Autolysis of cells in succinate buffer at pH 5. 0 and 50
0 c resulted in 8.5 % loss in cell protein after 4 hand
75 % loss after 24 h. When the temperature of autolysis was increased to 60 °c, the rate of loss of cell protein was significantly enhanced. At this temperature, 44 % of the cell protein was lost by 4 h, but further losses in the next 20 hours were only small (increase from 44 % to
58 %) . It was suggested that the higher temperature accelerated the loss of cell structure, thereby permitting a faster exit of protein from the cells (Trevelyan 1976). Heat-shocking of the cells at 70 °c for TABLE 1. A comparison of autolytic properties of some yeast species
Aeration Autolysis buffer Autolysis Total soluble nitrogen Total amino nitrogen Yeast species during cell temperature Recovered Optimum Recovered Optimum growth (degree C) (%) pH (%) pH
Baker's yeasF yes 0.5 M citrate, pH 5.0 53 28 5.0 17 5.0 yes 0.5 M phosphate, pH 5.0 53 25 5.0 17 5.0 yes 0.5 M arsenate, pH 5.0 53 36 5.0 22 5.0 yes 0.5 M citrate, various pHs 53 53 4.0 20 4.0 yesb 0.5 M citrate, various pHs 45 75 4.4 30 4.4
Candida lipolytica 49-49 yes 1.0 M citrate, various pHs 45 61 4.0 19 4.94
Yeast Jerez (Torulopsls sp.) 156 yes 0.5 M citrate, various pHs 45 57 3.98 21 3.98 Saccharomyces carlsbergensis 421 yes 0.5 M citrate, various pHs 45 83 5.01 - - no 0.5 M citrate, various pHs 46 70 4.67 - - yes 1.0 M citrate, pH 4.82 46 62 4.82 - - yes 1.0 M citrate, pH 7.0 46 50 7.0 -- yesc 0.5 M citrate, pH 5.01 45 70 5.01 - - yesd 0.5 M citrate, pH 5.01 45 85 5.01 --
a= Data from Vosti and Joslyn (1954a); other data from Vosti and Joslyn (1954b); cells autolysed for 24 h. b= After autolysis for 72 hours. Cultures grown at pH about 4.3-4.9. c= N d= Cultures grown at pH 3.0. O'\ 27
1 min prior to autolysis, substantially ·accelerated the rate of protein loss. After such treatment, some 57 % of the cell protein was lost after 4 hours of autolysis and by 24 hours, some 89 % had been lost (Trevelyan 1976) . Heat-shocking at higher temperature (75 °c) or lower temperature (60 °c) or at 70 °c for less time gave decreased rate of protein loss. The heat-shocking at 70
0 c for 1 min presumably facilitated loss in cell structure to permit protein exit from the cell, without significant loss to the autolytic machinery of the cell.
Inclusion of 7. 5 % ethanol in the autolytic buffer also accelerated the rate of protein loss. Under such conditions, 70 % of the cell protein had been lost by
4-4½ hours. Lower concentrations of ethanol (2.5 and 5.0
%) gave decreased autolytic kinetics (Trevelyan 1977).
Inclusion of 0.5 M sodium chloride in the autolysing buffer also accelerated protein loss (19 % after 4-4½ h), with less influences at lower concentration. It was concluded by Trevelyan (1976, 1977) that factors which destroy the membranous structure of the cell and decrease cell permeability will accelerate autolysis and protein loss. In an earlier less detailed study, Sugimoto (1974) also concluded that addition of ethanol (5 %) and sodium chloride (5 %) to the autolytic reaction mixture of baker's yeast encouraged autolysis leading to a greater recovery of Kjeldhal Nin the autolysate.
Al though experimental details were not given, Babayan et al. (1981) provided further evidence that the 28
addition of ethanol (3 %) to cell suspensions of baker's yeast encouraged their autolysis and release of amino nitrogen into the autolysate. Other additives which improved the autolytic reaction were ethylacetate, lauric acid and lecithin.
In a series of papers, c. Charpentier and coworkers (Feuillat and Charpentier 1982, Charpentier et al. 1986, Charpentier and Freyssinet 1989, Leroy et al. 1990) reported the release of total nitrogen from wine yeasts (S. cerevisiae) during autolysis in several different systems. These systems included autolysis in acetate buffer pH 5.0, alcoholic buffer (ethanol 10 %, pH
3.0 or ethanol 12 %, pH 3.5) and wine. Over a time course of 20 days, cell suspensions of a strain of wine yeast, S. cerevisiae or S. bayanus released 1500-1750 mg/1 of total nitrogen into the medium as a result of autolysis. Most release occurred during the first day, and thereafter up until day 6 when the kinetics of release levelled off. Best release occurred in suspensions held at pH 5.0, and it increased as the temperature of autolysis was increased from 37-55 °c. However, preheating the yeasts at 45 °c for 4 days before autolysis inactivated the autolytic system. It was concluded that the rapid first release of nitrogen was due to excretion or exsorption of the intracellular pool of amino acids and, thereafter, true autolytic release of intracellular protein occurred. The later reaction could be inhibited by heating the cells to 45 °c or higher 29
prior to their autolysis. It was also concluded that yeast autolysis could occur at the low pH (3.5) and ethanol concentrations of wine (Feuillat and Charpentier
1982).
An increase in the concentration of free amino acids in autolysates is a major consequence of yeast autolysis. This behaviour has been observed by authors working with model systems (Vosti and Joslyn 1954a,
1954b, Hough and Maddox 1970), beer (Schryver et al.
1927, Masschelein and Van de Meerssche 1976, Masschelein
1986) and wine (Feuillat and Charpentier 1982, Leroy et al. 1990). It is concluded that, during autolysis, proteins are degraded within the cell, thereby releasing amino acids to the extracellular medium. Thereafter, proteins and peptides already released by autolysis are subject to further breakdown to amino acids by proteases also released by the autolytic reaction. The profiles of amino acids found in autolysates have been reported by various workers (Kulka 1953, Farrer 1956, Masschelein and
Van de Meerssche 1976, Feuillat and Charpentier 1982, and
Masschelein 1986) , but it is not possible, as yet, to draw conclusions about the behaviour of particular amino acids or groups of amino acids. The nature of the amino acids present in yeast autolysates are affected by the rate and extent of the proteolytic processes and, presumably, the yeast strains involved.
Role of proteases JO
The loss in cell protein during autolysis and the
appearance of amino acids, peptides and proteins in the soluble autolysates, clearly implies a role of proteases
in the process. Such enzymes are not only active within
the cell, but would also act on the solubilized proteins and peptides. Consequently, some knowledge about the biochemistry of proteases in yeasts may assist in understanding the process of autolysis. The relatively recent conclusions that proteases are not merely involved in non-specific turnover of cell protein and that they also function to control fine aspects of cell metabolism, have lead to much increased interest in these enzymes and their overall involvement in cell biology. Presently, some 40-50 different proteases have been described in yeasts and only a few of them have been well characterised. The reviews of Jones (1984) Achstetter and Wolf (1985), and Wolf (1986) describe the occurrence of proteases in yeasts and their biological functions.
The proteases of yeasts are primarily located in the vacuole, but some are associated with the ~i tochondria, periplas~ic space, and membranes. The main biological function of intracellular proteases is general protein degradation inside and outside of the vacuoles as part of the protein turnover. However, additional functions include activation and inactivation of some enzymes as part o~ the regulatory mechanism of cell ~etabolism. Some proteases act with broad specificity and degrade a wide range of cell proteins, while others may have highly restrictive specificity. Less of cell compartmentali- 31
sation as a result of autolysis as well as degradation of membranes should allow the cell proteins to come into contact with a multiplicity of proteases. Consequently,
the process of protein degradation during autolysis should be quite complex and extensive but very much determined by the autolytic conditions of pH, temperature, and presence of agents, such as ethanol, salt and solvents that are likely to affect the activity of the various proteolytic enzymes.
Early studies that associated proteolytic activity with yeast autolysis have been reviewed by
Schryver et al. (1927), Vosti and Joslyn (1955), and
Farrer (1956). Essentially, these studies showed that yeast autolysates could liquefy gelatin and that more than one type of protease was involved in autolysis. The involvement of more than one protease activity was indicated by the fact that gelatin liquefaction by autolysates exhibited at least two pH optima, one at acid pH {pH 3.1) and one at neutral pH In addition, various peptidase activities were demonstrated in some autolysates. Since these early 0bservations, more defined research has implicated proteases in autolysis. Lenney and Dalbec (1967) report~d the partial purification and preparation of two proteinases from autolysates of S. cerevisiae. One of these protease, termed protease A, had a pH optimum at 3.7 for splitting acid denatured haemoglobin, while the other (protease B) exhibited an optimum pH of 6.2 for hydrolysis of urea denatured 32
protein. Both enzymes, however, had a similar pH optimum
(6.0-7.0) for the hydrolysis of gelatin. The protease
activity of the autolysate was substantially increased
when it was pre-incubated at pH 5.0 as compared with pH
7. 0. It was suggested that the enzymes were present in
the cell as zymogen forms.
In a series of papers Hata and coworkers (Hata
et al. 1967ab, 1972, Hayashi et al. 1968ab),
fractionated three proteinases from autolysates of baker's yeast. These enzymes were designated as proteases
A, B, and C. Proteinase A has an optimal pH at 2.0 for
casein hydrolysis and 3.0 for hydrolysis of acid dena tura ted haemoglobin. Proteinase B has an optimal pH of 9. 0 for casein digestion and 6. 0 for milk clotting activity. Proteinase C exhibited an optimal pH 6. 0 for casein hydrolysis. Proteinase A and B were endopeptidases and were considered comparable to the proteases A and B reported by Lenney and Dalbec ( 1967} . Proteinase C was
found to be a carboxypeptidase, and like the other two enzymes, as reported by Lenney and Dalbec (1967), occurred as an inactive form that was subsequently
activated.
Using a combination of colur.m chromatographic
techniques, Maddox and Hough (1970) separated the prot2olyt ic activity in au tolysa tes of S. car 1 sbergens is
into four fractions, designated as enzymes A, B, C, and
D. Electrophoretic data suggested that each fraction was
a single protein. The optimum pH for proteolytic activity 33 on acid denatured haemoglobin was 7.5, 6.2, 6.2, and 3.5 for enzymes A, B, C, and D respectively, while the corresponding temperature optima were 3 5 °c, 50 °c, 50
0 c~ and 60 °c. The products of proteolysis suggested that enzyme A was an exo-peptidase, while the other enzymes were endopeptidases. Isolation of these enzymes from autolysates suggested their role in autolysis, al though no firm evidence of their activity during autolysis was presented. The existence and properties of the proteases
A, B, and C in baker's yeast had been confirmed by Hata et al. (1972), but protease D was not mentioned.
In a study of the biochemistry of liquefaction of pressed yeast, Tohoyama and Takakuwa (1972) confirmed the occurrence of proteinases A, B, and C in this yeast, as previously reported by Hata and coworkers. They also found that the onset of liquefaction correlated with the solubilization of peptides and amino acids from the yeast, and that this corresponded with a significant increase in the activity of proteinase C.
The role of proteases in autolytic proteolysis was further studied by Behalova and Beran (1979). who examined changes in the different proteolytic activities during autolysis of mechanically disintegrated and intact cells of S. cerevisiae. Faster autolysis (proteolysis as measured by amino acid formation) occurred in the disintegrated yeast and was concluded that the three proteases A, B, and C were involved in the activation of each other and that this activation system depended upon 34
the environmental conditions of autolysis. The concept
that proteases occur in inactive forms that require
activation by proteolysis itself has been discussed by
Saheki and Holzer ( 197 5) and Saheki and Holzer ( 197 4) who also reported that proteases A and B were tryptophan synthase, inactivation enzymes.
Charpentier and coworkers (Feuillat and
Charpentier 1982, Charpentier et al. 1986, Charpentier and Freyssinet 1989, Leroy et al. 1990) have demonstrated the role of proteases in the autolysis of yeasts during wine and champagne fermentations. As the yeast ages on lees, its proteolytic activity increased and eventually passed into the wine where protein and peptide components are hydrolysed to amino acids. It was suggested that protease A, which is active at acid pH, is the main protease associated with these activities.
2.4.3 Nucleic acids
Nucleic acids comprise about 6-12 % of the yeast cell dry weight (Kihlberg 1S72). Trevelyan (1976, 1977) reported values of 6-8 % of the dry weight for S. cerevisiae. The bul1< (3.6-8.5 %} of it consists of RNA and about 0.2-1.2 % consists of DNA (Polakish and
Bartley 1966, McMurrough and Rose 1967, Ingledew 1977,
Trevelyan 1978). The DNA is mostly confined to the nucleus but small amounts are found in the mitochondria and as extrachromosomal elements. RNA consists of 35
messenger RNA, ribosomal RNA and transfer RNA (Mounolou
1971).
The degradation of nucleic acid in yeast cells during autolysis was first suggested by Haehn and Leopold in 1935, who noted that nucleic acid incubated with autolysed yeast was hydrolysed to free phosphoric acid, ribose and ammonia (Farrer 1956). Since then, several studies have described the breakdown of nucleic acids during yeast autolysis.
Vosti and Joslyn (1954a) reportPd that approximately 80 % of the nucleic acid in cells of baker's yeast was degraded during the first 20 hours of autolysis. This breakdown parallelled the release of soluble phosphate, suggesting that phosphate behaviour could indicate the reaction of nucleic acids. Like the release of amino nitrogen, phosphate release was affected by pH, temperature, buffer, yeast spe~ies and whether or not the yeast was cultured aerobically or anaerobically.
However, phosphate was solubilized more rapidly than nitrogenous constituents and, depending on conditions,
90-100 % of the cell phosphorus could be released by 4 hours (Vosti and Joslyn 1954b, Joslyn 1955, Joslyn and
Vosti 1955).
Hough and Maddox (1970) reported the progressive loss of DNA and RNA from cells of brewer's yeast during autolysis in distilled water at 45 °c. Some
80-85 % of the cell DNA had been lost after 14 hand was 36
recovered in the soluble autolysate. About 70 % of the cell RNA was lost b:r 14 h, but only about 30 % of this was recovered in the autolysate. They suggested that nucleic acid degradation would involve the combined action of nucleases, nucleotidases and nucleosidases, producing free bases, ribose and inorganic phosphate.
In a series of papers, Trevelyan ( 1976, 1977,
1978) examined the breakdown of nucleic acids in commercial baker's yeast durins autolysis under different conditions. When autolysed at 50 °c in succinate buffer at pH 5.0, the nucleic acid content of the cells decreased f rorn 6. 3 % to 0. 6 % (i. e. 90 % reduction) by
24 h. This percentage decrease was less at 55 °c and 60
0 c where it was believed that nucleases involved in the reaction were inactivated. The decrease in cell nucleic acid content was negligible during the first 4 h of autolysis, but this lag could be overcome by pre he a ting the cells at 70 °c for 1 min before autolysis or pretreating them with ethyl acetate. It was thought that such pretreatments served to disorganize the cell membranes, allowing nucleases to contact ti1eir substrates and permitting the degradation products to diffuse out of the cells. With such treatments, 90 % of the Ducleic acid
(mostly RNA) was degraded within 2 h ( Trevelyan 197 6) .
These rapid rates of nucleic acid degradation were also obtained by autolysis of the cells in the presence of ethanol ( 5-7. 5 %) or by drying and rehydrating the cells before au tolys is. Addition of salt ( 0. 5 M NaCl) to the 37
autolysis medium also promoted faster autolysis as measured by nucleic acid breakdown (Trevelyan 1977).
However, even after optimum conditions, a small percentage of nucleic acid remained resistant to
autolytic degradation and remained associated with the cellular material. It was later shown that this fraction was DNA. This fraction represented about 0. 3 % of the cell dry weight and this value remained relatively unchanged during autolysis, despite 95 % reduction in the RNA content (Trevelyan 1978).
The nucleic acid components were recovered in the autolysate of S. cerevisiae after incubation of the cells in a suspension containing 3 % ethanol at 55 °c
(Babayan et al. 1981). After 20 minutes, nucleic compounds in the autolysate represented 0. 04 % of the cell dry weight, while after 60 minutes, this value was
0.33 %.
In a study of autolysis during the ageing of yeast lees in champagne wines, Leroy et al. (1990) reported a decrease in the nucleic acid content of the cells with age (up to 25 months) and concomitant release of these acids into the wine. They also noted the /l A4 ct.i,Q- association of RNase activity with yeast cells in the early stages of the process.
Brewer's yeast undergoes autolysis during the post-fermentation lagering of beer (Masschelein 1986).
During this process, there is a massive release of 38
nucleotides (adenosine-5-monophosphate, cytidine-5-mono- phosphate and others) into the beer, indicating degradation of yeast nucleic acids (Masschelein and Van de Meerssche-1976).
As suggested by Hough and Maddox (1970) a
A ~ complex of different DNases and RNases is probably involved in the degradation of nucleic acids during 'A- autolys is. Various DNases and RNases have been described in yeasts (Chow and Resnick 1987, Karwan and Winterberger
1988, Brown 1989, Karwan and Kindas-Mugge 1989, Karwan et al. 1990), but their specific involvement in autolysis has not been examined.
2.4.4 Lipids
Lipid composition and metabolism in yeast cells have been reviewed by Ratray (1975), Arnold (1981a), Ratledge and
Evans (1989), and Kockova-Kratochvilova (1990). On the basis of location of their synthesis within cells or their secretion into the surrounding medium, lipids are classified into cellular lipids, extracellular lipids, capsular lipids, cell wall lipids, periplasmic space lipids, and plasma membrane lipids (Arnold 1981a, Kokova
Kratochvilova (1990).
The content and composition of lipids found in yeasts vary widely with species and on how the cultures are grown. Culture conditions of pH, dissolved oxygen tension, temperature and nutrient composition are factors 39
which affect cell lipid (Ratray 1975, Ratledge and Evans
1989, Kokova-Kratochvilova 1990).
Total lipids may comprise up to 18-20 % of the
cell dry weight in non-oleaginous yeasts, or 20-80 % in
oleaginous yeasts (Ratledge and Evans 1989). Ratray
(1975) and Ratledge and Evans (1989) cite values from
3. 5-20 % for S. cerevisiae, and O. 3-23 % for species
Candida. The composition of the lipids varies depending upon their cellular location. Thus, in S. cerevisiae the
cellular lipids consist of neutral lipids (67 %) and phospholipids (32 %) . Within the neutral lipids, the proportion of components are: triglycerides (26 %) , diglycerides ( 0 %) , sterol esters ( 38 %) , sterol ( 3 % )
and free fatty acids. Within the phospholipids, the proportions of the different components are: phosphatidylethanolamine (31.2 %) , phosphatidylinositol
(29.7 %) , phosphatidylcholine (25.3 %) , phosphatidylserine (6.2 % ) ' cardiolipin ( 3. 8 %) , and phospha tidic acid ( 2. 3 %) • The main fatty acids
comprising these lipids are stearic (c18 :ol, oleic
(c18 : 1 ), palmitic (c16 :o>, palmitoleic (c16 : 1 ), myristic
(C14:ol, lauric (c12 :ol, capric (Clo:ol (Arnold 1981a, Kokova-Kratochvilova 1990).
Isolated cell membranes of S. cerevisiae have a different composition to the cellular lipids. These
consist mostly of neutral lipid (92 %) and phospholipids
(8 %) . The neutral lipid is composed of triglycerides (36
%) , sterol ester (28 %) , free sterol (13 %) , and free 40
fatty acids and others (23 %) . The phospholipids consist mostly of phosphatidylcholine (42 %) , phosphatidyl ethanolamine (25 %) , phosphatidylinositol (16 %) , phos pha tidylserine ( 7 %) , cardiolipin ( 9 %) , and phospha tic acid (<1 %) (Arnold 1981a, Henscke and Rose, 1991).
Extracellular lipids, that is those excreted into the external medium, comprise (i) esters of polyol fatty acids, (ii) sophorosides of hydrocarboxylic acids,
(iii) acetylated sphingosines, and (iv) hydroxy acids
(Arnold 1981a, Kokova-Kratochvilova 1990).
Capsular lipids are only found in species with slime layers that consist mostly of polysaccharide, but may also contain significant amounts of lipids. Species producing this type of lipid are found in the genera of
Cryptococcus, Lipomyces, Hansenula, Rhodotorula,
Trichosporon, Sporobolomyces, and Candida. Polyol fatty acid esters have been reported to be secreted in the slime layer of Rhodotorula (Arnold 1981a, Kokova-
Kratochvilova 1990).
Al though the subject of some question, lipids may also compose part of cell wall of yeast (Fleet 1991).
In S. cerevisiae, lipid represents 2-4 % of the cell wall; it contains no phospholipids and sterols, but consists mainly of glycerides with unsaturated palmi toleyl and oleyl residues. However, composition of the wall 1 ipid varies with the species: for instance, C.
albicans contains O. 5-7 % wall lipid that consists of 41
mainly sterol esters, sterols, phospholipids, and triglycerides (Fleet 1991).
Periplasmic lipoprotein has been isolated from
S. cerevisiae, but its composition is not clear (Arnold
1981a).
Degradation of lipids during autolvsis
The early literature implies that some alteration to cell lipids must occur during autolysis, but no experimental data were presented (Joslyn 1955, Farrer 1956). These implications are based upon the assumptions that function of the cell membrane is impaired during autolysis, thereby by allowing diffusion of the products of autolysis to the external environment. Moreover, it has been known for a long time that some organic solvents, which are likely to affect membrane function and permeability, can initiate autolysis (Joslyn 1~55, Arnold
1981a). Further evidence that cell lipids are affected during autolysis comes from cytological observations which show changes in the organisation of intracellular lipid globules (see Section 2.3). Degradation of lipids during autolysis should be indicated by decreases in the amount of extractable lipid from the cells, increases in
the concentration of free fatty acids and glycerol in the
cell free autolysate, and changes in the composition of
lipid classes associated with the cells during autolysis.
Lipid metabolism, in general, in fungi has been reviewed 42
by Chopra (1984), but no rere~enc~ is made to the breakdown of lipids during autolysis.
In a study of the drying and rehydration of baker's yeast, Harrison and Trevelyan ( 1963) reported a loss in fermentation activity of the yeast and correlated this with substantial leakage of intracellular material from the cells on rehydration. It was suspected that the drying and rehydration processes affected the lipid composition and permeability of the cell membrane. They subsequently showed that the phospholipid content of the. treated cells had decreased by 10-20 % and that this was notably due to losses in phosphatidylcholine (lecithin) and phosphatidylethanolamine. The content of phosphat idylinosi tol was not affected. They also reported that similar changes to the phospholipid content of the yeast occurred when toluene was used to induce autolysis. In this circumstance, the contents of both phosphatidyl choline and phosphatidylethanolamine decreased, but not that of phosphatidylinositol.
Ischida-Ichimasa (1978) examined the degradation of lipids in S. cerevisiae during autolysis induced by the addition of toluene. The cell suspensions in acetate buffer were incubated in the presence· of toluene (10 %) at 30 °c for 5 h. During autolysis at pH 6.0, decreases were noted in the contents of phospholipid, sterol and sterol ester with concomitant increase in the concentrations of free fatty acids and triglycerides. On autolysis at pH 4.0, however, only the 43
phospholipid components decreased. Within the phospholipids, the main decreases at pH 4. 0 were phosphatidylcholine (94 % reduction), phosphatidyl ethanolamine (88 %) , and phosphatidylinositol (72 %) .
These decreases correlated with proportional increases in free fatty acids. These rates of decrease were less at pH
6.0. Thus, it seems that pH has an influence on the rate and type of lipid breakdown in yeast during autolysis.
Compressed baker's yeast ·1ndergoes a softening or liquefaction in about 10 days during storage at 30 °c.
The process is accompanied by major leakage of intracellular material and is considered to be a form of autolysis (Takakuwa and Watanabe 1981). Leakage of free fatty acids, phospholipids and total sterol was progressively but weakly observed up until day 6, after which the rate of leakage significantly increased. Two stages of leakage were noted. Up until day 6, leakage of phospholipids was mostly noted with little leakage in free fatty acids. After this time, the decrease in phospholipid content within the cells correlated with the release of free fatty acids. By comparison, there was much less release of sterol, sterol ester, triacylglycerol, diacylglycerol. Within t~e phospholipids of the cell, the greatest losses were due to the losses of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinosi tol-serine. There was little degradat
ion of lysophosphatidylcholine (Takakuwa and Watanabe
1981). 44
Liquefaction of compressed baker's yeast was
accelerated by exposing the cells to acetic acid vapour
at 30 °c. This was accompanied by a marked (30 %} decrease in the phospholipid content of the cells and a
corresponding increase in the proportion of free fatty acids. Little cha~ge was observed in the cellular contents of other lipid components (Watanabe and Takakuwa
1983). Exposure of the cells to formic acid gave a similar response. A similar but slower reduction in phospholipids was observed for cells incubated at 37 °c in acetate and other buffer when the pH was maintained below pH 6.0. A faster degradation of phospholipids occurred at pH 4. 0, then at ~H 6. 0. However, at pH 7. 0 and above pH 9. 0, the degradation of phospholipids was very weak and there was little production of free fatty acids. At pH 8.0, phospholipids were degraded but different components were reported to be affected than at low pH values.
In a study to determine the autolytic behaviour of yeasts during beer fermentation, Brown et al. {1982) examined the influence of ethanol concentration on the lipid composition of cells of brewer's yeast. Cells suspended in a so!ution of 20 % ethanol at 18 °c overnight exhibited up to 7 5 % reduction in the content of phospholipids. Little degradation of phospholipids was observed after this time. Longer incubation times were required to observe the effects of lower ethanol concentration. Thus, at 10 % ethanol, incubation for 1 45
week was needed to observe 70-80 % reduction in phospholipids. Within the phospholipid components, the relative rates of degradation were (fastest to slowest) phosphatidylcholine, phosphatidylethanolamine, and phos pha tidylserine. Little breakdown of phosphatidylinosi tol was observed. They also reported about 75 % breakdown of phospholipids when the yeast cells were stored for 5 hat 45 °c in either water or beer. The main products of phospholipid degradation were ethyl esters of c1 6 and C18 fatty acids.
The lipids of S. cerevisiae undergo changes when the cells are left in contact with wine during champagne ageing (Troton et al. 1989b). It is generally recognised that autolysis occurs during this ageing process. Over a period of 6 weeks to 19 years, the phospholipid component of the yeast lipid decreased by 57
% and 84 % , respectively, and this was accompanied by a marked increase in the proportion of triacylglycerol. The diacylglycerol content increased only marginally (Piton et al. 1988, Troton et al. 1989b). Such alterations resulted in a release of triacylglycerol into the wine
(Troton 1989a).
Chen et al. (1980) examined the release of free fatty acids during autolysis of cell suspensions (106-107 cell/ml) of brewer's yeast. Some 10 different free fatty acids were detected in the autolysates. Their concentrations ranged from about 10 ug/1 up to 900 ug/1, the most prevalent ones being octanoic (C 8 ), decanoic 46
( c10 ) , and dodecanoic ( c12 ) acids. Greatest release of the acids occurred when autolysis was conducted at 5-55
0 c and pH 6.0. Inclusion of ethanol (10-15 %) in the suspension promoted slightly greater release of the acids, but the influence of other organic solvents (e.g. ethylacetate, toluene) was only marginal.
In his review of the biochemistry of beer maturation, Masschelein (1986) reported the release of caprilic and caproic acid~ from suspensions of brewer's yeast after 15 days at different temperatures. Release occurred in the temperature range 0-20 °c with fastest rates occurring at 20 °c. Presumably, this release is a consequence of autolysis, and is considered to impact on beer flavour.
Lipases
A complex of lipolytic enzymes is involved in the breakdown of lipids. Different enzymes are required to break the different bonds in the lipid structure. Figures
1 and 2 indicate the range of lipolytic enzymes required for lipid hydrolysis.
Lipases hydrolyse esters of the primary alcohol at 1 or 3, 1 and 3, or 1, 2 and 3 positions in triglycerides (Figure 1) to give diglyceride, monogly ceride, and free fatty acids ( Boyce 1986) . In complete degradation, free fatty acids and glycerol are the end products (Chopra 1984). Phospholipids are hydrolysed by 47
(a)
0 I 0 H2C-O-C--R1 R2-!-o-JH 0 I I H2C-O-P-O-CH2 -cH2N+ (CH3 ) 3 I o-
( b)
Figure 1. Structure of (a) triglyceride (b) phosphatidyl choline (lecithin). Side chain in the bracket of lecithin structure is different in other phospholipids.
Source: Boyce (1986). 48
phospholipases as illustrated in Figure 2 (Fogerty 1971).
Several authors have reported the occurrence of phospholipase and lipolytic activities in yeasts and have suggested that they are active in lipid breakdown during autolysis. Treatment of yeasts with organic solvents tends to activate autolysis and it is considered that such treatment promotes the spatial interaction of membrane lipids with lipolytic and phospholipase enzymes
(Harrison and Trevelyan 1963, Ishida-Ichimasa 1978, Chen et al. 1980, Brown et al. 1982, Watanabe et al. 1983).
Harrison and Trevelyan {1963) suggested that a phospholipase C might be involved in lipid breakdown during autolysis of baker's yeast because the production of free fatty acids was much lower than their loss from the phospholipid fraction. However, subsequent authors could not demonstrate phospholipase C activity in various fractions of S. cerevisi~e and, in fact, presented evidence that phosphol ipase A and a lysophosphol ipase were mainly involved in phospholipid breakdown (Van den
Bosch et al. 1967, Letters 1968) These authors found that the decrease in phospholipid content was balanced by the production of free fatty acids, rather than by an increase in diglycerides. This profile is consistent with phospholipase A action. Ishida-Ichimasa (1978) also con firmed an absence of phopholipase C activity during the autolytic softening of baker's yeast and suggested that phospholipase A, lysophospholipase, and phospho lipase B might be involved. Further studies by Takakuwa 49
(Glycerophosphatides)
Phospholipase PhospholipaseI ! CH2OOCR1 Phosphatidic I acid R2COOCH Phosphatase 0 0 I I CH2OPO-X CH2OH CH2OPOH I I OH OH (Lysophospholipid) (Diacylglycerol) (Phosphatidic acid) + + Phosphoryl-X X
Phospholipase B
(Glycerylphosphoryl-X)
Figure 2. Sites of action of (a) phospholipase A, (b) phospholipase B, (c) phospholipase C, and (d) phospholipase D on glycerophosphatides and their hydrolytic breakdown products.
Source: Fogerty (1971). 50
and Watanabe (1981) and Watanabe and Takakuwa (1983) on the liquefaction of baker's yeast also suggest the action of phospholipase A and possibly phospholipase B in phospholipid degradation. In a study unrelated to autolysis, Nurminen and Suomalainen (1970) reported both lipolytic and phospholipase activities in various fractions of S. cerevisiae and noted a strong association of these enzymes with the cytoplasmic membrane - pre sumably the substrate which they attack.
It may be concluded from the few studies to date, that lipolytic and phospholipase enzymes are involved in the autolytic process by modifying lipid composition and, consequently, the permeability properties · of yeast membranes. However, more systematic detailed studies of these enzymes, their methods of action, and factors which affect their activity are required.
2.4.5 Vitamins
Yeast cells mainly contain water soluble vitamins of the vitamin B group. Some typical vitamin contents of S. cerevisiae per 100 g cell weight are as follows; thiamine (0.8 mg), riboflavin (4.5 mg), niacin (55.0 mg), folic acid ( 0. 4 mg) , pyridoxine hydrochloride ( 8. 3 mg) , pantothenic acid (9.4 mg), biotin (0.08 mg), p aminobenzoic acid (1.4 mg) and riboflavin (0.0004 mg) (Kinsella 1986). 51
There are very few publications about the release of vitamins during yeast autolysis and these are reviewed by Joslyn (1955) and Farrer (1956). In an early study, Trufanov and Kirsanova (1940) reported the release of thiamine and riboflavin during the autolysis of baker's yeast in buffer containing toluene and disodium sulfide as activator, at pH 4.8-5.0 and 35-45 °c.Maximum thiamine was released after 6-10 h of autolysis while maximum riboflavin release was noted at 12-24 h. Farrer
(1946) found that liberation of thiamine, riboflavin, and nicotinic acid from brewer's yeast autolysed at 52 °c in distilled water was very rapid and was complete in about 2½ hours. Farrer (1951) also found that pantothenic acid was released less rapidly from autolysing brewer's yeast. A rapid release occurred during the first hour after which the rate decreased. Complete liberation was not observed, and even after 45 h, only 7 5 % of the vitamin had been released.
2.5 Commercial Significance Of Yeast Autolysis
Yeast autolysis has several commercial implications, many of which have been recognized for a long time. In some instances, yeast autolysis has a beneficial outcome, whereas in other cases its occurrence is considered undesirable. In a commercial context, control of autolysis is important and provides a rational basis for studies into the chemistry and biochemistry of this 52
phenomenon. This Section gives an overview of the
comn1ercial implications of yeast autolysis.
2.5.1 Leavening power of compressed baker's yeast
A sig~ificant proportion of baker's yeast is retailed as
compressed blocks of biomass that still retain about 75 % moisture. Such yeast cells are biologically unstable and gradually lose fermenting/leavening power with storage
time. This loss of activity is faster at higher storage
temperature than at lower temperature (Trivedi 1986) and is associated with a softening and eventual liquefaction of the product (Vosti and Joslyn 1954a). Autolytic proteolysis and lipolysis have been connected with the liquefaction process (Harrison and Trevelyan 1963,
Tohoyama and Takakuwa 1972, Takakuwa and Watanabe 1981).
2.5.2 Autolysed yeast extracts
It is well known that the yeast cell wall is thick, tough and rigid. It resists intestinal digestion by animals, thereby limiting the availability of intracellular proteins and other constituents of yeasts as nutrients.
However, the preparation of autolysed yeast extracts overcomes this difficulty, and commercial procedures for
the production of such autolysates have been known for many decades ( Peppler 198 2) • These yeast autolysa tes are rich in proteins, peptides, amino acids, group B vitarr.ins, and nucleic acid components and have found wide 53
acceptance in the food and feed industries as versatile, multifunctional ingredients (Albrecht and Deindoerfer
1966, Smith 1968, Blake 1975, Schmidt and Nannan
1988) .They have very desirable flavour and aroma qualities (meaty character) and are added to a wide variety of foods (e.g. soups, sauces, canned vegetables, cereals, seasonings, snack foods) as flavour enhancers and for the purposes of nutrient fortification, especially, for increasing the content of B vitamins and amino acids (Peppler 1982, Dziezak 1987). The isolation of proteins/nuleo-protein complexes from yeast autolysates has received significant attention because of the possibility of obtaining specific fractions with functional properties suited to food processing (Kinsella
1986). Some of these proteins, for example, can have interesting antioxidant properties (Bishov and Henick
1972) as well as being of nutritional value.
2.5.3 Industrial fermentations
Yeast autolysis may occur at two stages during brewing operations. It can occur in the pitching yeast prior to inoculation into the wort (leading to decreased fermenta tive activity), and during the lagering or maturation phase, when the newly fermented beer containing residual or freshly inoculated yeasts is stored for several days or weeks. The extent of autolysis at this stage depends 54
on the time and temperature of storage, and is characterized by a release of amino acids, peptides, proteins, nucleic acids (Masschelein and Van de Meerssche
1976, Masschelein 1986), esters (Engan 1969), and free fatty acids {Clapperton ,(197 8ab, Van de Meerssche et al.
1979, Masschelein 1986) into beer. The release of these products may or may not be desirable and contribute to the development of "yeasty" flavour in the beer
(ClappertonA1978ab).
Wine fermentation
Yeast autolysis is recognized as an important, al though little researched, reaction in wine fermentations. High populations (107-109 cfu/ml) of yeasts develop during the alcoholic fermentation of grape juice. This population consists mostly of S. cerevisiae, but according to Fleet
(1990a), significant populations of indigenous yeasts
(e.g. Kloeckera apiculata, Candida stellata) are also present. While these later species die off during the early stages of fermentation, their dead biomass will represent part of the wine lees of sediment along with cells of S. cerevisiae. Yeast sediment in contact with the wine during the storage phases after alcoholic fermentation is prone to autolysis which is characterized by a release and an increase in the concentration of protein and free amino acids in the wine
(Kunkee and Amerine 1970, Fleet 1990a). Such autolysis has several effects. Presumably, it may affect the 55 sensory profile of the wine. It may serve as a source of nutrients that stimulates the growth of bacteria in wines. Such stimulation may be beneficial, if these bacteria ( lactic · acid bacteria) conduct a desired malolactic fermentation. However, it is also considered that these autolytic nutrients might encourage the growth of bacteria that spoil the wine.
In champagne or sparkling wine production, a secondary yeast fermentation is conducted in the bottle, subsequent to which the wine is aged for 6 months to many years in contact with the sedimented yeast. It has now been well demonstrated that the sedimented yeast cells autolyse during this ageing process liberating peptides, amino acids, lipid materials and nucleic acids into the wine and, consequently, affecting its sensory character
(Feuillat and Charpentier 1982, Piton et al. 1988, Troton et al. 1989a, Leroy et al. 1990).
Enzymes
Yeast autolysis is the first step in the release and subsequent commercial purification of enzymes such as invertase and ~-galactosidase for use in food processing and other applications. Aspects of the preparation of these commercial enzymes are discussed by Peppler (1979, 1982).
Pigments 56
The yeast Phaffia rhodozyma is characterized by the production of an intracellular pigment, astaxanthin, which has considerable commercial potential as an additive for food colouring (Johnson 1991). Autolysis has been explored as an initial process for releasing the pigment for subsequent purification (Okagbue and Lewis
1984).
Miscellaneous
In the area of agriculture, yeast autolysates were reported as an effective protein Pait containing insecticide to control fruit flies (Smith and Nannan 1988). 57
3. MATERIALS AND METHODS
3.1 Yeast Strains And Culture
Yeast strains used throughout this study were obtained from the culture collection within the Department of Food
Science and Technology, University of New South Wales.
The origins of these strains are shown in Table 2.
The strains were maintained by regular subculture on slants of Halt Extract Agar (Oxoid-CM59). Cultures were incubated at 25 °c for 3 days, then stored at 4 °c. All cultures were checked for purity by streaking onto plates of Malt Extract Agar before use in experiments.
Cells for autolysis experiments were grown in yeast extract (1.0 %) - glucose (5.0 %) broth. Inoculum cultures were grown in test tubes of medium (10 ml) for 48 h at 25 °c. These were used to inoculate 1.5 1 of medium in 2 1 conical flasks at 0.1 % inoculum. The large cultures were incubated for 48 hat 25 °c with shaking in a Gallenkamp orbital shaker at 100 rpm. Yeast cells were harvested by centrifugation at 3000 rpm for 10 min at 4
0 c (Centrifuge, Damon IEC B-20A). The cell pellets were washed three times with 150 ml of O. 9 % sterile sodium chloride solution by resuspension and recentrifugation.
The cells were suspended in 1400 ml of 0.1 M sodium phosphate buffer pH 4.5 for use in autolysis experiments.
All harvesting and washing operations were conducted under aseptic conditions. 58
TABLE 2. Yeast strain and their origin
Yeast strain 0 rig in
Saccharomyces oerevisiae 2180a Department of Food Science
and Technology, University
of California, Davis
HB350 French winerya
EC1118 Commercial wine yeast Lalvin
K/oeckera apiculata 202 Australian winery b
521 Australian winery b
412 Australian winery b
Candida stellata 8008 Australian winery b
800MEA Australian winery b
504 Australian winery b
a= Fleet, Lafon-Lafourcade and Ribereau-Gayon (1984). b= Heard and Fleet (1986) 59
3.2 Autolysis Of Yeast Cells
Cells were not stored before autolysis, and were used immediately after culture and harvesting. The cell suspension in phosphate buffer, pH 4.5, was incubated at 45 °c with orbital shaking at 100 rpm (Gallenkamp orbital shaker incubator). At intervals during autolysis for up to 10 days, samples of the suspension were aseptically removed for the determination of cell viability, dry weight and chemical composition. The suspension was thoroughly mixed before each sampling to ensure uniformity.
3.3 Cell Viability
Samples (J.O ml) of autolysing cells were serially diluted in sterile 0.1 % peptone solution and 0.1 ml of the dilutions spread inoculated over the surface of plates of Malt Extract Agar. Plates were incubated at 25
0 c for 3 days and then examined for colony counts, as well as verification of culture purity. Analyses were done in duplicate.
3.4 Cell Dry Weight; Soluble Autolysate
Samples ( 10 ml) of the autolysing suspension were filtered by vacuum through a pre-weighed 0.45 µm membrane
filter (Millipore-HP04700). The residue of cells retained by the membrane was washed three times with 10. 0 ml of distilled water, then dried at 60 °c for 24· h. The dry 60 weight of cells was determined by subtracting the weight of the filter. The filtrate and washings of this operation ( termed the autolysate) were transferred to a pre-weighed porcelain crucible, evaporated on a waterbath and then dried under vacuum (25 psi) at 70 °c until constant weight. The weight of autolysate was obtained by subtracting the weight of the crucible. Also, corrections were made for the weight of the 0.1 M phosphate buffer used for suspending the cells for autolysis, by drying similar volumes of the buff er and calculating its weight. Duplicate samples of the autolysing suspension were assayed.
3.5 Total Carbohydrate, Reducing Sugar, And Glucose
Samples of autolysate were obtained by filtration as described already and stored frozen until required for assay. Immediately on thawing, aliquots were used for the determination of total carbohydrate, reducing sugar and glucose. Total carbohydrate was measured by the anthrone reagent ( Spiro 1966, Weiner 197 8) . Samples ( 1. 5 ml) in test tubes were reacted with 5 ml of anthrone reagent (50
% w/v in 85 % sulphuric acid) at 95 °c in a waterbath for 20 minutes. The tubes of reactants were rapidly cooled and absorbance read at 625 nm. The concentration of total carbohydrate was calculated by reference to a standard assay using known concentrations of glucose (Ajax Chemicals-783). 61
Reducing sugars were determined following the method of Nelson and Somogyi ( Spiro 1966} . Aliquots ( J. 0 ml} in test tubes with caps were reacted with of 1.0 ml copper reagent and heated in a boiling waterbath for 30 min. The tubes of reactants were cooled and 1. 0 ml of arsenomolybdate reagent was added. After 60 min, 1.0 ml of distilled water was added to each tube and the absorbance measured at 520 nm. The concentration of reducing sugar was then calculated by reference to a standard assay with known concentrations of glucose.
Glucose was specifically measured using the enzymatic procedure based on glucose oxidase (Bergmeyer et al. 1974). The reagents were obtained in kit form from
Boehringer Mannheim GmbH, Biochernica (Sandhofer Stra~e
116 6800 Mannheim 31 - West Germany) and the method followed as described by this manufacturer.
3.6 Organic Acids (Davis et al., 1986)
The concentrations of organic acids in autolysates were determined by high performance chromatography (HPLC) consisting of the following instrumentation from Waters
Associates: Model 501 pump; a U6K injector; and model 480 variable wavelength detector operating of 210 nm. The acids were separated by passage through an Aminex non exlusion cation-exchange column (Bio-Rad HPX-87H} which was eluted at 65 °c with 0.1 % phosphoric acid at a flow rate of 0.5 ml/min. Autolysates were refiltered through a 62
0. 4 5 µm membrane before injection in to the column. The identity of the acids and their concentrations were determined by reference to the elution of solutions of standard concentrations of oxalic, citric, tartaric, malic, succinic, lactic, formic, acetic and propionic acids.
Acetic acid was also measured using an enzymatic procedure (Beutler 1984) The reagents were obtained as a kit from Boehringer Mannheim GmbH,
Biochemica and the method was followed as described by this manufacturer.
3.7 Protein
Total protein was estimated following the procedure of
Lowry et al. ( 19 51) using the Fol in-Ciocal teau reagent.
Bovine serum albumin (Calbiochem-12657) was used as a standard at concentrations of 5, 10, 15, 20 and 25 µg/ml.
3.8 Amino Acids
Amino acids were only determined in autolysates. Frozen autolysate was thawed, and filtered through an Amicon YMS ultrafilter to remove proteins of molecular weight greater than 5000. Amino acids in the filtrate were qualitatively and quantitatively determined by separation
using HPLC and detection by post-column derivatization
with ninhydrin. The HPLC instrumentation consisted of two pumps (Waters Associates Model M6000A) for delivery of elution buffer to the column, one pump ( Eldex) for post column ninhydrin addition, a manual injector (Waters
Associates Model U6K), an automated gradient controller
(Waters Associates Model 680), a temperature controller
(Waters Associates Temperature Control Module) for maintaining the ninhydrin reactor temperature at 110 °c and the column temperature at 43 °c, and a detector
(Waters Associates Model 440) measuring at 546 nm and 436 nm. The column used was a physiological amino acid analyser column of 12 cm X 4.6 mm, type ANW P150 (Waters
Associates ANW P150) packed with a 5 µm spherical ea tion exchange resin in the lithium f orrn. For gradient elution of the column, tri-lithium citrate buffer of pH
2.51 (buffer A) and lithium hydroxide-boric acid buffer of pH 10.08 (buffer B) were used as mobile phases and were prepared according to the directions of Waters
Associates manual.
The multi-step gradient program used to control the mobile phase mixture for column elution is shown in
Table 3.
The identities and concentrations of individual amino acids (ug/ml) were determined by reference to the
separation of a standard mixture of amino acids ( Pierce-
20077). Individual amino acids were also injected into
the column to verify peak location and identity. 64
Table 3. Gradient program for elution of amino acids by HPLC
Timea Flow rate Solvent composition Curve profileb (minutes) (ml/min) Buffer A Buffer B
0 0.4 100 0 -
50 0.4 50 50 6
60 0.4 23 77 7
87 0.4 0 100 8
100 0.4 0 100 6
101 0.4 100 0 6
a= Total run time was 120 minutes. b= Shown on automated gradient controller (Waters Associates Model 680).
3.9 Ribonucleic Acid
Ribonucleic acid (RNA) concentration was estimated by two methods, ( i) the optical density method as described by
Herbert et al. (1971), Boudrant et al. 1979, and
Miyasaka et al. (1980), and (ii) the orcinol method as described by Schneider (1957).
Optical density method
Cells from samples (1.0 ml) of the autolysing suspension were separated by centrifugation at 4000 rpm for 5 min.
The sedimented cells were washed three times, each with 5
ml of distilled water by resuspension and centrifugation.
The cell pellet was suspended in 5.0 ml of ice cold 0.25 N perchloric acid (Ajax-359), allowed to stand at 4 °c in 65
ice water for 30 min with occasional shaking and then centrifuged at 4000 rpm for 5 min to extract acid soluble compounds. The supernatant was discarded. Five millilitres of 0.3 M sodium hydroxide solution was added to the cells, and the mixture was shaken and incubated at
37 °c for 1 hour. One millilitre of 3.5 M perchloric acid was added, and the mixture shaken and centrifuged at 4000 rpm for 5 min. The optical density of the supernatant was read at 260 and 232 nm in the same cuvette against a distilled water treated by the same procedure (Varian
Techtron UV-Vis spectrophotometer; Model 635) . RNA concentrations were calculated according to this formula.
RNA (µg/ml)= (35 Absorbance - 15 Absorbance ) 260 232 X Dilution factor
The results were compared with standard solutions of RNA
(Boehringer Mannheim - 109223) at 50, 75, 100, 125, 150,
200 and 250 µg/ml dissolved in O. 3 M sodium hydroxide used in the procedure.
Filtered samples (2.0 ml) of soluble autolysates were analysed by the above procedure starting at the stage of sodium hydroxide addition.
Orcinol method
Cell pellets and autolysates were separated from the
autolysing suspension and treated and extracted with perchloric acid as described in the optical density method. One millilitre of the clarified extract was 66
pipetted into a test tube, and 4.5 ml of orcinol reagent was added. This mixture was heated in a boiling waterbath for 20 minutes, cooled to room temperature, and the absorption read at 660 nm against a blank which was 1.0 ml of O. 5 M perchloric acid similarly treated as the
samples. Standards of RNA solution (200 300 I 400 I 500 and 600 µg/ml) in O. 5 M perchloric acid were similarly treated with orcinol. In addition, solutions of D(-) ribose (Sigma-R7500) in distilled water were also used as reference standards. Finally, RNA concentrations in the samples were calculated based on the ribose standard curve, and corrected with the RNA standard curve.
3.10 Deoxyribonucleic Acid
The concentrations of deoxyribonucleic acid (DNA) in cells and soluble autolysates were determined by two methods, ( i) the diphenylamine method and (ii) a spectrophotometric dye method.
Diphenylamine (DPA) method (Herbert et al. 1971, Aigle et al. 198:·n
Washed cells ( 5 ml) from the autolysing suspension were
transferred to a homogenizer tube containing 2 ml of 1.5
M perchloric acid. About 1.5-2.0 g of glass beads of 0.5 mm in diameter ( Sigma-G9268) were added and the mixture was homogenized for 30 seconds at 4000 rpm in a Braun
Homogenizer (Brownwill Scientific, Rochester, N.Y.) to
disrupt the cells. The homogenate (decanted from glass 67 beads) was transferred to a test tube, incubated at 70 °c for 30 min with shaking at 100 rpm, and brought to a fixed volume with 1.5 M perchloric acid, and centrifuged.
The supernatant was used for reaction with DPA.
For the assay of DNA in autolysates, 5 ml of sample was pipetted into test tubes containing 3 ml of
4.0 M perchloric acid, incubated at 70 °c for 30 min with shaking at 100 rpm, and brought to 8 ml with 1.5 M perchloric acid.
An aliquot ( 2. 5 ml) of the DNA extracts was mixed with 2.5 ml of DPA (Sigma-D2385) reagent and incubated at 30 °c for 16-20 h. The absorbance was read at 600 nm against a blank which was 2. 5 ml of 1. 5 M percl-, loric acid mixed with 2. 5 ml of DPA reagent and similarly treated as for the sample. Deoxyribonucleic acid concentration in the sample was calculated by references to standard DNA. One millilitre of standard
DNA (Sigma-D8515) solutions (20, 40, 60, 80, 100, 120 and
140 µg/ml) were similarly treated with perchloric acid and measured using the DPA reagent.
Dye method (Labarca and Paigen 1980)
Washed cells ( 2 ml) from the autolysing suspension were pipetted into homogeniser tubes containing about 1.5-2.0 g of glass beads of O. 5 mm in diameter ( Sigma-G9268) , cooled to 4 °c and homogenised for 30 seconds at 4000 rpm in the Braun Homogeniser. Two millilitres of 0.05 M 68
phosphate buffer, pH 7.4, containing 2.0 M sodium chloride and 1. 0 ml of 5 µg/ml of the dye benzimidazol
H33258 (Calbiochem-382061) were added to each tube, with shaking, transferred to centrifuge tubes and then spun down at 3000 rpm (Clements Model B Universal Centrifuge).
The supernatant was read with a fluorescence spectro fluorometer (Hitachi 1000) at excitation wavelength of
356 nm and emission wavelength of 458 nm.
For the estimation of DNA in autolysates, samples ( 2. 0 ml) were mixed with about 2. 0 ml of 0 .1 M sodium hydroxide to achieve pH 7.4, and then reacted with the benzimidazol dye as described already. Standard solutions of DNA ( 25, 50, 100, 150 and 200 µg/rnl) in phosphate saline buffer, pH 7. 5, were similarly reacted with the dye and fluorescence determined. These values were then used to calculate the concentration of DNA in the samples.
3.11 Lipids
E x t r a c t i o n
Lipids were determined by a modified method of Watson and
Rose (1980). Two hundred and fifty millilitres of sample
(cell suspen$ion or autolysate separated from autolysing mixture) was frozen and then freeze dried.
For cell suspension samples, the dried material was collected into a 70 ml homogeniser tube. About 15 ml of methanol containing 50 mg/1 of buthylated toluene (BHT) (Sigma-B1378) and 20 g of glass beads (0.5 mm diameter) were added to the tube. This mixture was cooled to 4 °c, disrupted for 30 seconds at 4000 rpm in a Braun Homogeniser, then immediately cooled to 4 °c again. Thirty millilitres of chloroform containing 50 mg/1 of
BHT were added to give the ratio 2:1 of chloroform: methanol by volume. The mixture was stirred for 2 hat room temperature, and filtered through Whatman
44 filter paper under nitrogen gas. The residue was extracted a further two times with 45 ml of chloroform:methanol. All the extracts were pooled, washed by shaking with 0.25 volume of 0.88 % (w/v) sodium chloride solution, and left overnight at 4 °c. The organic phase, which was at the bottom, was transferred to a 100 ml round bottom flask, and dried by rotary evaporation at 30 °c, to give the lipid extract.
For autolysate samples, the freeze-dried material was immediately extracted three times with 45 ml of chloroform-methanol (2:1, by volume, containing 50 mg/1 BHT) as described already for the cell suspension.
The lipid extract was dried by rotary evaporation.
Lipid extracts were dissolved in 1.0 ml of chloroform containing 50 rng/1 BHT and used for analysis by thin layer chromatography (TLC).
Separation of individual lipids 70
Two microli tres of extract were spotted onto pre-coated thin layer plates of silica gel 60 (Merck-5721). The plates were developed with a mobile phase containing chloroform : methanol : isopropanol: 0.25 % w/v potassium chloride solution: ethylacetate (30 : 9 25 : 6 : 18, by volume) for separation of phospholipids (Hedegaard and
Jensen 1981). Neutral lipids were separated by a two stage one dimensional procedure using the mobile phase diethylether : benzene : ethanol : acetic acid ( 40 : 50
2 0.2, by volume) in the first stage, followed by elution with n-hexane diethyl ether (96 4 by volume) in the second stage after drying the first stage at room temperature (Sahasrabudhe 1979). Spot visualisation was done by spraying the thin layer plates with copper acetate 3 % (w/v) in phosphoric acid 8 % (v/v) reagent, then heating at 180 °c for 20 min (Hedegaard andJensen 1981) . The spots were scanned with an Atago densitometer Kemic-H fitted with a 590 nm wavelength filter to quantify the individual lipid spots.
The following lipid standards were similarly resolved by thin layer chromatography, neutral lipids including monoglyceride, diglyceride, triglyceride, ergosterol, cholesteryl ester and palmitic acid, and phospholipids including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, cardiolipin, phosphatidylglycerol and phosphatidylethanolamine. These standards were obtain('d from the Sigma Chemical Company.
3.12 Free Fatty Acids (Takakuwa and Watanabe 1981) 71
The unvisualized free fatty acid spots on TLC plates were scrapped off and put into screw-tefloned-cap tubes.
Four and half millilitres of sulphuric acid (2 % v/v, in methanol) , were added to each tube. Nitrogen gas was blown into the tubes to replace air, the tubes were capped tightly, and then heated overnight at 60 °c. Fatty acid methylesters formed were extracted three times with
5 ml n-hexane. The hexane-extracts {in the top part) were collected and dried to a residue by vacuum evaporation at room temperature. The residues {fatty acid methyl esters) were each dissolved in 0.2 ml iso-octane.
These esters were analysed by gas chromato- graphy (Varian 3300) through a glass column ( 2 m in length and 2 mm in internal diameter) packed with 10 %
DEGS-PS on 80/100 mesh Supelcoport {Supelco Inc., PA,
USA). The injection port temperature was 200 °c, detector temperature was 215 °c and column temperature was 100-200
0 c (gradient) with increasing temperature of 6 °c/min.
Initial column hold time was 2 min. The flow rate of gases through the s y .s-/~,-, was air 5 ml/ sec, hydrogen 5 c~,,,e, -::,111$ ml/10 sec andj(ni trogeq) 5 ml/15 sec. Fatty acid methyl ester standards were obtained from the Sigma Chemical
Company.
3.13 Glycerol
Glycerol was specifically measured using an enzymatic procedure (Anon 1987). The reagent were obtained in 72 kit form from Boehringer Mannheim GmbH, Biochemica and the method followed as described by this manufacturer.
3.14 Cytological Observations
Cytological observations of yeast cells during autolysis were carried out by microscopical analysis using scanning and transmission electron microscopy. The autolysis of yeast cells was conducted as described already.
Immediately after sampling, the suspension of autolysing cells was mixed with an equal volume of fixative solution of glutaraldehyde (6 % w/v) in O. 2 M cacodylate buffer, pH 7.4 and left at 4 °c for 3 h.
Scanning electron microscopy (Watson and Arthur 1977).
The aldehyde fixed cells were washed three times with 0.1
M cacodylate buffer and dehydrated by serial passage through ethanol (50 %, 70 %, 95 %, 100 % and 100 %) and, finally, twice in dry acetone. Each passage was conducted for 5 min. The cells were suspended in dry acetone.
About O. 5 ml of this suspension was filtered through a
0. 45 µm nylon membrane filter (Gelman Sciences Inc.
66606) , then treated in a critical point dryer ( Bio-Rad-
1654, Micro Science Division). The specimens were mounted on aluminium stubs and coated with _gold-palladium
( Polaron Equipment Ltd - SEM coating Unit E5000) . Cell specimens were viewed in a scanning electron microscope 73
using an accelerating voltage of 20 kV (Cambridge
Instrument - Stereoscan 360).
Transmission electron microscopy
Fixation and embedding of samples were carried out following a modified method of Spurr (1969), Hayat and
Giaquinta ( 1970) , Bain and Gove (1971), Kopp (1975) and Streiblova (1988).
The aldehyde fixed cells were washed four times with O.1 M cacodylate buffer. For further fixation, the cell pellet was covered with 2-3 drops of 2 % w/v osmium tetroxide in 0.1 M cacodylate buffer pH 7.2, and left for 4 hat room temperature, then suspended in 2 % agar at gel point. Small blocks of about one cubic millimetre were cut from the agar after gelling, washed once with cacodylate buffer for 15 min and twice with 2 % sodium acetate each for 5 min, and then stained with 2 % uranyl acetate for 60 min. The blocks were dehydrated by serial passage through ethanol ( 50 % , 70 % , 95 % , 100 % and 100 %), and then dry acetone (two changes), each for
15 min (using a Lynx microscopy tissue processor). The blocks were then treated for embedding in epoxy resin by passage through epoxy resin : acetone (1:1) for 1-2 h, in epoxy resin acetone ( 9: 1) for over night, in 100 % epoxy resin for 24 h (twice). Finally, they were put into gelatin capsule moulds containing 100 % epoxy resin and polymerised at 70 °c for two days (Spurr 1969). Thin sections of about 90 nm thickness of the resin embedded 74
specimens were obtained by ultracutting using a glass knife, and were mounted on 200 mesh copper grids. Post staining of the specimens was carried out with a 4 % aqueous solution of uranyl acetate followed by lead citrate (Reynolds 1963) . Grid specimens were viewed in a transmission electron microscope (Hitachi 7000).
Measurement of cell wall thickness
Using vernier callipers (M.T.I. Qualos), the thickness of the walls was measured from the transmission electron micrographs of thin sections. For each stage of autolysis
(0, 5, and 10 days), wall thickness was measured on five different cells at five different locations on the wall, excluding the budding area. The mean thickness of the wall at each autolysis stage was calculated from the 25 measurements. The data were adjusted for the magnificat ion factors. 75
4. RESULTS
4.1 Comparative Survey Of Autolysis In Several Species And Strains Of Yeasts
4.1.1 Introduction
Studies on the autolysis of yeasts have focussed on one species, namely, S. cerevisiae (Vosti and Joslyn 1954a,
Joslyn 1955, Joslyn and Vosti 1955, Farrer 1956 and
Arnold 1981b). Although different authors have used different strains of this species, their conditions of study were not comparable, thereby making it difficult to conclude if there were any major differences between strains with respect to autolytic behaviour.
The autolytic behaviour of K. apiculata and C. stellata, two species that make significant contributions to wine fermentation (Fleet 1990a), have not been reported previously.
As a preliminary survey to more detailed studies presented in the next chapter, this chapter compares the autolytic reactions in three strains each of
S. cerevisiae, K. apiculata and C. stellata.
4.1.2 Results
Cell viability
For all species and strains, cell viability was lost during autolysis. Starting with initial populations of 76
106-108 cfu/ml, no viable cells were detected after autolysis for 5 days (Table 4).
Solubilization of cell biomass
For all strains, the weight of insoluble cell biomass decreased during autolysis (Table 5). The dry weight of the cellular material decreased by about 25-30 % for each of the yeasts, although a little higher loss (33 %) was noted for K. apiculata 521.
This loss of cell weight was reflected as an increase in soluble biomass that occurred in the supernatants (autolysates) of the autolysing suspensions.
For most yeasts, approximately 21-23 % of the initial cell material was recovered in the soluble form.
Kloeckera apiculata 20~, K. apiculata 521 and C. stellata 504 yielded slightly higher soluble material in the supernatant (25-27 %) . It is noteworthy for all yeasts, that a small amount (<2 %) of cell material was solubilized by the operations of cell suspension in buffer, followed by separation of the supernatant, in the absence of any autolysis (0 day data).
It is evident from the data for all yeast strains that the loss in dry weight of the cells during autolysis was not fully recovered as soluble material in the supernatants. For example, with S. cerevisiae 2180a, loss in cell weight approximates 27 % after autolysis for 5 days, but only about 23 % of this weight was recovered as 77
TABLE 4. The viability of cells of Saccharomyces cerevisiaeJ K/oeckera apiculata, and Candida stel/ata after autolysis for five days
Autolysis Viable counts Yeast species and strains time
(days) (cfu/ml)
Saccharomyces cerevisiae 2180a 0 4.8 X 10 8
5 a
HB350 0 1.0 X 10 8
5 a
EC1118 0 1.ox10 8
5 a
Kloeckera apiculata 202 0 1.0X10 6
5 a
521 0 1.7X10 8
5 a
412 0 1.2 X 10 7
5 a
Candida stellata 8008 0 1.7X 10 8
5 a
800MEA 0 1.6X10 7
5 a
504 0 5.2 X 10 7
5 a
a= Viable counts not detectable in 0.2 ml of sample, i.e., less than 5 cfu/ml. 78
soluble material in the supernatant (autolysate).
Reproducibility of data on dry weight analysis
Because of the importance that dry weight determinations have in calculating the extent of autolysis, Table 6 and
Table 7 show the reproducibility of data for insoluble and solubilized cell material. For the insoluble cell material, the mean deviation calculated from 54 determinations was ±2.7 % while that for the solubilized material, calculated from 15 determinations, was ±4.6 %.
Carbohydrates
Table 8 shows the solubilization of carbohydrate during autolysis of nine yeast strains. For the three strains of
S. cerevisiae, the total carbohydrate solubilized represented only 1. 5-2. 0 % of the initial cell weight.
Similar proportions of solubilization were noted for K. apiculata 412, C. stellata 8008, c. stellata 800MEA.
However, notably higher proportions, ( 4-6 %) were solubilized from K. apiculata 202, K. apiculata 521 and
C. stellata 504. Only trace amounts (less than 0.15 %) of carbohydrate were recovered as reducing sugars or glucose.
Organic acids
Table 9 shows the organic acids recovered from autolysates. Figure 3 illustrates the separation of acids
-.J -.J
I.O I.O
-
-
weight weight
----
1.9 1.9
biomass biomass
1.5 1.5
0.9 0.9
1.3 1.3
0.6 0.6
1.4 1.4
1.4 1.4
0.7 0.7
0.7 0.7
-----
25.0 25.0
21.1 21.1
22.8 22.8
20.6 20.6
27.1 27.1
22.7 22.7
25.0 25.0
21.6 21.6
23.0 23.0
Initial Initial
cell cell
% %
of of
------·-· ------·-·
------·-
biomass biomass
---
______
·------
1.4 1.4
weight weight
2.7 2.7
0.1 0.1
1.9 1.9 0.1 0.1
3.4 3.4
1.7 1.7
2.6 2.6
1.6 1.6
0.1 0.1
0.1 0.1
3.9 3.9
3.4 3.4
0.1 0.1
0.1 0.1
0.1 0.1
0.1 0.1
0.1 0.1
days days
(mg/ml) (mg/ml)
Soluble Soluble
Dry Dry
five five
tor tor
autolysis autolysis
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0
0.0 0.0 0.0 0.0
0.0 0.0
0.0 0.0
(%) (%)
29.6 29.6
25.5 25.5
26.3 26.3
25.0 25.0
32.6 32.6
30.3 30.3
25.7 25.7
25.7 25.7
26.7 26.7
---
Loss Loss
after after
------
-·----
-
-
biomass biomass
strains strains
and and
---
------
-
7.6 7.6
5.1 5.1
6.8 6.8
8.4 8.4
7.4 7.4
5.5 5.5
7.4 7.4
5.3 5.3
7.6 7.6
5.5 5.5
9.7 9.7
Cell Cell
weight weight
---
10.8 10.8
12.0 12.0
16.1 16.1
11.4 11.4
14.4 14.4
11.0 11.0
15.0 15.0
(11'!9£~1) (11'!9£~1)
Dry Dry
species species
yeast yeast
5 5
5 5
0 0
5 5
0 0
5 5
5 5
5 5
0 0
5 5
0 0
5 5
5 5
0 0
0 0
0 0
0 0
0 0
time time
(days) (days)
different different
Autolysis Autolysis
of of
replicates. replicates.
biomass biomass
S0a S0a
412 412
504 504
SOOMEA SOOMEA
EC1118 EC1118
8008 8008
HB350 HB350
521 521
21 21
202 202
three three
cell cell
of of
of of
strains strains
mean mean
cerevlsiae cerevlsiae
and and
the the
apiculata apiculata
Solubilization Solubilization
stellata stellata
5. 5.
species species
represent represent
Yeast Yeast
Candida Candida
Kloeckera Kloeckera
Saocharomyces Saocharomyces
Data Data
TABLE TABLE --- 80
TABLE 6. Reproducibility of data on dry weights of yeast biomass during autolysis for five days
Autolysis Dry weight of biomass Yeast species and strain ± time Percent mean deviation
(days) (mg) a
Saccharomyces cerevisiae 2180a 0 15.0 ± 2.7 % 5 11.0 ± 3.0 % H8350 0 7.4 ± 2.7 %
5 5.5 + 2.4%
EC1118 0 7.4 + 2.7 %
5 5.5 ± 2.4%
Kloeckera ap/culata 202 0 7.6 ± 2.6 %
5 5.3 + 2.5 %
521 0 14.4 ± 3.3 % 5 9.7 + 2.1 %
412 0 11.4 ± 2.9%
5 8.4 ± 3.2 %
Candida ste/lata 8008 0 16.1 ± 2.7 %
5 12.0 ± 2.2 %
800MEA 0 6.8 ± 2.9%
5 5.1 ± 2.6% 504 0 10.8 ± 3.1 %
5 7.6 ± 2.6 %
Total of percent mean deviation ± 48.6 %
Average of percent mean deviation + 2.7% a = Data from Table 5. 81
TABLE 7. Reproducibility of data on dry weights of yeast biomass solubilized after autolysis for five da}'S
Dry weight of biomass Yeast species and strain solubilized ± Percent mean deviation
(mg)a
Saccharomyces cerevisiae 2180a 3.4 ± 1.9 %
H8350 1.6±6.3%
EC11 ~8 1.7±2,0%
Kloeckera apiculata 202 1.9:: 8.8 %
521 3.9 ± 4.3%
412 2.6 ± 6.4%
Candida stellata 8008 3.4 ± 2.9%
800MEA 1.4 ± 4.7 %
504 2.7 ± 3.7%
! j Total of percent mean deviation ± 41.0 %
: Average of percent mean deviation + 4.6%
a = Data from Table 5. TABLE 8. Concentration of carbohydrate in soluble autolysates of yeasts after autolysis for five days
-----·------·------r------r-----~--- Autolysis Initial Total carbohydrate Reducing sugars Glucose I Yeast species an d strains time cell weight %Initial %Initial % Initial ---· (days) (mg/ml) (ug/ml) cell weight (ug/ml) cell weight (ug/ml) cell weight Saccharomyces ~revisiae 2180a 0 15.0 4.6 0.03 3.0 0.02 1.4 0.01 5 284.8 1.90 9.6 0.06 11.5 0.08
H8350 0 7.4 68.7 0.92 2.5 0.03 1.7 0.02 5 133.2 1.79 6.5 0.09 9.6 0.13
EC1118 0 7.4 1.4 fJ.02 2.1 0.03 2.2 0.03 5 116.1 1.57 6.2 0.08 7.0 0.09
Kloeckera apicu/, :1ta 202 0 7.6 23.3 0.31 3.7 0.05 2.4 0.03 5 462.6 6.09 7.7 0.10 10.0 0.13
521 0 14.4 6.9 0.05 2.7 0.02 2.6 0.02 5 603.1 4.19 14.1 0.10 17.2 0.12
412 0 11.4 7.9 0.07 2.5 0.02 2.2 0.02 5 242.7 2.12 6.0 0.05 8.6 0.08
Candida stel/ata 8008 0 16.1 2.4 0.01 3.0 0.02 0.3 0.00 5 168.2 1.04 10.2 0.06 10.0 0.06
800MEA 0 6.8 13.8 0.20 4.5 0.07 2.6 0.04 5 110.6 1.63 6.1 0.09 6.5 0.10
504 0 10.8 15.7 0.14 1.2 0.01 5.8 0.05 5 611.2 5.64 13.3 0.12 14.4 0.13
0) Data represent the mean of three replicates. l"v 83
in autolysates by HPLC. The concentrations of total organic acids found in autolysates of most strains were approximately 3-5 % initial cell weight, with the except ion of that for S. cerevisiae HB350 which was 7.5 % and·
that for K. apiculata 202 and 412 which were less than 2
% • With a few exceptions, the same acids were found in autolysates of all of the strains. Lactic acid was not found in the autolysates of all strains of S. cerevisiae. Formic acid was not found in the autolysates of K.
apiculata 412 and C. stellata 800MEA, and citric acid did not occur in C. stellata 800MEA autolysates.
There was some variation in the concentration of individual acids released by different strains. Propionic and succinic acids were the two main acids found in the autolysates of all three strains of S.
cerevisiae. The next important acid in terms of concentration, varied with the strain. However, citric acid was found in the lowest concentrations for all three strains. Propionic and succinic acids were the two main acids found in autolysates of K. apiculata 202, but for
K. apiculata 521 the two main acids were lactic and malic acids, and for K. apiculata 412 they were oxalic and propionic acids. Propionic and succinic acids were the main acids released by all three strains of C. stellata, but after these, the next most significant acids varied with the strains. Notably, no citric and formic acids were found in autolysates of C. stellata SOOMEA, but were found for the other two strains. An unidentified peak 84.
(presumably an organic acid) was found in the autolysates of s. cerevisiae and, especially, K. apiculata.
Protein
The protein released from yeast cells after autolysis for
5 days is shown in Table 10. Depending on strain, this protein represented 10-12 % of the initial cell dry weight for S. cerevisiae, approximately 13 % for K. apiculata and 9-11 % for C. stellata. All of the strains, excluding S. cerevisiae 2180a, gave 0.1-0.3 % release of protein by washing with buffer (0 day data).
Amino acids
Amino acids found in the autolysates of yeasts after five days of autolysis are shown in Table 11. Figure 4 shows separation of the amino acids in autolysates of yeasts by HPLC. In general, total amino acids were found at concentrations of about 8. 0 % of the initial cell weight in S. cerevisiae, 5.0-8.0 % in C. stellata, and
2.0-6.0 % in K. apiculata. Most strains of the same species produced similar amino acids. Table 12 shows the five main amino acids in decreasing concentration obtained in the autolysates of each yeast strain. The strains of S. cerevisiae produced the same main amino acids, phenylalanine, glutamic acid, leucine, alanine and
arginine but at different concentration. The same amino TABLE 9. Concentration of organic acids in autolysates of yeasts after autolysis for five days
Saccharomyces cerevisiae J(loeckera apiculata Candida stellata Acid -- Ac Id Acid 2180a H8350 EC1118 202 521 --412 8008 800MEA 504 (ug/mg) a (u_9{mg)a (ug/mg)a --
Propionic 14.5 40.9 13.3 Prop ionic 4.5 1.9 3.3 Propionic 11.0 20.0 11.5
Succinlc 5.5 9.0 7.0 Succinic 3.4 2.6 2.3 Succinic 8.8 10.3 12.1
Malic 3.1 6.2 5.5 Acetic 2.6 3.3 1.2 Oxalic 5.5 7.9 1.7
Formic 3.1 2.8 3.2 Oxalic 2.1 2.8 6.8 Malle 3.7 7.5 5.3 Acetic 3.0 4.4 2.6 Malic 1.6 4.3 1.6 Citric 2.5 - 3.4 Oxalic 2.3 6.9 3.5 Lactic 1.3 5.2 0.6 Acetic 2.5 5.8 7.4 Citric 0.7 0.8 0.8 Formic 1.1 2.8 - Formic 1.9 - 2.3 Tartaric 0.7 4.0 3.9 Tartaric 0.7 2.2 1.0 Tartaric 1.8 2.4 3.1 Lactic - -- Citric 0.7 1.5 2.1 Lactic 1.4 1.2 1.2
Total (ug/mg) a 32.9 75.0 39.8 Total (ug/mg)a 18.0 26.6 18.9 Total (ug/mg) a 38.9 54.9 48.0
Percent initial Percent initial Percent initial cell weight 3.29 7.50 3.98 cell weight 1.80 2.66 1.89 cell weight 3.89 5.49 4.80
0) a= Calculated as weight of acid in autolysate per mg of initial dry weight of cells. Ul Data represent the mean of three replicates. 86
1
)
7 2
4
8
9 9
5
X
(a) ( C)
1
5
5
( b) ( d)
Figure 3. Resolution of organic acids in yeast autolysates using HPLC. Representative e.lution profiles. Standard mixture of acids. (b) Autolysate of Saccharomyces cerevisiae 2180a. (c) Autolysate of Kloeckera apiculata 202. (d) Autolysate of Candida stellata 8008. 1. Oxalic acid (1263 ug/ml) 2. Citric acid (4101 ug/ml) 3. Tartaric acid (4758 ug/ml) 4. Malic acid (4954 ug/ml) 5. Succinic acid (2523 ug/ml) 6. Lactic acid (4366 ug/ml} 7. Formic acid {5611 ug/ml} 8. Acetic acid (7536 ug/ml) 9. Propionic acid (7144 ug/ml}, and X. Unidentified peak. 87
TABLE 10. Concentrations of prot~in in autolysates of yeasts after autolysis for five days
Autolysis Protein Yeast species time % Initial (days) (ug/mg) a cell weight
Saccharomyces cerevisiae 2180a 0 0.2 0.02
5 104.4 10.44
H8350 0 1.2 0.12
5 109.1 10.91
EC1118 0 1.7 0.17
5 117.0 11.70
Kloeckera apiculata 202 0 2.4 0.24
5 129.9 12.99
521 0 1.0 0.10
5 132.3 13.23
412 0 1.5 0.15
5 125.8 12.58
Candida ste/lata 8008 0 1.2 0.12
5 100.9 10.09
800MEA I 0 2.8 0.28 5 87.2 8.72 I 504 0 2.4 0.24
5 107.9 10.79
a= Calculated as weight of protein in autolysates per mg of initial dry weight o, cells. Data represent the mean of three replicates. 88
acids were also found in the autolysates of K. apiculata and c. stellata, with the exception of K. apiculata 521 and 412 and C. stellata 800MEA. Kloeckera apiculata 521 yielded lysine instead of alanine, and K. apiculata 412 produced isoleucine instead of arginine, while C. stellata 800MEA produced lysine instead of phenylalanine.
Ribonucleic acid
Table 13 shows the concentrations of RNA in yeast cells and in autolysates before and after autolysis for five days. The concentrations of RNA were estimated using two different methods, namely, the optical density method and the orcinol method.
There were only minor differences between the
RNA concentrations measured by both methods. The orcinol method consistently gave slightly higher RNA concentrat ions (Table 13).
The RNA content of most strains at the beginning of autolysis was in the range 3. 5-4. 5 % of initial cell weight. Two strains gave values outside this range. These were K. apiculata 412 with RNA of 5.7-6.0 % of cell dry weight and C. stellata 504 with 2.4-2.8 %. The RNA content of the cells decreased by 90-98 % after autolysis with the exception of K. apiculata 412 (87-89 %) and C. stellata 504 (75-77 %) and this was reflected in RNA material being recovered in the soluble part of the auto- TABLE 11. Concentrations of amino acids in autolysates of yeasts after autolysis for five days
Saccharomyces cere_yj_si~ Kloeckera apiculata Candida stellata Amino acids 2180a HB350 EC1118L Amino acids 202 521 412 Amino acids 8008 800MEA 504 (ug/mg) a (ug/mg) a (ug/mg)a
Phenylalanine 9.3 8.6 7.3 Glutamic acid 12.8 5.5 2.0 Arginine 13.0 12.8 I 10.2 Glutamic acid 8.3 9.4 7.9 Alanine 12.2 2.8 1.5 Phenylalanine 8.2 3.5 2.6 Leucine 7.2 7.2 7.4 Leucine 6.6 3.1 2.4 Glutamic acid 7.5 5.4 5.4 Alanine 6.5 7.6 9.2 Arginine 4.8 7.2 1.2 Alanine 6.6 5.4 2.3 Arginine 6.3 9.1 10.2 Phenylalanine 4.0 3.6 2.9 Le•Jcine 6.3 3.5 5.4 lsoleucine 5.6 5.2 4.5 Serine 3.3 2.0 0.8 Tyrosine 3.8 1.6 1.7 Valine 4.5 4.8 3.6 Valine 2.7 2.3 0.5 lsoleucine 3.7 2.4 1.5 Aspartic acid 4.4 3.8 3.5 lsoleucine 2.7 2.0 1.6 Valine 3.6 2.1 1.9 Serine 3.8 4.3 3.2 Glycine 2.6 1.6 0.7 Serine 3.4 2.0 2.3 Tyrosine 3.8 4.3 3.1 Cystine 2.6 - 0.8 Histidine 3.3 2.7 1.4 Threonine 3.7 3.9 3.2 Aspartic acid 2.3 1.7 1.2 Aspartlc acid 3.2 1.5 1.7 Lysine 3.4 5.3 6.0 Threonine 2.1 1.7 1.0 Threonine 3.2 1.4 1.5 ,i-aminobutyric acid 3.2 2.3 2.3 Histidine 2.0 1.2 0.3 Glycine 3.0 1.5 1.7 Glycine 2.8 3.2 2.6 'o-aminobutyric acid 1.7 2.5 - ~-aminobutyric acid 2.5 0.5 2.8 Histidine 1.7 1.6 3.3 Methionine 0.8 0.5 - Lysine 2.2 2.8 5.3 Methionine 1.3 1.2 1. 1 Taurine 0.5 0.5 0.2 Methionine 1.1 0.5 0.3 Taurine 0.4 0.3 0.3 Lysine - 3.9 - Cystine 1.0 - - Tyrosine - 1.9 1.1 Taurine 0.4 1.3 0.8
------>------
T o ta I (ug/mg) a 76.2 82.1 78.7 T o t a I (ug/mg) a 63.7 44.0 ~8.2 T o t a I (ug/mg) a 76.0 50.9 48.8 a= Calculated per mg of initial dry weight of cells. CD Data represent the mean of three replicates. I.O 90
\_
~ 4 12 2 14 " II
I) L_ 10 ( b)
I) J!)
1 ~ 12 14 _;/' ~ i 8 -'\ '·:;n u q~' •
II
JO Jj\
Figure 4. Separation of amino acids In autolvsates of yeasts bv HPLC. (a) Standard mixture of amino acids. (b) Autolysate of Saccharomy_oes cerevisiae 2180a. (c) Autolysate of Kloeckera apiculata 202. (d) Autolysate 6f Candida ste//ata 8008. 1. Taurine. 2. AsQartlc acid. 3. Threonlne. 4. Sertne. 5. Glutamlc acid. 6. Glvclne. 7. Alanine. 8. Valine. 9. L-ystlne. 10. Methionine. 11. lsoleuclne. 12. Leuclne. 13. Tyrosine. 14. Phenylalanine. 15.t- amlnobutyrlc acid. 16. Hlstldlne. 17. Lysine. 18. Ammonia. 19. Ethanol amine. 20. Arglnlne. 91
TABLE 12. Main amino acids found In autolysates of yeasts after al.itolysis for five days a
Saccharomyoes cerevisiae
2180a HB350 EC1118
Phenylalanine Glutamic acid Arginine
Glutamic acid Arglnine Alanine
Leucine Phenylalanine Glutamic acid
Alanine Alanine Leucine
Arginine Leucine Phenylalanine
Kloeckera apiculata
202 521 412
Glutamic acid Arginine Phenylalanine
Alanine Glutamic acid Leucine
Leucine Lysine Glutamic acid
Arginine Phenylalanine lsoleucine
Phenylalanine Leucine Alanine
Candida stellata
8008 800MEA 504
Arginine Arginine Arginine
Phenylalanine Alanine Glutamic acid
Glutamic acid Glutamic acid Alanine
Alanine Lysine Phenylalanine
Leucine Leucine Leucine
a= Amino acids of each strain in decreasing order of concentration found in autolysate. 92
lysate. However, not all of the RNA apparently lost from the cell was recovered in the autolysates. For example, while the cellular content of RNA in S. cerevisiae 2180 decreased by 96-98 % after autolysis for 5 days, only ,fl,L 80-85 % was recovered in~autolysate.
For all strains, a very small percent {0-0.3 %) of the cell RNA was extracted into the autolysing buffer by suspension and centrifugation (0 day data).
Deoxyribonucleic acid
Table 14 shows the concentrations of DNA in yeast cells and autolysates before and after autolysis for five days.
The DNA concentrations in both cells and autolysates were estimated by two different methods, namely, the diphenylamine method and the benzimidazol dye method.
Despite several repetitive analysis, the dye method always g4ve much lower values for the concentrations of
DNA in the cell than the diphenylamine method. Using the diphenylamine method, most yeast strains before autolysis contained DNA at 0.2-0.5 % of initial cell weight, with the exception of S. cerevisiae EC1118 which contained 1.2
%. With the dye method, yeast cells before autolysis contained DNA at 0-0.08 % of initial cell dry weight.
After autolysis, the DNA content of the cells had decreased by 36-55 %, with the exception of S.
cerevisiae 2180 and C. stellata 8008 which gave lower decreases of 21 % and 29 %, respectively. Such percent- TABLE 13. Concentrations of ribonucleic acid (ANA) in cells and autolysates of yeast after autolysis for five days
Autolysis Initial Cell ANA Cell ANA recovered in autolysates Yeast species and strains cell % Decrease % Increase time weight % Initial in % Initial based on initial cell weight ANA content cell weight ANA content (ug/ml) of cells (ug/ml) et cells ,days) (mg/ml) a b a b a b a b a b a b - Saccharomyces cerevisiae 2180a 0 15.0 550.9 559.9 3.67 3.73 4.5 6.0 0.03 0.04 5 11.8 21.6 0.08 0.14 97.9 96.1 449.1 479.0 2.99 3.19 80.7 84.5
H8350 0 7.4 288.7 318.4 3.90 4.30 14.1 19.3 0.19 0.26 5 13.7 26.6 0.19 0.36 95.3 &1.6 230.6 267.8 3.12 3.62 75.0 78.0
EC1118 0 7.4 288.2 313.0 3.89 4.23 5.7 7.4 0.08 0.10 5 9.3 19.1 0.13 0.26 96.8 93.9 268.3 296.7 3.63 4.01 91.1 92.4
Kloeckera apiculata 202 0 7.6 292.2 300.6 3.84 3.96 6.8 12.1 0.09 0.16 5 10.1 19.6 0.13 0.26 96.5 93.5 254.3 274.1 3.35 3.61 84.7 87.2
521 0 14.4 580.3 639.4 4.03 4.44 4.0 11.5 0.03 0.08 5 38.7 60.8 0.27 0.42 93.3 90.5 499.2 564.5 3.47 3.92 85.3 86.5 412 0 11.4 652.1 688.6 5.72 6.0~ 7.3 12.6 0.06 0.11 5 71.2 87.1 0.62 0.76 89.1 87.4 560.6 608.6 4.92 5.34 84.8 86.6
Candida stellata 8008 0 16.1 661.3 737.4 4.11 4.58 7.8 8.1 0.05 0.05 5 14.7 21.5 0.09 0.13 97.8 97.1 516.2 584.4 3.21 3.63 76.9 78.2
800MEA 0 6.8 238.0 262.5 3.50 3.86 15.0 17.7 0.22 0.26 5 17.2 28.3 0.25 0.42 92.8 89.2 204.0 231.7 3.00 3.41 79.4 81.5
504 0 10.8 259.9 303.2 2.41 2.81 28.2 36.8 0.26 0.34 5 60.6 76.9 0.56 0.71 76.7 74.6 194.9 237.7 1.80 2.20 64.1 66.3
a= Measured by optical density method.
b= Measured by orcinol method. \,0 Data represent the mean of three replicates. w 94 ages of DNA decrease were not recovered in the soluble autolysates, where the recovery values ranged from 1.8
% (S. cerevisiae EC1118) to 14.4 % (S. cerevisiae HB350) (diphenylamine method). When · the dye method was used to determine the DNA concentration, much higher percentages of DNA decrease were recorded after autolysis, ranging from 38 % for K. apiculata 202 to 100 % for K. apiculata 521, 412 and C. stellata 8008 and 504. However, such high losses were not detected by recovery in the autolysate where recovery values of 10-47 % were obtained.
For all strains, 0.03-0.34 % of DNA was extracted by the autolysing buffer during suspending and spinning down of the cells as shown in day 0 data.
Lipid
Table 15 shows the total lipid content in cells and autolysates of three yeast species before and after autolysis for five days. The total lipid content of the cells was approximately 3.2-4.0 % of cell dry weight. For
S. cerevisiae, this decreased by 48.7 % during autolysis while for K. apiculata and C. stellata the decreases were
22. 2 % and 30. 5 % , respectively. A greater proportion
(33.2 %) of the initial cell lipid was found in the autolysate of S. cerevisiae than for K. apiculata (8.1
%) and C. stellata (22.8 %) . TABLE 14. Concentrations of deoxyribonucleic acid (DNA) in cells and autolysates of yeasts after autolysis for five days ------·--- Autolysis Initial ~ Cell DNA Cell DNA recovered in autolys~tes __ Yeast species and strains cell % Decrease % Increase time weight % Initial in % Initial based on initial cell weight DNA content cell weight DNA content _ (ug/ml) of cells (ug/ml) of cells (days) (mg/ml) a b a b a b a b a b a b I Saccharomyces cerevisiae 2180a 0 15.0 23.4 8.4 0.16 0.06 0.1 0.3 C C 5 18.6 0,8 0.12 0.01 20.5 90.5 i.3 1.5 0.01 0.01 5.1 14.3
HB350 0 7.4 16.0 6.1 0.22 0.08 0.1 0.3 C 0.01 5 10.2 1.5 0.14 0.02 36.3 75.4 2.4 1.2 0.03 0.02 14.4 14.8
EC1118 0 7.4 91.4 3.2 1.24 0.04 0.1 0.3 C C 5 53.4 1.5 0.72 0.02 41.6 53.1 1.7 1.4 0.02 0.02 1.8 34,4
K/oeckera apicu/ata 202 0 7.6 19.0 3,4 0.25 0.04 0.1 0.3 C C 5 8.9 2.1 0.12 0.03 53.2 38.2 1.9 1.3 0.03 0.02 9.5 29.4
521 0 14.4 44.9 1.9 0.31 0.01 0.1 0.3 C C 5 20.2 0.2 0.14 C 55.0 - 5.9 1.2 0.04 0.01 12.9 47,4
412 0 11.4 47.6 4.8 0.42 0.04 0.1 0.3 C C 5 26.5 0.0 0.23 C 44.3 - 1.9 0.8 0.02 0.01 3.8 10.4
Candida ste/lata 8008 0 16.1 66.5 4.3 0.41 0.03 0.1 0.3 C C 5 47.1 0.5 0.29 C 29.2 88.4 4.5 1.7 0.03 0.01 6.6 34.9
800MEA 0 6.8 33.4 4.1 0.49 0.06 0.1 0.3 C C 5 19.2 0.6 0.28 0.01 42.5 - 1.2 0.8 0.02 0.01 3.3 12.2
504 0 10.8 41.3 3.0 0.38 0.03 0.1 0.3 C C 5 20.7 0.0 0.19 C 49.9 100.0 5.3 0.9 0.05 0.01 12.6 20.0
I a= Measured by diphenylamine method. c= Percent initial cell weight calculated was negligible (<0.005 %). b= Measured by benzimidazol method. Data represent the mean of three replicates. u,\0
0\ 0\
1.0 1.0
initial initial
~---
cells cells
on on
8.1 8.1
content content
22.8 22.8
33.2 33.2
--
Increase Increase
of of
-
-
-
% %
lipi,j lipi,j
. .
based based
---
-
-·-
-
----
cells cells
content content
in in
30.5 30.5
48.7 48.7
22.2 22.2
of of
Decrease Decrease
---
lipid lipid
% %
lipids lipids
days days
·------· ·------·
cells. cells.
-
-
of of
five five
Total Total
-
Initial Initial
weight weight
1.76 1.76
1.85 1.85
0.88 0.88
2.26 2.26
3.25 3.25
0.93 0.93
0.14 0.14
3.08 3.08
3.96 3.96
0.71 0.71
0.61 0.61
3.43 3.43
for for
% %
cell cell
weight weight
dry dry
autolysis autolysis
1.4 1.4
9.3 9.3 7.1 7.1
8.8 8.8
6.1 6.1
17.6 17.6
18.5 18.5
22.6 22.6
32.5 32.5
34.3 34.3
30.8 30.8
39.6 39.6
initial initial
(ug/ma)a (ug/ma)a
after after
of of
mg mg
yeasts yeasts
(C) (C)
per per
A A
A A
A A
A A
A A
C C
A A
C C
C C
C C
C C
C C
11 11
or or
(A) (A)
C C e
Autolysate Autolysate
autolysates autolysates
of of autolysates
5 5
5 5
0 0
5 5
5 5
and and
0 0
5 5
5 5
0 0
0 0
0 0
0 0
time time
(days) (days)
soluble soluble
Autolysis Autolysis
cells cells
and and
in in
cells cells
lipids lipids
in in
replicates. replicates.
total total
8008 8008
2180a 2180a
202 202
of of
lipids lipids
three three
of of
of of
mean mean
weight weight
cerevisiae cerevisiae
the the
as as
species species
Concentrations Concentrations
apiculata apiculata
stellata stellata
15. 15.
represent represent
Calculated Calculated
Yeast Yeast
Kloeckera Kloeckera
Candida Candida
Saccharomyces Saccharomyces
Data Data
a= a= TABLE TABLE 97
Figures 5-9 illustrate separation of the lipid classes by TLC. The phospholipids found in the cells were represented by phosphatidylcholine, phosphatidyl serine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Phosphatidyl ethanolamine was found only in K. apiculata and C. stellata (Table 16). Phosphatidylglycerol, phosphatidyl serine and phosphatidylinositol were the three most prevalent phospholipids, in descending order of concentration, in all three yeast species. The concen trations of these three phospholipids as well as those of the other phospholipids notably decreased during autolysis. No phospholipids were detected in the autolysates of any of the yeasts. The neutral lipids found were represented by monoglyceride, ergosterol, free fatty acids, diglyceride, triglyceride and cholesteryl ester (Table 16). The proportions of these different lipid components varied with the yeast species. Thus, the triglyceride was the most prevalent component in S. cerevisiae, but in K. apiculata and C. stellata cholesteryl ester was present at the highest concentrations. For all lipid classes, the concentration in the cells decreased during autolysis. They were recovered in low concentrations in the autolysates. Some were extracted by buffer washing of the cells (0 day data) . 98
PEA r PFA
PG FG CAR CAR
PI PS PS
PC PC
0 1 2 3 4 5 6 MIX MIX
Figure 5 Typical separation of phospholipid standards by thin layer chromatography. 0= origin; PC= phosphatidylcholine, PS = phosphatidylserine; PI= phosphatidylinositol; CAR= car diolipin; PG= phosphatidylglycerol; PEA= phosphatidylethanolamine; Mix= standard mixture of phospholipids. 99
CE
TG
X X
DG
PAL/ PAL
E
MG MG
0 I , 2 3 4 5 6 MIX MIX
Figure 6. Typical separation of neutral lipid standards by thin layer chromatography. 0= origin; MG = monoglyceride; E= ergosterol; PAL= palmitic acid; DG= diglyceride; TG= tri glyceride; CE= c holesteryl ester; X= impurities; Mix= standard mixture of neutral lipids. IPFA1 ro C2\R PI PS
PC
Io r· I sr sr sr dOc dOc d5c d5c dOa dOa d5a d5a
Figure 7 Representation of thin layer chromatograms of phospholipids in cell biomass (dOc, d5c) and autolysate (dOa, d5a) fractions of of Kloeckera apiculata 202 during autolysis for five days. O=- origin ; PC= phosphatidylcholine ; PS= phosphatidylserine ; PI= phosphatidyl1nositol; CAR= cardiolipin; PG= phosphatidylglycerol; PEA= phosphatidylethano lamine
---" 0 0 1 01
CE
'IG
X
[G PAL
E
u
u
X
0 ST ST ST dOc dOc d5c d5c
Figure 8. Representation of thin layer chromatograms of neutral lipids in cell biomass frastion of Kfoeckera apicufata 202 during autolysis for five days. 0= origin; MG= monogly ceride; E= ergosterol; PAL = palmitic acid; DG = diglyceride; TG= triglyceride; CE= cholesteryl ester; U = unidentified spots; X = impurities. 102
CE
X
rx; PAL
E
u
~ u
0 sr ST aoa dOa. dSa
Figure 9. Represen tation of thin layer chromatograms of neutral lipids In autolysate fraction of Saccharomyces ceravisiaa 2180a during autolysis for five days. 0= origin ; MG= monoglyceride; E= ergosterol; PAL= palmitic acid; DG = diglyceride; TG = trigly ceride : CE= cholesteryl ester; U= unidentified spots; X= impurities. ~
0
w
-
1.5 1.9
1.3
CE
4.2 8.8
7.6 2.6
3.3
3.5
14.0 12.2
acid.
1.3
1.9
TG
7.6 2.6 4.5 5.2
3.6 0.8 6.6 3.3
3.0
11.0
palmitic
phosphatidyl
-
as
1.3
8.3 4.3 2.2 DG 2.1
5.5
3.6 8.0 6.9
ester.
lipids
PEA=
(ug/mg)a
1.1
1.4
7.0
3.6 8.6
2.6
0.3 0.9 0.5 0.4
3.5 2.5 2.2
0.5
FFAb
Neutral
Determined
-
-
E
1.1
0.9 0.7
0.4 0.9
0.6
0.8
0.1
b==
CE=cholesteryl
1.6 1.4
1.6
0.6 0.6
0.3 0.1 0.3 0.3 0.6 0.2
0.7
0.£
0.1
MG
cells.
phosphatidylglycerol;
of
- -
- -
-
0.3
0.1 0.1
0.3
days
PEA
PG=
I
five
TG=triglyceride;
weight
-
-
--
PG
2.1
2.5
3.0
0.6
0.4
for
dry
-- -
-
0.3
0.9
0.7 0.3
0.2
CAR
initial
autolysis
I
of
CAR=cardiolipin;
(ug/mg)a
-
-
-
PI
after
1.0
0.2 0.3
0.8
0.8
0.6
mg
DG=diglyceride;
Phospholipids
per
-----
-
------
yeasts
PS
1.8
1.3
0.5
2.0
0.9 0.6
0.4
acids;
of
fatty
-
------
-
1.3
PC
0.9 0.5
0.3
0.2
0.7
autolysates
phosphatidylinositol;
autolysates
(C)
FFA=free
PI=
A A
A A
A
C
or
(A)
and
soluble
Cell
Autolysate
cells
and
in
5 C
5 C 0 5
5 0 5 A 0 C
5 C 0 C
0
0
time
(days)
cells
Autolysis
E=ergosterol;
in
classes
replicates.
phosphatidylserine;
lipids
lipid
8008 202
2180a
three
of
of
PS=
of
weight
mean
cerevisiae
as
MG=monoglyceride;
the
species
Concentration
apiculata
stel/ata
16.
represent
phosphatidylcholine;
Calculated
Yeast
Candida
K/oeckera
Saccharomyces
Data
PC=
ethanolamine;
TABLE
a= 104
Free fatty acids
With the extraction and GLC detection methods described in Section 3.12, no free fatty acids were detected in the autolysates.
4.2 Chemical Changes During The Autolysis Of Saccharo myces ~rev1s1ae 2180a, Kloeckera ~piculata 202, And Candida stellata 8008
4.2.1 Introduction
The previous chapter screened the changes in several strains and species of yeasts after autolysis for 5 days.
For more detailed information, this chapter has focussed only on three yeast strains, one for each species above mentioned, to see the kinetic changes during autolysis for 10 days.
4.2.2 Results
Cell viability
The viability of yeast cells rapidly decreased during autolysis (Table 17). Starting with initial cell populations of 107-108 cfu/ml, no viable cells of either
K. apiculata or C. stellata were detected after 24 h. The viability of S. cerevisiae had decreased by 87 % after one day, and thereafter no viable cells were detected. 105
TABLE 17. Decrease in the viability of cells of Saccharomyces cerevisiae , Kloeckera apiculata , and Candida stel/ata. during autolysis for 10 days
Autolysis i Viable counts Yeast species time
(days) (cfu/ml)
Saccharomyces cerevisiae 2180a 0 2.3X 10 8
1 0.3 X 10 8
5 a
10 a
Kloeckera apicu/ata 202 0 1.3X 10 7
1 a I I 5 I a
10 a
Candida stel/ata 8008 0 2.3 X 10 8
1 a
5 a
10 a I a= Viable counts not detectable in 0.2 ml of sample, i.e., less than 5 cfu/ml. 106
Solubilization of cell biomass
The decrease in cell dry weight and the soluble biomass recovered from autolysates at intervals during autolysis for 10 days are shown in Table 18 and Figure 10.
Loss in ce]l weight and production of soluble autolysate increased with time of autolysis. Cells of
K. apiculata gave the greatest loss of dry weight (33 %) after autolysis for 10 days. The other two yeasts, S. cerevisiae and C. stellata, gave slightly less (26-30 %) loss of weight. Reduction in cell dry weight was reflected in increases in the weight of autolysate.
However, not all of the loss in cell weight was recovered in the autolysate and, depending on yeast species, 5-10 % of the cell weight was not found in the autolysate.
Carbohydrates
Carbohydrates were solubilized from the cells during autolysis (Table 19; Figure 11). After 10 days, the total carbohydrates solubilized represented 7. 4 % of the cell dry weight of K. apiculata, and lesser values of 3 .1 % and 2.7 %, respectively, for S. cerevisiae and C. stellata. Only trace amounts of carbohydrate (less than
0.15 % of cell dry weight) were solubilized as either reducing sugars or glucose. Most carbohydrate was solubilized during the first five days of autolysis. 107
TABLE 18. Solubilization of cell biomass of different yeast species during autolysis for 10 days
Autolysis Cell biomass Soluble biomass Yeast species time Dry % Initial Loss Dry % Initial weight weight of weight weight of (days) (ug/ml cell biomass (%) (ug/ml cell biomass
Saccharomyces ceravisiaa 2180a 0 6.4 100.0 0.0 0.0 0.0
1 5.7 89.1 10.9 0.4 8.3
5 4.8 75.0 25.0 1.1 17.2
10 4.7 73.4 26.6 1.4 21.9
Kloeckara apiculata 202 0 3.6 100.0 0.0 0.1 a
1 3.0 83.3 16.7 0.4 11.1
5 2.6 72.2 27.8 0.8 22.2
10 2.4 66.7 33.3 1.0 27.8
Candida stallata 8008 0 7.9 100.0 0.0 0.0 0.0
1 7.0 88.6 11.4 0.5 8.3
5 5.7 72.2 27.8 1.4 17.7
10 5.5 69.6 30.4 1.6 20.3
a= The value was less than 0.05 %. Data represent the mean of three replicates. 108
120------a
80
eo
40
20
0 .., b .c 100 .....O> Q) 80 ~ ,- 60 ,- Q) 0 40 .... .,.....,ta 20 .,... 0 .,...C C .., 100 C Cl) 0 80 L Cl) a. 60
40
20
0 0 2 4 6 8 10 12 Time of autolysis (days)
Figure 10. Solubilization of cell dry weight ( •) and recovery of soluble biomass ( -0- ) of (a) Saccharomyces cerevisiae 2180a, (b) Kloeckera apiculata 202, and (c) Candida stellata 8008 during autolysis for 10 days. TABLE 19. Concentrations of carbohydrates in autolysates of yeasts during aJtolysis for 10 days
Autolysis Initial Total carbohydrate Reducing sugars Glucose Yeast species time cell %Initial %Initial %Initial weight cell cell cell (days) (mg/ml) (ug/ml) weight (ua/ml) weight Cua/ml) weight
Saccharomyces cerevisiae 2180a 0 6.4 3.3 0.05 1.7 0.03 1.0 0.02
1 59.4 0.93 2.6 0.04 1.8 0.03
5 159.1 2.49 5.8 0.09 5.8 0.09
10 198.5 3.10 6.1 0.10 5.8 0.09
Kloeckera apiculata 202 0 3.6 6.9 0.19 2.7 0.08 0.2 0.01
1 55.1 1.53 3.6 0.10 3.2 0.09
5 211.5 5.88 4.0 0.11 4.1 0.11
10 267.9 7.44 5.0 0.14 4.3 0.12
Candida stallata 8008 0 7.9 4.2 0.05 1.6 0.02 0.5 0.01
1 52.7 0.67 4.9 0.06 3.1 0.04 5 127.2 1.61 4.9 0.06 5.0 0.06
10 209.4 2.65 6.1 0.08 5.6 0.07 ..... 0 1.0 Data represent the mean of three replicates. ....
0
....
12
10
,
8
10
(-O-)
for
Candida
autolysates
and
in 2180a
autolysis
(days)
6
-o-),
(
during
202
autolysis
cerevisiae
carbohydrate
of
-6-)
(
4
total
Time
apiculata
8008
of
Saccharomyces
stellata
Kloeckera
Recovery days.
of
2
11.
Figure
0
2
4 8
6
0
a, M Q)
a,
a, :'l u
a, c:: u c:: t)
~
~ .,i
~
...... c: ....
•rl
•rl
-rt ·ri 111
Organic acids
Organic acids were recovered in the autolysates of three yeast species (Table 20; Figure 12). These acids represented a mixture of oxalic, citric, tartaric, malic, succinic, lactic, formic, acetic and propionic acids.
After autolysis for 10 days, the solubilized acids, collectively, represented approximately 4.9-5.6 % of the cell weight of S. cerevisiae and C. stellata, and 2.5 % of the weight for K. apiculata. Release of the acids into the autolysate had reached their maximum value by 5 days for K. apiculata, but continued until 10 days for both
S. cerevisiae and C. stellata. At 10 days, propionic acid was the most prevalent organic acid in autolysates of S. cerevisiae, representing 1.69 % of the initial cell weight. This was followed by succinic (1.18 %) , acetic
(0.87 %) and formic (0.63 %) acids, with the remainder each representing less than O. 5 % of the weight. In K. apiculata, the main organic acids recovered were succinic
(0.6 %) , followed by propionic (0.5 %), while the others formed O .1-0. 4 % of the initial cell weight. Propionic, succinic and oxalic acids were the main acids recovered in the autolysates of C. stellata and represented 0.8-1.4
% of the cell weight. Other acids were found at less than
0. 4 % of the cell weight. Notably, no lactic acid was found in the autolysates of S. cerevisiae, although small amounts were found in the autolysates of the other two yeasts. TABLE 20. Concentrations of organic acids in autolysates of yeasts during autolysis for 10 days
- . ------.....------Saccharomyces cerevisiae Kloeckera apiculata Candida stellata Acid -----·--·-·-·-2180a Acid 202 Acid 8008 Autolysis time (days) Autolysis time (days) Autolysis time (day~J__ _ ]---1--J---5-] ___!Q_ -o 0 1 5 10 0 1J 5 10 ~p.i.g)_~-r- ---1-- -- (ug/mg) a (ug/mg) a
Propionic 15.7 16.5 16.9 Propionic - 1.9 4.9 4.9 Propionic - 6.6 11.9 13.7 Succinic 2.4 5.1 11.8 Succinic - 5.8 6.0 6.0 Succinic - 2.9 7.1 11.9 Malic •. 2 2.4 3.9 Acetic - 2.9 3.5 3.7 Oxalic - 2.3 6.1 8.1
Formic 4.3 , 5.5 , 6.3 Oxalic - 2.9 3.3 3.3 Malic - 0.7 3.8 3.8 Acetic I I 2.4 2.8 8.7 Malic - 0.8 2.1 2.5 Citric - 2.2 2.2 2.3 Oxalic 2.4, 3.5 , 5.1 Lactic - 1.0 1.4 1.4 Acetic - 2.5 3.1 3.9 Citric I I 0.4 0.8 2.0 Formic - 1.0 1.2 1.2 Formic - 1.4 1.8 2.2 Tartaric I I 0.8 I 1.2 I 1.2 Citric - 0.2 0.6 1.0 Tartaric - 0.5 1.8 1.8 Lactic Tartaric - 0.8 1.2 1.2 Lactic - 1.6 1.8 1.8
------· ------1-+L _l_ _ _ Total (ug/mg)a - 29.6 I 37.8 I 55.9 Total (ugl'!lID a - 17.3 24.2 25.2 Total (ug/mg)a - 20.7 39.6 49.5
Percent initial Percent initial Percent initial _ ceUweight __ L ·_ _J 2.96_1 3.78 I 5.59 -~~~eig~t - 1.73 2.42 2.52 cell weight - 2.07 3.96 4.95 a = Calculated as weight of acids in a, ,tolysates per mg of initial dry weight of cells...... Data represent the mean of three replicates...... I\J 6
+J J: 5 C, .,.. G> 3 ,- 4 ,- QI 0 ,- .,..«s 3 +J ·r- C ·r-
+J 2 C G) 0 I.. Q) Q. 1
0 ...... ______..______.______.______.______.______, 0 2 4 6 8 10 12 Time of autolysis (days)
Figure 12. Recovery of total organic acids in autolysates of Saccharomyces cerevisiae 2180a (-0-), 202 ( and Candida .... Kloeckera apiculata -6-), .... stellata 8008 (-0-) during autolysis for 10 w days. 114
Acetic acid
As a check on the reliability of HPLC for measuring the concentration of organic acids, changes in the concentration of acetic acid during autolysis were also determined by an enzymatic method (Table 21; Figure 13).
The data are in approximate agreement with those precent ed in Table 20, and show significant, progressive release of the acid during autolysis. Particularly noteworthy, as also evident in Table 20, was the increased production of acetic acid in the autolysates of S. cerevisiae at 10 days.
Protein
The protein concentration in autolysates increased progressively with time (Table 22; Figure 14), although most had been released during the first five days. In relation to initial cell weight, slightly higher concentration of protein was released by K. apiculata.
After 10 days approximately 12-13 % of the cell dry weight had been solubilized as protein.
Amino acids
Table 23 shows amino acids recovered in the autolysates of the three yeasts. All the three yeasts produced nearly similar amino acids but at different concentrations.
After 10 days, total free amino acids released repre sented 12.0 % of initial cell weight for S. cerevisiae, 115
TABLE 21. Concentration of acetic acid in autolysates of yeasts during autolysis for 10 days
Autolysis Acetic acid Ye as t species time % Initial cell ' - (days) (ug/mg) a weight
Saccharomyces cerevisiae 2180a 0 0.0 0.00
1 2.6 0.26
5 3.2 0.32
10 9.1 0.91
Kloeckera apiculata 202 0 0.0 0.00
1 3.0 0.30
5 3.7 0.37
10 4.0 0.40
Candida stellata 8008 0 0.0 0.00
1 2.6 0.26
5 3.4 0.34
10 4.2 0.42
a= Measured with kit reagent and the acetic acid recovered was calculated as weight per mg initial dry weight of cells. Data represent the mean of three replicates......
.....
°'
12
___J
____
10
__l._
____
of
_L_
8
8008
Kloeckera
stellata
(-0-),
autolysates
days.
_____
(days)
6
10
__L
Candida
2180a from
for
and
acld
autolysis
_____
of
(--ir),
cerevisiae
4
autolysis
acetic
Time
202
of
during
-0-)
(
apiculata
Saccharomyces
Recovery
2
13.
Figure
l.-'U-----~-----L_
0
1.0
0.0 0.2
0.4
0.8 0.6
~
QI
QI
s::
s:: Q) u as Q)
u M
.µ
.µ
""
rl .c: rl
g.. rl -~
•ri
•ri
·ri 117
TABLE 22. Concentration of protein material in autolysates of yeasts during autolysis for 1O days
Autolysls Tyrosine-containing material Ye as t species time % Initial (days) (ug/mo) cell weight ! i Saccharomyces cemvislae 2180a 0 0.0 0.00
1 32.6 3.26
5 93.2 9.32
10 116.5 11.65
Kloeckera aplculata 202 0 5.5 0.55
1 79.7 7.97
5 115.4 11.54
10 131.9 13.19
Candida stallata 8008 0 0.0 0.00
1 43.0 4.30
5 108.9 10.89
10 122.9 12.29
a= Calculated as weight of tyrosine-containing ma1erlal In autolysates per mg dry weight of cells. Data represent the mean of three repflcates.
-·~ ....
CD
....
12
10
of
8008
Kloeckera
8
stellata
(--6-),
autolysates
days.
(days)
10
Candida
2180a
for
from
6
and
autolysis
of
-D-),
(
cerevisiae
protein
autolysis
Time
202
of
4
during
)
0-
(
Saccharomyces
apiculata
Recovery
1~
2
Figure
0
2
4
0
6
8
14
10
12
L.
•
CJ
C •
C
a.
+l
.,
G) CJ
~
o» ..., • ...
.,..
...
,...
,... ,...
+l
.c ... 119
11.0 % for C. stellata, and 9.0 % for K. apiculata.
Table 24 shows the five main amino acids in decreasing order found in autolysates after autolysis for
10 days. The autolysates of the three species examined contained the same five main amino acids, though the order of their concentration varied with the species.
These acids were phenylalanine, glutamic acid, leucine, alanine, and arginine.
Ribonucleic acid (RNA)
The ribonucleic acid (RNA) content of yeast cells progressively decreased during autolysis and was recovered in the autolysate (Table 25; Figure 15). The concentration of RNA was measured by the orcinol and optical density methods. Both methods gave similar data.
The initial RNA content of the cells ranged between 4-6
% depending on the species. For the three yeasts, this content decreased by approximately 85-90 % during autolysis. Ribonucleic acid material was recovered in autolysates, al though 10-15 % less than that initially present in the cells.
Deoxyribonucleic acid (DNA)
As measured by the diphenylamine method, the content of cell DNA decreased progressively during autolysis and by day 10, it had decreased by approximately 42 % for both TABLE 23. Concentrations of amino acids ir, autolysates of yeasts after autolysis for 10 days.
Saccharomyces cerevisiae Kloeckera apic11/ata Candida stallata 2180a 202 8008 Ami.,o acids Autolysis time (days) Amino acids Autolysis time (days) Amino acids Autolysis time (days) 0 1 5 10 0 1 5 10 0 1 5 10 (ug/mg) a (ug/mg) a (ug/mg) a
Phenylalanine - 1.7 10.5 14.0 Glutamic acid - 13.6 14.4 14.9 Arginine - 3.2 14.1 13.2 Glutamic acid - 2.6 11.2 14.7 Alanine - 12.5 13.9 13.9 Phenylalanine - 2.7 10.1 14.8 Leucine - 2.1 9.8 12.1 Leucine - 6.7 7.3 9.9 Glutamic acid - 2.1 8.2 12.5 Alanine - 3.5 7.5 9.1 Arginine - 4.7 5.2 7.5 Alanine - 3.2 7.4 11.5 Arginine - 0.8 7.3 8.5 Phenylalanine - 4.2 4.9 6.2 Leucine - 2.0 7.2 8.0 lsoleucine - 1.2 5.8 8.4 Serine - 3.3 3.9 4.5 Tyrosine - 1.8 4.5 4.6 Valine - 1.1 5.2 7.1 Valine - 2.8 3.4 4.2 lsoleuclne - 1.2 4.3 4.5 Aspartic acid - 1.0 5.4 7.4 lsoleucine - 2.7 2.9 3.9 Valine - 1.3 3.8 4.5 Serlne - 0.9 4.2 5.6 Glycine - 2.8 3.0 3.8 Serine - 1.1 4.1 5.5 Tyrosine - 1.1 4.6 6.2 Cystine - 2.6 3.1 3.4 Histidine - 1.2 3.1 3.7 Threonine - 1.0 4.2 5.2 Aspartic acid - 2.1 2.5 2.8 Aspartlc acid - 0.8 4.2 4.5 Lysine - 1.5 5.0 5.2 Threonine - 2.2 2.5 2.9 Threonine - 1.0 3.9 3.9 '1-aminobutyric acid - 0.8 3.5 4.3 Histidine - 2.0 2.2 2.7 Glycine - 1.3 3.2 3.4 Glycine - 1.2 3.5 4.2 'l5~aminobutyrlc acid - 1.5 1.8 2.6 11-aminobutyric acid - 1.1 3.4 3.5 Histidine - 0.3 1.7 2.0 Methionine - 0.6 1.0 1.1 Lysine - 4.1 2.9 3.0 Methionine - - 1.8 2.1 Taurine - 0.4 0.5 0.6 Methionine - 0.5 1.6 2.2 Taurine -- 0.3 0.6 Cystine - - 1.3 2.1 Taurine - - 0.3 0.8
Tot a I (ug/mg)a - 20.8 91.5 116.7 T o t a I (ug/mg) a - 64.7 72.5 84.9 Tot a I (ug/mg)a - 28.6 87.6 106.2
a= Calculated per mg of initial dry weight of cells...... N Data represent the mean of three replicates. 0 121
TABLE 24 Main amino acids in autolysates of yeasts after autolysis for 10 days
Yeast species Amino acids a
Saccharomyces cerevisiae 21 eoa Phenylalanine
Glutamic acid
Leuclne
Alanine
Arginine
Kloeckera apiculata 202 Glutamic acid
Alanine
Leucine
Arginine
Phenylalanine
Candida stellata 8008 Arginine
Phenylalanine
Glutamic acid
Alanine
Leucine a= Amino acids of each strain in decreasing order of concentration found In autolysate.
~ ~
l'v l'v
l'v l'v
b b
0.0 0.0
0.0 0.0
0.0 0.0
24.8 24.8
72.6 72.6
69.6 69.6
68.1 68.1 25.8 25.8
75.3 75.3
73.8 73.8
26.2 26.2
76.4 76.4
initial initial
cells cells
on on
content content
Increase Increase
of of
a a
0.0 0.0
0.0 0.0
% %
0.0 0.0
ANA ANA
24.2 24.2
75.1 75.1
75.2 75.2
72.3 72.3 69.5 69.5
67.7 67.7
24.1 24.1
77.3 77.3
25.1 25.1
based based
autolvsates autolvsates
in in
b b
1.38 1.38
1.05 1.05
3.79 3.79
4.80 4.80
2.91 2.91
4.71 4.71
2.23 2.23
3.84 3.84
0.71 0.71
2.96 2.96
0.21 0.21
0.00 0.00
Initial Initial
weight weight
% %
a a
cell cell
1.30 1.30
4.69 4.69
4.58 4.58
3.61 3.61
2.78 2.78
2.06 2.06
3.71 3.71
0.66 0.66
2.89 2.89
0.20 0.20
0.96 0.96
0.00 0.00
recovered recovered
0.0 0.0
b b
ANA ANA
15.2 15.2
34.8 34.8
73.9 73.9
72.7 72.7
26.3 26.3
109.2 109.2
100.6 100.6
235.2 235.2
230.8 230.8
280.2 280.2
(ug/ml) (ug/ml)
0.0 0.0
a a
days days
14.6 14.6
94.9 94.9
32.3 32.3
69.5 69.5
72.3 72.3
24.1 24.1
100.7 100.7
229.8 229.8
224.5 224.5
270.8 270.8
263.5 263.5 276.4
10 10
for for
b b
0.0 0.0
0.0 0.0
85.4 85.4
78.1 78.1
84.4 84.4
29.3 29.3
81.8 81.8
23.4 23.4
89.7 89.7
85.8 85.8
27.6 27.6
in in
content content
cells cells
autolysis autolysis
Decrease Decrease
of of
a a
0.0 0.0 0.0
0.0 0.0
0.0 0.0
ANA ANA
% %
87.4 87.4
84.8 84.8 29.8 29.8
29.8 29.8 83.4 83.4
81.0 81.0 25.5 25.5
90.9 90.9
86.6 86.6
during during
b b
ANA ANA
1.29 1.29
0.86 0.86
4.75 4.75
3.36 3.36
0.73 0.73 4.50 4.50
0.49 0.49
0.74 0.74
0.57 0.57
4.01 4.01
yeasts yeasts
I I
Initial Initial
weight weight
of of
% %
--
Cel Cel
cell cell
1.10 1.10
4.54 4.54 5.80 5.80 5.88
0.69 0.69
3.19 3.19
4.32 4.32 0.73 0.73
0.41 0.41
2.70 2.70 2.90 0.64 0.64
3.84 3.84
0.51 0.51
14.2 14.2
54.0 54.0
42.0 42.0
63.1 63.1
72.5 72.5
100.2 100.2
autolysates autolysates
346.8 346.8
220.5 220.5
287.9 287.9
and and
(ug/ml) (ug/ml)
a a b a
12.9 12.9
16.0 16.0 18.2
30.0 30.0 35.8
50.5 50.5
35.8 35.8
53.9 53.9
96.1 96.1
67.5 67.5
232.8 232.8 245.3
331.4 331.4
211.1 211.1
284.0 284.0
cells cells
in in
7.3 7.3
4.9 4.9
2.5 2.5
cell cell
Initial Initial
weight weight
(mg/ml) (mg/ml)
(ANA) (ANA)
1 1
1 1
1 1
5 5
5 5
0 0
5 5
0 0
0 0
acid acid
10 10
10 10
10 10
time time
(days) (days)
Autolysis Autolysis
replicates. replicates.
method. method.
ribonucleic ribonucleic
8008 8008
202 202
2180a 2180a
of of
three three
of of
method. method.
density density
mean mean
orcinol orcinol
optical optical
cerevisiae cerevisiae
the the
by by
by by
species species
Concentrations Concentrations
apiculata apiculata
stellata stellata
25. 25.
represent represent
Measured Measured
Measured Measured
Yeast Yeast
Candida Candida
Kloeckera Kloeckera
Saccharomyces Saccharomyces
Data Data
b= b=
a= a= TABLE TABLE 123
1~------. a e
,
+J .c .,..C, b Q) 3 ,- 5 ,- Q) 0 • ,- .,...«s 3 .,...+J 2 .,...C
+J C Q) 0 0 I... C Q) a.. 6
6 •
3
2
6 8 10
T i m e o f a u t o I y s i s (d a y s)
Figure 15. R i bonuc 1 e i c acid dee rease i n ce 11 s ( ... , * ) and increase in auto 1 ysates ( -0- , -b- ) of Saccharomvces cerevisiae 2180a (a), K1oeckera aoicu1ata 202 (b) and Candida ste11ata 8008 (cl durinQ autolysis for 10 days, measured by oot i ea 1 dens i tv method ( -A- , -fr ) and ore i no 1 ( + , -o-) method. 124
s. cerevisiae and c. stellata and 24 % for K. apiculata. Deoxyribonucleic acid material was recovered in the autolysates at concentrations reflecting the decrease found in the cells. The dye method for measuring DNA, gave much lower values for the DNA content of cells and autolysates than the diphenylamine method. This also showed a progressive decrease in DNA content of the cells during the 10 days and increase in DNA material in the autolysate. However, the percentage changes were much higher than those found with the diphenylamine method.
Thus by the dye method, the DNA content of the cells decreased by 72-88 % (depending on yeast) and only 33-55
% of DNA material was recovered in the autolysates (Table
26; Figure 16).
Lipids
Table 27 and Figure 17 show changes in the total lipid content of yeast cells during autolysis. Lipids represented 3-4 % of the cell dry weight and this decreased during autolysis. After autolysis for 10 days, cells of S. cerevisiae showed a greater decrease (81.1 %) in lipid content than those of K. apiculata (31.8 %) and
C. stellata (42. 7 %) • Soluble lipid material was recovered in the autolysates of the three yeasts, the concentration of this lipid increasing with time of autolysis. Highest concentration of lipid in the autolysate at day 10 was found with S. cerevisiae (46.0
%) , while that in K. apiculata and C. stellata were, TABLE 26. Concentrations of deoxyribonucleic acid (DNA) in cells and autolysates of yeasts during autolysis for 10 days ------~------.-----~- Autolysi In :ial Cell DNA DNA recovered in autolysates Yeast species time c 911 % Decrease % Increase we ight % Initial in % Initial based on initial cell weight DNA content cell weight DNA content (ug/ml) of cells (ug/ml) of cells (daysU_(m 1/ml) a b a b a b a b a b a b
Saccharomyces cerevisiae 2180a 0 6 .4 9.4 3.3 0.15 0.05 0.0 0.0 0.0 0.0 0.00 0.00 0.0 0.0
1 8.4 1.6 0.13 0.03 10.6 51.5 0.7 0.3 0.01 0.05 7.4 9.1
5 6.4 0.4 0.10 0.01 31.9 87.8 2.6 0.6 0.04 0.07 27.7 18.2
10 5.5 0.4 0.09 0.01 41.5 87.8 3.5 1.1 0.05 0.08 37.2 33.3
K/oeckera apicu/ata 202 0 .6 7.1 1.7 0.20 0.05 0.0 0.0 1.6 0.5 0.04 0.01 0.0 0.0 6.6 0.9 0.18 0.03 7.0 47.1 2.1 0.8 0.06 0.03 7.0 17.6
5 5.6 0.3 0.16 0.01 21.1 82.4 2.9 1.1 0.08 0.05 18.3 35.3
10 5.4 0.2 0.15 0.01 23.9 88.2 3.1 1.2 0.09 0.05 21.1 41.2
Candida stel/ata 8008 0 .9 10.1 5.3 0.13 0.07 0.0 0.0 0.0 0.0 0.00 0.00 0.0 0.0
1 8.6 2.1 0.11 0.03 14.9 60.4 1.3 1.8 0.02 0.02 12.9 34.0
5 6.7 2.0 0.08 0.03 33.7 62.3 3.2 2.2 0.04 0.03 31.7 41.5
10 5.9 1.5 0.07 0.02 41.6 71.7 4.0 2.9 0.05 0.04 39.6 54.7
a= Measured by diphenylamine method. Measured by benzimidazol method. b= .... Data represent the mean of three replicates. l'v lT1 126
,------, 0.26 • 0.20
0.06
+> 0.00 .c b .,...C) Q) 0.20 ~ .- .- 0.15 Q) 0 .- 0.10 .,...«I .,...+> 005 .,...C: +> C: o.oo Q) C 0 L Q) a. 0.20
0.1 f;
0.10
0.06
0.00 0 2 " e 8 10 12 Time of autolysis (days)
Figure 16. Deoxyribonucleic acid changes in cells (• , • ) and recovered from autolysates ( -0-, -0-) of (a) Saccharomyces cerevisiae 2180a, (b) Kloeckera apiculata 202, and (c) Candida stellata 8008 during autolysis for 10 days, measured by diphenylamine method ( • , -0-) and benzimidazol ( • , -0- ) method. 127
respectively, 20.8 % and 26.6 % of initial total lipid· content of cells.
Table 28 shows the main classes of lipids found in the cells and autolysates of the three yeasts. There were significant variations between the three species. In
S. cerevisiae, triglyceride and diglyceride were the main constituents. Their contents in the cells decreased during autolysis, with some appearance in the autolysates. There were lesser, but significant contents of cholesteryl ester and free fatty acids, which also decreased during autolysis and appeared in the autolysates. Minor amounts of monoglyceride and ergosterol were also detected in the cells/autolysate of
S. cerevisiae.
In K. apiculata, free fatty acids followed by diglyceride and cholesteryl ester and lesser amounts of triglyceride were the main lipid classes in the cells and the concentration of these components did not decrease significantly until day 10 when they were also found in autolysates. Small amounts of monoglyceride and ergosterol were also found in the cells and autolysates.
The cholesteryl esters were the most significant lipid component in cells and autolysates of
C. stellata. This class was followed by the triglyceride, diglyceride and free fatty acids, and minor amounts of monoglyceride and ergosterol.
Phosphatidylglycerol and phosphatidylserine 128
TABLE 27. Changes in the concentrations of total lipids in cells and autolysates of yeasts during autolysis
Autolysis C e 11 (C) Tota I Ii pi d s Yeast species time or %Initial % Decrease % lncreasej Autolysate cell in based on I (A) weight lipid content initial lipid ' a of cells content (days) (ug/mg) of cells
Saccharomyces cerevisiae 2180a 0 C 37.6 3.76 - 1 27.4 2.74 27.1 5 17.2 1.72 54.3 10 7.1 0.71 81.1
0 A 2.1 0.21 - 1 9.0 0.90 18.4 5 15.7 1.57 I 36.2 10 19.4 1.94 46.0 I
Kloeckera apiculata 202 0 C 41.8 4.18 - 1 39.9 3.99 4.5 5 35.3 3.53 15.6 ' i 10 28.5 2.85 31.8 I
I 0 A 4.1 0.41 - I 1 4.4 0.44 0.1 5 6.5 0.65 5.7 10 12.8 1.28 20.8
Candida stel/ata 8008 0 C 39.8 3.98 - 1 32.3 3.23 18.8 5 25.2 2.52 36.7 10 22.8 2.28 42.7
0 A 0.3 0.03 1 5.7 0.57 13.6 5 8.8 0.88 21.4 10 10.9 1.09 26.6 I
a= Calculated as weight of lipids in cells and soluble autolysates per mg of initial dry weight of cells. Data represent the mean of three replicates. 129
15.------, a
2
1
0 ~ .c 0) b .,.. 4 Cl) ~ ,- 3 ,- Cl) 0 ....., 2 ...... ,..+J .,..C
~ C C Q) 0 .. L.. Cl) Q.. 3
2
1
2 e 8 10 12 Time of autolysis (days) Figure 17. Total lipid changes in cells ( •) and auto lysates ( a-) of (a) Saccharomyces cerevisiae 2180a, (b) Kloeckera apiculata 202, and (c) Candida stellata 8008 during autolysis for 10 days. 130
were the main phospholipids in the cells of all three yeasts followed by slightly lesser amounts of phosphat idylcholine, phosphatidylinositol and cardiolipine. Phos phatidylethanolamine was found in small amounts only in
K. apiculata. The concentration of these phospholipids in the cells decreased during autolysis for all yeasts, but none were found in the autolysates.
Glycerol
Glycerol was recovered in the autolysates of all three yeasts. Highest concentrations were produced in the autolysates of S. cerevisiae. Most of the glycerol was released into the autolysate during the first day of autolysis (Table 29; Figure 18).
4.3 Effect Of Cell Concentration On The Kinetics Of Yeast Autolysis
4.3.1 Introduction
Vosti and Joslyn (1954a) and Joslyn and Vosti (1955) reported that there was no effect of cell density on the process of autolysis. Apart from this study, there have been no other observations on the effect of cell density on yeast autolysis. This chapter examines the ef feet of cell concentration on the kinetics of death and cell weight changes during autolysis. TABLE 28. Changes in the concentrations of lipid classes in cells and autolysates of yeasts during autolysis for 10 days
Autolysis Cell (C) Phospholipids (ug/mg) a Neutral lipids (ug/mg) a t time or Ye as species l: (days) Autolysate (A) PC PS PI CAR PG PEA MG E FFA DG TG CE
Saccharomyces ceravisiae 2180a 0 C 0.9 1.4 1.3 0.3 2.6 - 0.7 0.7 4.1 9.4 11.6 4.6 1 0.8 1.0 0.8 0.2 1.3 - 0.5 0.6 3.1 5.6 9.5 4.0 5 0.5 0.4 0.3 0.2 0.0 0.4 0.5 1.6 5.0 5.9 2.4 10 0.5 0.3 0.3 0.2 0.0 - 0.3 0.4 1.2 3.0 5.3 1.6 0 A ------0.2 0.3 - 1.6 1 ------0.2 0.1 1.3 3.5 1.8 2.1 5 ------0.3 0.2 2.6 4.2 4.8 3.6 10 ------0.4 0.3 3.1 6.1 5.3 4.2 Kloeckera apiculata 202 0 C 1.2 1.9 0.8 0.8 2.9 0.1 1.7 0.9 11.5 8.2 4.0 7.8 1 1.1 1.6 0.6 0.7 2.5 0.1 1.6 0.8 11.3 8.0 4.0 7.6 5 0.7 1.2 0.6 0.6 1.4 0.1 1.6 0.7 10.9 6.8 3.3 7.4 10 0.4 1.1 0.5 0.6 1.4 0.1 1.6 0.6 8.6 4.3 3.3 6.0 0 A ------0.4 1.0 - 2.7 1 ------0.1 - 0.5 1.0 - 2.8 5 ------0.1 0.1 0.7 2.1 0.5 3.0 10 ------0.1 0.2 2.6 4.8 0.6 4.5 Candida stellata 8008 0 C 1.0 2.1 0.7 0.3 2.7 - 1.7 0.5 3.5 3.8 5.1 18.4 1 0.8 1.7 0.6 0.3 2.2 - 1.5 0.5 3.1 2.9 4.6 14.1 5 0.4 0.3 0.4 0.2 0.3 - 1.1 0.3 3.0 2.7 2.4 14.1 10 0.3 0.2 0.4 0.2 0.3 - 0.8 0.3 2.0 2.3 2.1 13.9 0 A ------0.3 -- - 1 ------0.1 - 0.6 0.9 0.4 3.7 5 ------0.5 0.2 0.7 0.9 2.4 4.1 10 ------0.8 0.2 1.7 1.2 2.8 4.2 a= Calculated as weight of lipids in cells and autolysates per mg initial dry weight of cells. b= Determined as palmitic acid. PC=phosphatidylcholine; PS=phosphatidylserine; Pl=phosphatidylinositol; CAR=cardiolipine; PG=phosphatidylglycerol; ..... PEA=phosphatidylethanolamine; MG=monoglyceride; E=ergosterol; FFA=free fatty acids; DG=diglyceride; TG=triglyceride; w CE= cholesteryl ester...... Data represent the mean of three replicates. 132
TABLE 29. Concentration of glycerol in autolysates of yeasts during autolysis for 10 days
Autolysis Glycerol Ye as t species time % Initial (days) (ug/mg)a cell weight
Saccharomyces cerevisiae 2180a 0 1. 1 0.11
1 5.1 0.51
5 5.9 0.59
10 6.1 0.61 ·'
Kloeckera apiculata 202 0 1. 1 0.11
1 1.3 0.13
5 2.2 0.22
10 2.4 0.24
Candida stellata 8008 0 1. 1 0.11
1 3.4 0.34
5 3.4 0.34
10 3.4 0.34
a= Calculated as weight of glycerol in autolysates per mg dry weight of cells. Data represent the mean of three replicates......
w w
12
10
of
8008
Kloeckera
8
stellata
(-0-),
autolysates
days.
(days)
10
Candida
2180a
from
for
6
and
autolysis
-fr)
(
cerevisiae
glycerol
of
autolysis
202
of
Time
4
during
(-0-)
Saccharomyces
apiculata
Recovery
18.
2
Figure
0
1
0.7
0.6
0.5 0.3 0.4 0.2 o. 0.0
CD x a,
a,
0
.,
C
+>
L i 0
.c a,
.,.. r- .t,l
,-
+> ....
~
.,.. .,..
a. 134
4.3.2 Results
Effect of cell concentration on the kinetics of cell death during autolysis
Table 30 shows the loss in cell viability of three yeast species during autolysis for 10 days. Two conclusions are evident. First, the rate of cell death during autolysis was different for the three species. It was notably slowest for S. cerevisiae. Viable cells were still detected after five days but not 10 days. For K. apiculata, all of the cells died off within the first 24 h. For C. stella ta, they had died off between days one and three. Second, the kinetics of these changes occurred in proportion to the initial cell concentration, indicating that they were no unusual influences of cell concentration on death during autolysis.
Effect of cell concentration on the kinetics of cell weight changes and cell solubilization during autolysis
The kinetics of decrease in dry weight of yeast cells and the kinetics of increase in solubilized material during autolysis for 10 days were not affected by the initial cell concentration (Table 31; Figure 19). The changes recorded were proportional to the initial concentration of cells. Thus, after autolysis, approximately 10 fold less of cell material was found in the autolysates of cell suspensions diluted 10 fold before autolysis. This was evident for autolysates measured after three, five or
10 days. This conclusion was found for the three species 135
TABLE 30. The effect of cell concentration on the kinetics of cell death during autolysis
Percent Autolysis Saocharomyces Kloeckera apicu/ata Candida stellata initial cerevisiae biomass time 2180a 202 8008 concen- tration (day~) Viable counts (cfu/ml)
7 .., 100 0 6.2 X 10 1.0 X 10 1.4X 10 8
1 0.2 X 10 7 a 4.7 X 10 4
$- 3 4.2X. 10 a a
5 2.5 X 10r a a
10 a a a .., 50 0 2.1X10 7 0.6 X 10 7.1 X 10 7
1 1.1X10 7 a 5.8X 10 4
3 1.2 X 106 a a
5 3.1 X 10 a a
10 a a a
7 25 0 1.1X10 2.8X 10' 3.1 X 10 7
1 3.2x10" a 1.6X 10 3
3 6.0 X 10"" a a
5 2.0 X 10 a a
10 a a a
10 0 4.6 X 10 6 1.1X10 ' 1.2X 10 7 1 3.8 X 10'- a 3.0X 10 2
3 7.0 X 10 3 a a
5 a a a
10 a a a
a= Viable counts not detectable in 0.2 ml sample, i.e., less than 5 cfu/ml. 136
examined.
4.4 Observations Of Yeast Autolysis With The Electron Microscope
4.4.1 Introduction
Using light microscopy, Vosti and Joslyn ( 1954) , Joslyn
(1955), and Joslyn and Vosti (1955) recorded changes to the morphology and cytology of yeast cells during autolysis. Their observations were restricted to S. cerevisiae (baker's yeast), and they concluded that the basic shape of the cell was retained during autolysis.
This suggested that the cell wall remained intact, and was not fragmented, despite obvious changes to cytoplasmic constituents. Such changes included loss of protoplasm, enlargement of the vacuole and reduction in number and size of intracellular granules and globules, and alteration to the mitochondria. Based on examinations with the light microscope, other authors have briefly noted that the cell wall is not physically disrupted during autolysis (Schryver et al. 1927, Farrer 1956,
Arnold 1972, Babayan et al. 1981, Arnold 1981b). Such conclusions have been confirmed by examinations using the electron microscope, although some chemical and structural reorganisation of the wall appears to occur
(Babayan et al. 1981, Charpentier et al. 1986, Piton et
al. 1988).
This chapter reports observations of s. 137
TABLE 31. The effect of cell concentration on the kinetics of cell weight changes and cell solubilization during autolysis
Percent Autolysis Saccharomyces Kloeckera apiculata Candida stel/ata initial ceravisiae biomass time 2180a 202 8008 concen- Cell Soluble Cell Soluble Cell Soluble tratlon dry weight autolysate dry weight autolysate dry weight autolysate (days) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml)_
100 0 2.54 0.00 4.86 0.07 7.32 0.00
1 2.26 0.12 4.01 0.41 6.25 0.44
3 1.98 0.43 3.66 0.81 5.70 1.28
5 1.92 0.48 3.49 0.89 5.31 1.46
10 1.76 0.60 3.30 1.10 4.79 1.91
50 0 1.18 0.00 2.67 0.00 3.95 0.00
1 1.02 0.07 2.24 0.26 3.43 0.28
3 0.92 0.19 2.05 0.41 2.98 0.66
5 0.89 0.22 1.99 0.48 2.79 0.87
10 0.84 0.25 1.86 0.58 2.56 1.08
25 I 0 0.58 0.00 1.21 0.00 2.00 0.00 I I 1 0.51 0.00 1.00 0 11 1.69 0.13
3 0.43 0.10 0.94 0.19 1.50 0.35
I 5 0.42 0.11 0.90 0.23 1.42 0.41
10 0.41 0.13 0.85 0.28 1.33 0.48
' . 10 0 0.24 0.00 0.47 0.00 0.80 0.00 : 1 0.21 0.00 0.39 0.05 0.69 0.00
3 0.19 0.04 0.37 0.08 0.62 0.13
5 0.18 0.05 0.35 0.09 0.58 0.16 I I 10 0.17 0.06 0.33 0.10 0.53 0.20 i I i i
Data represent the mean of triplicate analyses. 138
100 a d
80 60 ~-- .., 40 ______. .c 0: 20 ·------4 Cl) 3: 0 .. • •
100 b Cl) e u 80 II, .,.. .., 60 C 40 -- ...... , • C 20 A. ... Cl) _,, u .. ~ I... 0 • Qi Q_ 100 C f
80 60 .____. 40 • ... 20
0 0 2 4 6 10 0 2 4 6 8 10 Time of autoly:sis (days)
Figure 19~ Effect of cell concentrations ( -It , 100 %; • , 50 \; * . 25 %; -If"", 10 %) on the kinetics of cell weight chanqes (a-c) and cell solubili zation (d-f) of related cell concentrations ( -0- , 100 ,; -0- , 50 %; -& , 25 \; -V- , 10 \ ) durinq autolysis of Saccharomyces cerevisiae 2180a (a,d), Xloeckera apiculata 202 (b,e) and Candida stellata 8008 (c,f). 139
cerevisiae, K. apiculata and c. stellata during autolysis, using the electron microscope.
4.4.2 Results
Scanning electron micrographs of autolysing yeasts
Figures 20-22, show scanning electron micrographs of cells of S. cerevisiae, K. apiculata, and C. stellata at
0, 5 and 10 days of autolysis. The micrographs presented are representative of many fields that were examined. For all three species, basic cell shape was retained during autolysis and there was no obvious disruption of the cell wall. Bud scars were clearly seen on the surfaces of cells at the three stages of autolysis and, overall, the topography of the cells was similar before and after autolysis.
Transmission electron micrographs of sections of autolysing yeasts
Figures 23-25 show electron micrographs of thin sections of S. cerevisiae, K. apiculata, and C. stellata at O, 5 and 10 days of autolysis. At O days, the cytoplasmic membrane was located adjacent to the inner layer of the cell wall, and the cytoplasmic contents completely or almost completely filled the entire cell. For both S.
cerevisiae and c. stellata, there was a distinct shrinkage and pulling away of the cell membrane from the wall during autolysis (5 day and 10 day micrographs). In 140
a
b
C
Figure 20. Scanning electron micrographs of Saccharomyces cerevisiae 2180a after autolysis for (a ) 0, (b) 5, and (c) 1 0 days Cell magnification 10000 times. 1 41
a
C
Figure 21. Scanning electron mi crographs of Kloeckera apiculata 202 after autolysls for (a) 0, (b} 5, and (c} 10 days Cell magnification 10000 times. 142
C
Figure 22. Scanning electron micrographs of Candida stel/ata 8008 after autolysis for (a) 0, (b) 5, and (c) 10 days Cell magnif1cat1on 10000 times 143
the case C. stellata, there was an obvious reduction in the density of cytoplasmic contents. This reduction also occurred with S. cerevisiae, but was not as evident. The behaviour of K. apiculata was different to the other two species in that shrinkage and pulling away of the cell membrane from the wall was not as apparent and had occurred only to a limited degree after day 10. Also, the decrease in density of the cytoplasmic material was not as strong.
For all three species, the cell wall remained intact or entire during autolysis and showed no evidence of disintegration. Its layered composition aJ so appeared to remain unchanged. However, wall thickness of each of the species had decreased by approximately 20 % after autolysis for 10 days (Tables 32, 33). This decrease was statistically significant at the 5 % level. For S. cerevisiae and C. stellata, but not K. apiculata, the decrease in wall thickness as a consequence of autolysis was also evident at 5 days. 144
a
b
c,
Figure 23. Transm1ssIon electro n m1crog raphs of thin secti ons of Sacc haromyces cerevisiae 2180a after autolys1s for Ia) 0 1bl 5 and (c) 10 days Cell magnifications are 1a 1) 12000, {a2) 10000 . (b 1) 10000. (b2\ 10000 1c 1) 10000, and (c 2) 12000 times 145
Figure 24. Transmission electron m1 crographs of thin sections of K!oeckera ap/culata 202 after autolysis for (a) o. (b) 5 and (cJ 10 days Cell magnifications are (a 1) 15000, (a2) 12000, (b 1) 10000, /b2) 10000. (c 1) 12000, and (c2) 15000 times 146
Figure 25. Transm1ss1on electron m1 crographs of thin sections of Candida sta/Jata 8008 after autolysis for (al 0, (b) 5. and (c) 10 days. Cell magnifications are (a1) 10000, (a2) 10000. (b 1) 7000. (b2l 8000, (c 1) 3500, and (c2) 8000 times. 147
TABLE 32 •. Changes in the thickness of the cell wall during autolyis for 10 days
Autolysis Micrograph Cel I wal I thickness Yeast species time Magni- Replicates of measurement Average Number fication Corrected 0 Mean (days) ()(1000) (mm)a (mm) (um) (um)
Saccharomyces 0 1 10 284 276 280 278 278 279 0.28 cer6Visiae 2 12 3.54 3.44 3.60 3.48 3.44 3.50 Q29 2180a 3 9 262 2.30 2.40 2.70 2.70 2.54 0.28 0.29 4 10 3.40 3.00 2.90 3.28 3.00 3.12 0.31 5 9 2.92 2.80 3.16 3.00 2.70 2.92 Q32
5 1 10 2.50 2.60 2.50 2.16 2.10 2.37 0.24 2 10 2.38 2.50 2.38 2.40 2.48 2.43 Q24 3 10 2.58 2.60 2.46 2.50 2.46 2.52 0.2!1 0.26 4 10 2.62 2.76 2.88 2.68 2.74 2.74 Q27 5 12 3.38 3.22 3.32 i.94 3.00 3.17 0.26
10 1 10 2.00 2.30 2.18 2.20 2.20 2.18 0.22 2 10 2.54 2.40 2.26 2.10 2.30 2.32 0.23 3 10 2.40 2.38 2.40 2.02 2.30 2.30 0.23 0.23 4 10 2.50 2.52 2.54 2.52 248 2.51 0.25 5 10 2.30 2. 18 2.32 2.20 2.40 2.28 0.23
Kloeckera apiculata 0 1 12 2.76 2.76 2.74 3.04 2.62 0.23 202 2 15 3.22 3.78 3.70 3.70 3.52 27813.50 Q24 3 20 4.22 4.60 4.78 4.38 4.48 4.49 0.22 0.23 4 17 3.88 3.90 3.80 3.92 3.92 3.88 0.23 5 30 6.50 6.78 6.90 6.90 6.46 6.71 0.22 I 5 1 12 , 3.12 280 2.70 2.60 2.60 276 0.23 2 12 280 2.92 2.78 2.80 2.94 2.85 0.24 I 3 12 262 270 2.80 2.62 2.60 2.67 0.22 0.23 I 4 10 2.38 2.50 240 220 248 2.39 0.24 I 5 10 2.30 2.26 2.32 2.26 2.26 2.28 0.23 I I' 10 1 20 3.72 3.68 3.34 3.80 3.20 3.55 0.18 I 2 15 2.54 270 258 2.80 2.60 2.64 0.18 'I ' 3 15 2.60 2.62 260 278 280 268 0.18 0.18 4 15 2.82 268 2.60 2.78 2.62 2.70 0.18 5 17 290 2.80 3.20 3.38 3.00 3.04 0.18
Candida stellata 0 1 15 4.28 4.60 4.28 4.56 4.46 4.44 0.30 8008 2 15 4.76 4.18 5.00 5.08 4.72 4.75 0.32 I' 3 12 3.92 3.70 3.76 3.78 3.80 3.79 0.32 0.31 4 10 3.10 248 2.94 3.30 3.00 296 0.30 5 12 3.58 3.70 3.68 3.92 3.90 3.76 0.31
5 1 7 1.96 2.00 2.10 2.18 1.88 202 0.29 I 2 30 8.08 7.80 7.82 7.90 8.10 , 7.94 0.26 I 3 10 2.42 2.90 2.60 2.80 2.50 264 0.26 I 0.27 4 10 2.48 2.70 242 2.76 2.78 2.63 0.26 I 5 10 2.80 2.78 292 284 2.70 2.81 0.28
10 1 8 1.88 2.02 2.08 1.92 1.98 1.98 0.25 I 2 15 3.38 3.18 3.38 3.68 3.82 3.49 I 0.23 3 20 ! 5.10 4.80 5.00 I 4.78 4.70 4.96 I 0.25 0.24 3.86 I 4 17 'i 3.69 3.72 3.94 4.06 3.88 0.23 5 20 I 4.68 4.46 4.76 4.82 I 4.80 4.70 ! v.24 I ' I I I I I I
a= Thickness of the wall on micrographs of cell sections was measured with vernier callipers. Five measurements were made at different locations of the wall of each cell (excluding region of budding). For each autolysis time, measurements were made on five different cells. Data of the twenty five measurements were analysed. b= Corrected for magnification of micrograph. 148
TABLE 33. Summary of changes in the thickness of the cell walls during autolysis for 10 days
Autolysis Cell wall thickness
Yeast species Corrected mean + time Percent mean Percent
deviation decrease
(days) (um)a
Saccharomyces cerevislae 2180a 0 0.29 ± 4.8 % 0.0
5 0.25 ± 6.8 % 13.8
10 0.23 ± 2.6% 20.7
I Kloeckera apiculata 202 0 0.23 ± 2.6 % I 0.0 I I 5 0.23 ± 2.6 % 0.0
10 0.18±0.0% 21.7
Candida stellata 8008 0 0.31 ± 2.6 % 0.0
I 5 0.27 ± 4.4% 12.9
10 0.24 ± 3.3 % 22.6
I
a= Data from Table 12. 14'=1
5. DISCUSSION
The autolytic behaviour of three yeast species has been
examined in this study. As noted clearly in Section 2. 2
and as is evident from the key reviews on yeast autolysis
(Schryver et al. 1927, Joslyn and Vosti 1955, Joslyn
1955, Farrer 1956, Babayan and Bezrukov 1985), nearly all previous studies on this subject have concerned only one
species, namely, S. cerevisiae. Because of the general biochemical and physiological diversity that occurs between different yeast species, it is reasonable to expect that there might be some variation in their autolytic properties. Consequently, three different species were selected for examination in this thesis. The selection of K. apiculata and C. stellata was based on previous studies in this laboratory (Heard and Fleet
1986, Fleet 1990a) that demonstrated a significant contribution of these species to wine fermentations, in addition to the well established role of S. cerevisiae.
There is general acceptance that the autolysis of wine yeasts after completion of alcoholic fermentation can influence wine quality (Kunkee and Amerine 1970, Feuillat and Charpentier 1982) . Consequently, knowledge about the autolytic properties of K. apiculata and C. stellata would carry some significance to the wine industry. In other contexts, K. apiculata is a very common food spoilage yeast and autolytic reactions could be
considered as part of their spoilage activity (Fleet 1990b, 1992). 150
5.1 Cell viability
Autolysis of washed cell suspensions of the three yeasts
at 45 °c was characterized by a rapid decrease in cell viability. The rate at which viability decreased was different for the three species. The cells of K.
apiculata died off more rapidly (within 24 h) than those of C. stt1llata and S. cerevisiae, while the viability of
the cells of C. stellata decreased faster than those of
S. cerevisi ae (Tables 17, 30) . No viable eel ls of C.
stellata were detectable after 1-3 days, whereas at least
5 days were needed for complete loss of viability of cells of S. cerevisiae. No biochemical or physiological reasons can be advanced at this stage to explain this difference in behaviour. However, Beuchat (1981) reported that in O .1 M potassium phosphate buffer, cells of K.
apiculata heated at 45 °c for 20 min decreased in population by 87 %, while cells of S. cerevisiae heated at 50 °c for the same time lost only 50 % of their population. Unfortunately, no comparable data were reported on C. stellata. From the heat resistance studies of Beuchat (1981) and others (Fleet 1992), it is evident
that most yeasts would be killed by exposure to a
temperature of 45 °c and higher. Resistance to heat
inactivation varies between yeast species, and the data suggest that K. apiculata is more sensitive to heat
inactivation than S. cerevisiae. 151
In their studies on the autolysis of baker's yeast, Joslyn and Vosti {1955) reported the loss in cell viability. Yeast samples stored at 34 °c contained only 8 % of viable cells after 24 h, while at 45 °c, no viable cells were detected after this time.
It is interesting to record that the relative rates at which the three yeasts lost viability on autolysis follows the same pattern of viability loss during wine fermentations as reported by Fleet (1990a).
In these fermentations, there is successive growth and death of these yeasts in the sequence, K. apiculata, C. stellata, and S. cerevisiae.
According to the available evidence, loss in cell viability is a prerequisite to autolysis (Joslyn and
Vosti 1955, Arnold 1981b). However, dead cells do not necessarily autolyse. This is demonstrated by the data of
Vosti and Joslyn (1954a) who noted that cells of baker's yeast incubated in citrate buffer, pH 4.4 for 24 hat 34
0 c, showed little evidence of autolysis although greater than 90 % of the cells were dead. When the cells were incubated at 45 °c, autolysis occurred, and it was suggested that, apart from cell death, other reactions
(e. g. lipolysis and proteolysis) within the cell needed to be activated before autolysis would commence. If cells are exposed to very high temperature (e. g. >60 °c),
they are quickly killed, but will not autolyse because enzymes responsible for autolytic reactions, such as 152
proteolysis and lipolysis are inactivated (Joslyn and
Vosti 1955, Trevelyan 1976).
Joslyn and Vosti (1955) concluded that. "storage conditions that killed yeast cells did not necessarily produce autolysis, while conditions necessary to cause extensive autolysis were so severe that none of the cells was viable after 24 h".
5.2 Solubilization of cell biomass
Solubilization of cell biomass and loss of cell dry weight are a key features of yeast autolysis. Various authors have reported losses in cell weights of 10-62 % for S. cerevisiae during autolysis (Farrer 1946, Hough and Maddox 1970, Arnold 1972, Trevelyan 1976, 1977,
1978). It is not possible to make direct comparisons between these studies and the data of Tables 5, 18 because of the different conditions used to conduct autolysis. However, the following conclusions can be drawn from the present study. For the thr~e species examined, greatest loss of cell weight occurred during the first 48 h (Figure 19) after which the kinetics of loss decreased. By 10 days, the cells had lost 25-33 % of their dry weight. From the kinetics shown in Figure 19, it seems unlikely that cells would autolyse to the extent
that 50 % of their dry weight would be lost if autolysis time was extended. However, further studies examining the autolytic reaction beyond 10 days are needed to establish 153
this fact. Trevelyan (1976, 1977, 1978) has reported cell weight losses of 50 % or more during autolysis of S. cerevisiae, but his conditions of autolysis included those of a higher temperature ( 50 °c) , preliminary heat shocking of cells at 60-75 °c, and the addition of agents, such as salt, ethanol, and ethyl acetate . • As expected, the loss in cell weight was reflected in the recovery of soluble biomass in the autolysate. Howe~er, for all three species and all autolytic trials, the weight of cell material lost and that recovered in the soluble fraction did not balance.
About 5 % or more of the cell weight was not recovered in the soluble form. It is likely that this difference could be attributed to losses by gas (Co2 , NH 3 , or H2 ) and water evolution during the autolytic process. Additional studies would be needed to substantiate this possibility.
On comparison of the three species, there is some indication that cells of K. apiculata undergo slightly more (about 5 %) autolytic solubilization than those of S. cerevisiae and C. stellata, especially K. apiculata strains 202 and 521. With regard to differences between strains, the three strains of S. cerevisiae, showed similar autolytic solubilization. There were some small differences in autolytic solubilization between the different strains of K. apiculata and C. stellata
(Table 5) . Apart from the minor variations noted, there were no gross differences between species or strains in 154
the extent to which cells were solubilized during
autolysis.
The kineti~s of cell solubilization during
autolysis were not affected by the initial concentration
of biomass used for the reaction (Table 31; Figure 19).
This conclusion supports previous observation by Vosti
and Joslyn (1954a) and Joslyn and Vosti (1955) who
studied the influence of this variable on the autolysis
of baker's yeast.
5.3 Carbohydrates
The solubilization of cell carbohydrate during autolysis
has been mentioned by several researchers (Joslyn 1955,
Hough and Maddox 1970, Babayan et al. 1981, Llauberes
~to..l.. 1987, Charpentier and Freyssinet 1989).
Two major conclusions can be drawn from the
data on the autolytic release of carbohydrates by the
yeasts examined in this study. First, virtually all of
the carbohydrate solubilized was probably polysaccharide.
Only small amounts (<1 %) were detected as either glucose
or reducing sugars a fact also noted by Hough and I • Maddox (1970}. Second, there was interesting variation
among the nine yeasts examined in the autolytic release
of carbohydrate. For all three strains of S. cerevjsiae as well as two strains of c. stellata, and one strain of K. apiculata the amount solubilized after 5 days was
approximately 2 % or less. Particularly noteworthy was 155
the greater amounts (4-6 %) released by K. apiculata strains 202 and 521 and C. stellata 504 (Table 8). The stronger release of carbohydrate by K. apiculata 202 was confirmed in more detailed studies (Table 19) where, after 10 days, almost 7.5 % of the cell weight was solubilized as carbohydrate. Since most of the released carbohydrate was polysaccharide, it probably originated from the cell wall. According to the conclusions of Fleet
(1991) and Charpentier and Freyssinet (1989), (1,3)-13- glucanases are probably active during autolysis, hydrolysing certain glucosidic linkages to cause the release of mannoproteins. Maddox and Hough ( 1971) have suggested that both glucanases and mannanases could be involved in the autolytic breakdown of yeast cell walls.
Such conclusjons apply only to S. cerevisiae and may not necessarily be extrapolated to include K. apiculata 202 to explain its the higher release of carbohydrate. The specific composition and structure of the cell wall ~f K. apiculata is not known (Phaff 1971, Fleet 1991).
Confirmation that mannoproteins are solubilized by autolysis would require isolation and purification of the released polysaccharide followed by structural studies. Such studies have been reported by Llauberes
e.f A...e. (1987) and Charpentier and Freyssinet
(1989} who provided strong evidence that the bulk of the released carbohydrate was mannoprotein. It is noteworthy that Maddox and Hough (1971) also suggested that the released polysaccharide was probably a glycoprotein. 156
Preliminary attempts in the present study (results not
reported in thesis) to isolate the polysaccharide released from S. cerevisiae were unsuccessful. Although a precipitate was obtained on treatment of the autolysate with ethanol, insufficient material was recovered for chemical analyses. Large scale autolysis would be needed
to recover enough material for isolation and structural studies and this is suggested as a direction for further research. In addition, it would be worthwhile to examine the autolysates for the activity of glucanase and mannanase enzymes that might be active in cell wall degradation during autolysis. It is now well established that isolated cell walls of a range of yeast species undergo self-degradation on incubation and that this behaviour is due to strongly associated (1,3)-13- glucanases. Interestingly, complete solubilization of the walls by this mechanism has never been observed (Fleet
1991). Proteases might also be involved in this type of wall hydrolysis (Fleet 1991).
5.4 Organir acids
Several authors have noted that the pH of the autolysate changes during the course of autolysis (Joslyn 1955).
However, the trends observed have not been consistent and seem to be influenced by the conditions used to culture the yeast, the pH and temperature of autolysis and also
the influence of buffer in which the autolysing cells are
suspended. In some instances, a pH decrease has been 157
observed during autolysis whereas in other cases the pH has been observed to incr~ase. A pH decrease is generally associated with acid production while a pH increase is related to protein degradation and the liberation of ammonia. Studies on the types of acids produced during yeast autolysis have not been previously reported.
Peppler (1982) noted that organic acids represented about
1.6-4.8 % of the dry weight of commercial yeast autolysates, but the types of acids were not mentioned.
This study has convincingly demonstrated that several organic acids are released or produced in the autolysates during autolysis. This finding occurred for all the strains examined. The total acids present in the autolysates represented between 2.5-7.5 % of the initial cell weight. There was notable variation·between strains, with one strain of S. cerevi si ae, namely strain number
HB350 (a wine yeast) showing the acids to represent about
7.5 % of the initial cell weight. Compared with S. cerevisiae and C. stellata, the strains of K. apiculata gave autolysates with less acid (Table 9). Propionic and succinic acids were particularlJ significant in the au tolysa tes of S. cerevisi ae and C. s tel la ta, but there was less of these acids in au toly3a t2s of K. api cul a ta.
Lactic acid was not found in the autolysates of any of the strains of S. cerevisi ae, but was present in autolysates of the other species. The high concentration of lactic acid in the autolysates of K. apiculata 521 is notable. An unidentified substance (presumably an acid) 158
was found in the autolysates of S. cerevisiae and K.
apiculata (Figure 3). Further studies with a greater range of reference acids than those used for the experiments reported in Figure 3 would be necessary to identify this substance. Thus, it may be concluded that there was some variation betwe£n species in the profile and kinetics of release/production of individual acids.
The biochemical mechanism that give rise to these acids in the autolysa~es can only be the subject of speculation at this stage. Enzymes of the glycolytic pathway and of the tricarboxylic acid cycle could lead the formation of lactic, formic, acetic, citric, succinic, and malic acids from hexose sugars (Gancedo and
Serrano 1989). In the early stages of autolysis, glucose might originate from the breakdown of reserve polysaccharides, such as glycogen and trehalose (Joslyn
1955, Kokova-Kratochvilova 1990). The phenomenon of autofermentation in the early stages when reserve carbohydrates were metabolized was reported in the early literature (Joslyn 1955, Farrer 1956, Ebbut 1961).
During the subsequent stages of autolysis, glucose could come from the degradation of cell wall glucans and be subject to a rapid metabolism by residual glycolytic and tricarboxylic acid cycle enzymes. Such activity might explain why almost no glucose was found in autolysates
(Tables 8, 19). Ribose, a pentose sugar, would be a product of RNA degradation during autolysis. Its metabolism by enzymes of the hexose monophosphate pathway 159
could also lead to the production of acetic and lactic acids (Gancedo and Serrano 1989). Acetic and propionic acids might also be expected to be produced by the metabolism of lipids (Chopra 1984) which, as discussed in a subsequent section, are broken down during autolysis.
Organic acids convey significant sensory properties. Consequently, their production by autolysis in wine, beer and other fermentations may be of commercial significance. Obviously, further research is needed to establish what factors affect the production of organic acids during yeast autolysis and to establish the biochemical mechanisms of their production. Such research should include more detailed analyses of the fate of storage carbohydrates during autolysis and examination of autolysates for the activity of key metabolic enzymes.
5.5 Proteins and Amino Acids
During autolysis, proteins, peptides (protein material) and free amino acids accumulate in the autolysate. The Folin-Ciocalteau reagent was used to measure the concentration of released protein material. This reagent works by reacting with tyrosine residues (Herbert et al. 1971) but a limitation of its use to measure protein material occurs when free tyrosine residues, also,, are present in samples. In such cases, misleadingly higher values for protein concentration will be recorded. The autolysate contained very low concentrations of free tyrosine, ranging from non-detectable values for the autolysates of Kl. apiculata to approximately 6 ug/mg for those of S. cerevisiae (Table 23). At best, interference from this source would increase protein release values by about 0.5% of the total cell weight, giving an error of only 5% or less in final calculations.
According to the data of Tables 10 and 22, about 10-13% of the cell weight was solubilized into the autolysate as protein/protein material. The three strains of K. apiculata gave slightly higher release than those of S. cerevisiae and C. stellata, but the differences were only marginal. Moreover, the variation in protein release between strains of the one species was also very marginal. 160
Very few other authors have reported protein
concentrations in yeast autolysates; consequently, it has been difficult to make comparisons with the protein release values given in Tables 10 and 22. Hough and
Maddox (1970) recovered protein between 13-19 % of cell weight in autolysates of brewer's yeast, and these values are in approximate agreement with those in Tables 10 and
22. They noted that the protein content of the autolysates decreased as autolysis progressed and suggested that this was due to the action of proteases or peptidases breaking down the proteins/peptides to amino acids. Other authors (Hough and Maddox 1970, Charpentier and Freyssinet 1989) have also noted that released proteins would undergo further degradation on extended autolysis. This trend was not observed in the present study {Figure 14) although the concentration of free amino acidsin the autolysates increased with time {Table
23). This increase, of course, could originate from the breakdown of intracellular proteins rather than those already solubilized. It is possible that the autolytic conditions of the present study (pH 4.5, 45 °c) were not conducive to the activity of released peptidases or exoproteases that would give rise to the amino acids.
Trevelyan (1976, 1977, 1978) has reported that the protein content of cells of baker's yeast decreased by more than 50 % during autolysis. Assuming that protein represents 50 % of the cell dry weight, then about 25 % of protein or less ( allowing for some breakdown to amino 161
acids) should appear in the autolysate. This extrapolated value is higher than tho5e reported in Tables 10 and 22 but, as pointed out already, Trevelyan' s conditions of autolysis were different to those used in the present study and gave, in general, a greater degree of cell solubilization.
Proteases are particularly active during autolysis and a complex of different proteases has been reported in autolysates of S. cerevisiae (Lenney 1956,
Hata et al. 1967ab, 1972, Hayashi et al. 1968ab,
Tohoyama and Takakuwa 1972, Behalova and Beran 1979,
Kelly-Treadwell 1988). These enzymes can be distinguished by their optimum pH and optimum temperature for activity. The conditions used in this study, (pH 4.5, 45 °c) would tend to favour the activity of protease A, an endoprotease that operates ~t acid pH. It is unlikely that proteases B and C, that have been described in yeasts, would be active under the above conditions (Hough and Maddox 1970). Protease B especially is not active at 45 °c (Maddox and Hough 1970). It is likely that the protease D described by Hough and Maddox (1970) and
Maddox and Hough (1970) is similar to the protease A •
Free amino acids in the autolysates represented about 8-12 % of the initial cell weight of the yeasts
(Table 23) . Somewhat higher values (19-27 %) were reported by Hough and Maddox ( 1970) for the release of amino acids into the autolysate of brewer's yeast. The 162
activity of proteases and peptidases, especially exo acting enzymes, will determine the concentration of amino acids in the autolysates. Such activity is likely to vary with the species of yeast and the conditions of autolysis. While considerable information is known about the proteases of S. cerevisiae (Hata et al. 1967ab, 1972,
Hayashi et al. 1968ab, Tohoyama and Takakuwa 1972,
Behalova and Beran 1979, Kelly-Treadwell 1988), there is a little equivalent information about the pro~eases of K. apiculata (Lagase and Bisson 1990) and C. stellata (Fleet
1992. The lower concentration of free amino acids in the autolysates of K. apiculata could be due to the fact that it might have decreased exo-protease activity.
The release of amino acids into yeast autolysates has been reported by Kulka (1953), Joslyn
(1955), Masschelein and Van de Meerssche (1976), Feuillat and Charpentier (1982), and Masschelein 1986). The principal amino acids found by these workers are shown in
Table 35, which are not completely matched with the main ones found in Table 12 and 24. The range of amino acids found in the autolysates will depend on the specificity and activity of the proteases associated with the particular yeast species, the nature of the proteins in the species and the conditions of au tolysis (pH, temperature) which affect the activity of proteases.
Hence, it is not unexpected to find some variation between species and strains (Tables 12, 23, 2~).
Further research that aims to correlate the
~ ~
0\ 0\
w w
acid acid
2days 2days
acid acid
C, C,
at at
20 20
yeast yeast
(1986) (1986)
acid acid
Prollne Prollne
Alanine Alanine
Glutamic Glutamic
Arglnlne Arglnlne
o<-amlnobutyrlc o<-amlnobutyrlc
8weeks 8weeks
lagering lagering
acid acid
C, C,
Methionine Methionine
Lysine Lysine
Glycine Glycine
Glutamic Glutamic
Arglnine Arglnine
0 0
Beer Beer
2days 2days
Masschelein Masschelein
C, C,
Flocculent Flocculent
10 10
Prollne Prollne
Glycine Glycine
Alanine Alanine
Tyrosine Tyrosine
«-amlnobutyrlc «-amlnobutyrlc
acid acid
authors authors
(1978) (1978)
. .
b b
acid acid
lagerlng lagerlng
several several
at at
by by
Beer Beer
(1982) (1982)
acid acid
Prollne Prollne
Glutamlc Glutamlc
Arglnlne Arglnlne
Alanine Alanine Meerssche Meerssche
acid acid
1-aminobutyric 1-aminobutyric
8weeks 8weeks
yeast yeast
4years 4years
condition condition
ageing ageing
and and
De De
C, C,
reported reported
Feuillat Feuillat
C, C,
condition condition
0 0
S.bayanus S.bayanus
as as
Van Van
Serine Serine
Alanine Alanine
35 35
Aspartlc Aspartlc
Threonine Threonine
Glutamic Glutamic
Wine Wine
Charpentier Charpentier
a a
acid acid
and and
autolysis autolysis
Flocculent Flocculent
Autolysis Autolysis
Lysine Lysine
Valine Valine Aspartic Aspartic
Leucine Leucine
Glycine Glycine
Autolysls Autolysls
yeast yeast
acid acid
Masschelein Masschelein
during during
days days
tank tank
3 3
acid acid
acid acid
anaerobic, anaerobic,
beer. beer.
C, C,
bottom bottom
21 21
released released
Beer, Beer,
Leucine Leucine
Proline Proline
Glutamic Glutamic
Arginine Arginine
o<-aminobutyric o<-aminobutyric
Alanine Alanine
Proline Proline
Glutamic Glutamic
Serine Serine
Threonine Threonine
yeast yeast
acids acids
(1953) (1953)
h h
Traditional Traditional
84 84
Kulka Kulka
amino amino
acid acid
Brewer's Brewer's
buffer buffer
C, C,
tank tank
Valine Valine
of of samples Different
+ +
30 30
Main Main
phenylalanine phenylalanine
= =
acid acid
+ +
b b
middle middle
4.5, 4.5,
34. 34.
Phosphate Phosphate
pH pH
and and
Phenylalanine Phenylalanine
a a
Valine Valine
Proline Proline
Alanine Alanine
Tyrosine Tyrosine
LeucinE:! LeucinE:!
Methionine Methionine
Alanine Alanine
Glutamic Glutamic
Table Table o(-aminobutyric o(-aminobutyric 164
kinetics of protein and amino acid release during autolysis with the activities of specific proteolytic enzymes is necessary to gain a better understanding of the dynamics of protein degradation and its control. The influences of pH and temperature on autolytic proteolysis would warrant particular attention because of the varying effects, which so far have been reported ins. cerevisiae. In this context, more research that examines the proteolytic systems in yeasts other than S. cerevisiae would be worthwhile.
5.6 Nucleic acids
Ribonucleic acid
The RNA content of S. cerevisiae has been reported as 6-8
% (Trevelyan 1976, 1977). The values of 3.5-4.0 % found in Tables 13 and 25 fall within the lower values of this range, and were determined by two different methods, namely, the optical density method and the orcinol method. Both methods gave equivalent values, with the orcino: method (which measures ribose) always giving just slightly higher values. There are no values in the literature for the content of RNA in K. apiculata and C. stellata, although values of 7-15 % (as nucleic acid) have been reported for C. utilis (Maul et al. 1970, Ohta et al. 1971, Sinskey 1975, Peppler 1979).
It has been well established by previous authors that the RNA of s. cerevisiae undergoes 165
extensive and rapid degradation during autolysis
(Trevelyan 1977). Depending upon the method of autolysis, it is generally 6-95 % degraded, with the degradation products appearing in the autolysate (Trevelyan 1976,
1977, 1978). This same trend was observed in the present study (Tables 13, 25) where, after 5-10 days, some 75-95
% of the RNA of all the yeasts examined was degraded.
According to Table 25, the bulk of the RNA had been degraded before 5 days. Slightly higher percentages of degradation were noted with the experi:nents reported in
Table 13, than Table 25. The reasons for this discrepancy are not evident, but it is known that RNA degradation is very susceptible to temperature influences, the RNase involved being most active in the temperature range 45-50
0 c (Ohta et al. 1971, Trevelyan 1976, 1977). It is possible that the autolysis experiments reported in Table
13 were conducted at slightly higher temperature than those reported in Table 25.
About 10-15 % of the RNA apparently degraded in the cell was not recovered in the autolysate. This observation was confirmed by both methods of RNA estimation, and is consistent with the earlier report of
Hough and Maddox ( 197 0) who noted an approximate 30 % discrepancy between the cell RNA degraded and that recovered in the autolysate. A likely explanation for this result is that some of the RNA degradation products may have been entrapped or associated with insoluble cell 166
wall material which was removed from the autolysate before analysis.
The degradation products of RNA are likely to be a complex mixture of oligo-ribonucleotides, ribonucleotides, ribonucleosides, and various bases
(adenine, guanine, cytosine, uracyl) , as well as ribose
(Hough and Maddox 1970, Kokova-Kratochvilova 1990) .
Because of the significant content of RNA in yeast cells and the wide spread commercial use of yeast autol:rsates as food ingredients, it would be worthwhile to know more about the nature of the RNA degradation products, their proportion in the autolysate and the factors that affect the concentrations of the individual fractions.
Further studies are needed to establish this information, as well as examine the types and properties of RNases involved in the autolytic reaction. It is possible, for example, that different yeast species or strains give autolysates with different concentrations of the final breakdown products.
Deoxyribonucleic acid
The DNA content of cells of S. cerevisiae has been reported to be approximately 0.2-1.2 % (Polakis and
Bartley 1966, McMurrough and Rose 1967, Trevelyan 1978).
Similar data have not been reported for K. apiculata and
C. stellata. As measured by the diphenylamine method, the method used by most authors (Herbert et al. 1971, Aigle 167
et al. 1983), the DNA content of the yeasts examined
range between 0.1-0.5 %, with the exception of S.
cerevisiae EC1118 which gave a value of 1.2 %. These are
within the range 0.2-1.2 % as reported for brewer's,
baker's, and Torula yeasts. The DNA content of the cells
as me3sured by the dye method (Labarca and Paigen 1980)
gave consistently low results (0.01-0.09 %) that did not
agree with the values reported in the literature. The
reasons for this discrepancy need further investigation.
The principles of the two methods as well as their
relative merits for determination of DNA in yeasts have been discussed by Labarca and Paigen (1980), Aigle et al.
(1983) and SubdeL and Krizus (1985). For the purposes of
this discussion, the DNA content as measured by the diphenylamine method will be considered as giving the most reliable data.
Unlike RNA, the DNA of the cells was not extensively degraded during autolysis and, depending on
the yeast, values of approximately 25-50 % were obtained
(Tables 14, 26) . Trevelyan ( 197 8) has also observed in baker's yeas c using the di phenyl amine method of assay,
that the DNA was not totally degraded by autolysis.
These findings are at variance with those of Hough and
Maddox (1970) who reported some 80-85 % DNA loss during
the autolysis of brewer's yeast and equivalent recovery
in the autolysates. In the present study, data on the
recovery of DNA material in the autolysates were not
consistent. While Table 26 shows approximately equivalent 168
recovery of the DNA in the autolysate, this was not so
for Table 14 where, for all of the strains examined, only
a small proportion of that initially present in the cells was found. Additional studies are required to resolve
these various discrepancies. Part of the problem probably resides in the fact that only low concentrations of DNA occur in the cells as well. as autolysates, creating difficulties for accurate analyses. According to Subden and Krizus (1985) accurate temperature control for the
8Xtraction and analysis of DNA is important in obtaining consistent data. Methods for the quantitative measurement
0f DNA in yeast cell need further evaluation and verification for reli~bility, for assays of DNA both inside and outside of the cell.
However, it is conclusive that some of the cell
DNA undergoes degradation during autolysis. Presumably, a complex of DNases is involved, and the nature and activity of these enzymes during autolysis as well as the identities of the DNA degradation products, warrant further study.
5.7 Lipids
The lipid content of S. cerevisiae has been reported as values ranging from 3. 5-20 % , and for species Candida, values between 0.3-23 % have been published (Ratray
1975, Ratledge and Evans 1989). No values for the lipid content of K. apiculata or Kloeckera species could be 169
found in the literature. The lipid contents for the strains of S. cerevisiae, K. apiculata, and c. stellata examined in this thesis were between 3-4 %, which represents the lower range of the values generally reported for "non oleaginous" yeasts. These lower values were consistently found (Table 15, 27) and were also noted in triplicate analyses. The lower values for the yeasts examined might be due to the fact that their lipid contents were calculated from summation of the amounts found for the individual lipid classes after their separation by thin layer chromatography. In this approach, the intensity of the lipid spot was taken as an index of concentration on comparison with the intensity of spots of known concentrations of standards. In this way, the lipids as measured, are relatively pure and free of contaminating protein or carbohydrate. This approach contrasts to the gravimetric methods that usually been used to calculate total lipid content (Kates and Paradis
1973, Takakuwa and Watanabe 1981). Nevertheless, a minor underestimation of the lipid content by the method used in this thesis is acknowledged as there were small spots
(e.g. Figures 8, 9) of unidentified lipid fractions that were not taken into account for the calculation of total lipid content because of the lack of authentic standards/reference lipids. Arnold (1981a) has oil specifically commented .Jthe difficulty of comparing data on lipid content and composition in yeasts because of the variation in lipid extraction methods used, variations 170
in methods of analysis, and the significant influences of
the conditions under which the yeast is cultured.
The composition of the total cellular lipid varied with the species (Tables 16, 28), particularly with respect to the proportion of the different classes of neutral lipids. Thus, triglycerides and diglycerides were the most prevalent in the lipids of S. cerevisiae, while in K. apiculata there were higher proportions of free fatty acids and cholestryl ester as well as diglyceride. In C. stellata, cholesteryl ester and, to a lesser extent triglyceride, were predominant. For S. cerevisiae, the proportions of the different lipid classes have been reported by several authors, but there is considerable variation in the values because of the important influence of the cultural environment {Ratray
1975, Watson and Rose 1980, Ratledge and Evans 1989).
Despite the difficulty of comparing data on lipid content and composition with respect to the literature, it was conclusively established for all three yeast species that cell lipid was degraded during autolysis. The extent of this degradation was much greater for S. cerevisiae {about 50 % after 10 days) than for the other two species {about 30-40 %) , and this could reflect the differences in composition of the lipids. As noted already, the lipids of S. cerevisiae were richer in content of triglycerides and diglycerides than those of the other species, and the contents of these components clearly decreased during the autolysis of this yeast 171
(Tables 16, 28). In contrast, the concentrations of free fatty acids a~d cholesteryl ester which were major components of the neutral lipids of K. apiculata were not as extensively degraded. Similarly, the cholesteryl ester of C. stellata were not extensively degraded during autolysis despit~ their predominance in that yeast.
Hardly any studies have been reported the degradation of neutral lipids during autolysis. Ishida-Ichirnasa ( 1978) briefly noted a c.lecrease in the content of sterol and sterol ester of baker's yeast, but this decrease depended on pH.
Consistent with some previous reports, the phospholipids of the yeasts were degraded during autolysis (Harrison and Trevelyan 1963, Takakuwa and
Watanabe 1981, Watanabe and Takakuwa 1983). Particularly noteworthy was the degradation of phosphatidylglycerol, especially for S. cerevisiae and C. stellata (Tables 16,
28). This has not been reported by other authors who have noted the specific degradation of phosphatidylethanol amine, phosphatidylcholine, and also the resistance of phosphatidylinositol to ctUtolytic breakdown.
A mixture of neutral lipids appeared in the autolysates, but no phospholipids were recovered from this source. The release of free fatty acids and triglycerides into beer and wine, presumably as a result of yeast autolysis, has been reported by Clapperton
(1978ab), Ahvenainen (1982), Piton et al. (1988), Troton
et al. ( 1989ab) . The recovery of lipid in the autolysa tes 172
was much less than that expected from the decrease in lipid content of the cells. This would suggest the complete degradation of some lipids to glycerol and free fatty acids of autolysates.
Glycerol was consistently recovered from the autolysate of all three yeasts (Table 29) and its content increased with autolysis time as did the content of free fatty acids. Higher concentrations of glycerol as well as those of free fatty acids were found in the autolysate of
S. cerevisiae, reflecting the greater lipid breakdown observed in this species. Attempts to examine the nature of the free fatty acids in autolysates were not successful probably because of their very low concentrations which was less than the detection limit of the GLC assay used. Larger batches of yeast would need to be autolysed to produce sufficient concentration of free fatty acids for separation and analyses.
In summary, the results of lipid analyses show that there are significant and interesting changes to these components in yeasts during autolysis. This is not unexpected in view of previous reports in the literature, and also in view of the extensive changes to cell membranes and cell permeability that seem characteristic of autolysis. Lipid chemistry, lipid analysis, and lipid metabolism are complex subjects (Ratray 1975, Chopra
1984, Ratledge and Evans 1984} and demand specialized, dedicated, attention. Further specific studies on the changes to lipids during yeast autolysis are needed to 173 confirm and clarify the reactions that occur. Concomitant studies on the activity of relevant lipolytic enzymes are also needed to provide some understanding of the biochemical mechanisms involved. Some studies have already implicated the role of phospholipases in autolytic lipolysis (Harrison and Trevelyan 1963, Takakuwa and Watanabe 1981, Watanabe and Takakuwa 1983) and these require verification. Because of the specific difficulties and demands associated with lipid extraction, fractionation, and analyses, much larger scale autolytic studies than those conducted in this thesis are needed to provide sufficient amounts of lipid materials for analyses.
5.8 Cytological changes
Several conclusions can be drawn from the electron microscopic studies reported in this study. First, the cell wall of all three species examined remained intact and was not disrupted during autolysis. This finding confirms previous observations with S. cerevisiae (Joslyn and Vosti 1955, Avakyants 1982, Babayan et al. 1981,
Charpentier et al. 1986) and extends the conclusion to include C. stellata and K. apiculata. It would appear, therefore, that the alkali-insoluble, acid-insoluble (1,3)-~-glucan of the wall is not degraded during autolysis since this is the wall component that confers wall rigidity and integrity (Fleet 1991). However, other components of the wall, such as the alkali soluble glucan 174
and the mannoprotein may undergo alteration. Such alterations are suggested by the fact that the thickness of the walls is decreased during autolysis, as indicated in Tables 32 and 33 and, also, as reported by other authors (Charpentier et al. 1986, Avakyants 1982).
Solubilization of mannoprotein during autolysis
(Valentine et al. 1984) could arise by glucanase action on the alkali-soluble glucan since it is believed that this glucan could anchor the mannoprotein to lhe wall
( Fleet 1991) . The protein component of the mannoprotein might be degraded since proteases are thought to be active during autolysis ( Sanz et al. 198 5, Fleet 1991) .
The release of mannoprotein from the outer surface of the wall could lead to rougher surface appearance as possibly noted in some scanning electron micrographs of S. cerevisiae and C. stellata {Figures 20 and 22).
Charpentier et al. ( 1986) noted the development of ridges on the wall surface (scanning eJectron micrographs) of S. cerevisiae after autolysis.
In line with the cytological and chemical alterations to the wall infrastructure during autolysis, some authors propose that the wall porosity must increase so as to permit passage of intracellular macromolecules, such as proteins and nucleic acids to the extracellular environment {Joslyn 1955, Arnold 198lb,Babayan et al.
1981).
The micrographs of Figure 23 ( S. cerevisiae) are consistent with those published by (Avakyants 1982) 175
which also showed that there was a major decrease in cytoplasmic density and vesicularization of membraneous material during autolysis of cells of S. cerevisiae. In addition, plasmolysis was observed as a definitive shrinkage of the cell membrane from the inner layer of the cell wall. These obser7ations were extended in this study to include the behaviour of C. stellata (Figure
25). However, the cytoplasmic reorganization of K. apiculata was different, with plasmolysis and vesicularization occurring to a lesser extent (Figure
24). These cytological observations support the chemical findings of this study that the autolytic behaviour of K. apiculata differs, to a limited extent, from that of S. cerevisiae and C. stellata. 176
6. CON LU SI ONS
1. All nine strains of yeast examined exhibited rapid loss of viability and autolytic breakdown when cell suspensions were incubated at 45 °c and pH 4.5. The autolytic breakdown was characterized by the release of carbohydrate, organic acids, proteins, amino acids, nucleic acids, lipids, fatty acid, and glycerol into the soluble autolysate. The cell wall and basic cell shape of all species remained entire throughout autolysis, but the thickness of the wall was significantly decreased and there was detachment of the plasmalemma from the inner layer of the wall. In addition, there was extensive loss of cytoplasmic contents and membranous vesicularization.
There were minor variations between strains of species examined. Between the species, K. apiculata exhibited some autolytic properties which were already different from those of S. cerevisiae and C. stellata.
2. There were differences between the three species in the kinetics of decrease in cell viability during autolysis. For all species, cell viability decreased rapidly, this being fastest for K. apiculata, followed by
C. stellata and S. cerevisiae. This order may reflect the relative sensitivities of the different species to heat inactivation.
3. Depending on species and strain, cell dry weights decreased by maximum values of 25-35 % during autolysis.
This loss was reflected in the recovery of biomass in the 177
autolysate, but about 4-6 % of the cell dry weight loss could not be accounted for in the autolysates. It is suspected that this discrepancy in mass balance might be due to weight losses through gas production and water evolution, and further studies are needed to test for this possibility. From data presented in the literature, it seems that higher cell weight losses by autolysis are obtained, but specific autolytic inducing agents such as organic solvents and salt, and possibly variations in pH and temperature, are needed to achieve these values.
Further studies of the specific effects of such agents/conditions on the kinetics and degree of yeast autolysis would be worthwhile.
4. Carbohydrate material was not a major product that was solubilized from the cells during autolysis.
Nevertheless, small but significant (2-6 %) amounts were found in autolysates. Most significantly, this existed as polysaccharide material, with very little free glucose or reducing sugar being found. Further studies are recommended to characterize the nature of this polysaccharide, especially from K. apiculata and c. stellata. From data presented in the literature for s.
cerevisiae, this polysaccharide is probably a mannoprotein degradation product from cell wall. Studies that examine the activities of glucanases and mannanases during autolysis may assist in understanding the fate of
the cell wall during autolysis and the release of associated polysaccharide components. The release of such 178
components has practical significance in wine and beer fermentation where they could affect the filterability and other properties of the product.
5. The detection of significant concentration (3-8 %) of organic acids in the autolysates was a novel finding, and there were interesting differences in the concentrations of individual organic acids between species (and strains in some cases) . Organic acids have well known sensory attributes: consequently, their release could assume commercial significance in wine and beer fermentations as well as in industrial preparations of yeast autolysates used as food ingredients. The biochemical mechanisms that lead to the autolytic production of these acids demand examination as well as those autolytic variables that are likely to affect their production.
6. Proteins and amino acids are well known major products of the autolysis of S. cerevisiae. This finding was confirmed in this study and extended to include yeasts of the species K. apiculata and C. stellata. The kinetics of protein release into autolysates differed to some previously published data in that less quantities were found and that extended autolysis did not show progressive breakdown of the released protein into amino acids. These differences highlight the need for carefully designed research that correlates the kinetics of autolytic release and breakdown of proteins with the activity of specific proteases. Such research would need to take into consideration that the protease activity in 179 yeasts is complex, consisting of many different ·enzymes that are active under different environmental conditions.
7. The characteristic breakdown of RNA during autolysis was confirmed in all yeast species and found to be rapid and extensive. The chemical nature of the end products (ribose, nucleotides, nucleosides, bases) is not well known, especially in terms of their relative proportions and the autolytic variables that affect these concentrations. In addition, the enzymes responsible for RNA degradation during autolysis require study.
Investigations of the degradation of DNA during autolysis were made difficult by the low concentrations of this component that occurs in yeasts, and some uncertainty about the reliability of the methods available for DNA measurement. The efficacy and reliability of these methods need to be resolved. Nevertheless, it was clear that DNA was partially degraded during autolysis and, as for RNA, more information is required about the chemical nature of the end products and the types of DNase activities involved.
8. Cell lipids were degraded during autolysis and to some extent, this was evidenced by the production of glycerol in the autolysates. In addition, small concentrations of neutral lipids, including free fatty acids were detected in autolysates. The composition of the cellular lipids in terms of proportions of neutral lipids and phospholipid components varied with the yeast species. But in all cases degradation was a feature of autolysis. Previous 180
studies with S. cerevisiae have convincingly demonstrated the breakdown of phospholipids during autolysis. This observation was confirmed in the present study as well as being extended to conclude the other two species, K. apiculata and C. stellata. In addition, it was evidPnt for all yeasts that their components of neutral lipids underwent degradation. Lipid chemistry, lipid analyses, and lipid metabolj sm are quite complex and require more detailed and specialized study with respect to yeast autolysis. Because of the low concentrations of the lipids and specific lipid classes that can occur in yeasts, larger scale autolysis experiments would appear to be necessary to produce sufficient materials for reliable quantitative analyses. The types of specific lipases and phospholipases associated with autolytic lipolysis have not been studied and require examination along with factors that affect their activity. membrane alteration seems to be a key feature of autolysis and this would certainly involve the activities of such enzymes and changes to lipid composition.
9. Electron microscopic studies verified that the yeast cell underwent autolysis. This was more evident for S. cerevisiae and C. stellata than for K. apiculata. Particularly obvious was the shrinkage of the plasmalemma from its usual proximity to the cell wall. Also very evident for all yeasts was the fact that the cell wall was not desintegrated during autolysis and that at retained the original cell shape. Nevertheless some 181
changes to wall composition or organisation are suggested from the measurements that indicate a decrease in overall thickness. Decrease in cell wall porosity is implied by the ability of molecules to diffuse od o£the cell. More information is required about wall composition and structure and their changes during autolysis. 182
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