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Arginine Metabolism in Malolactic Wine Lactic Acid Bacteria and Its Oenologicallmplications
A Thesis Presented i n Partial Fulfilment of the Requirem ents for the Degree of Doctor of Philosophy in
Microbiology at
Massey University Palmerston North
New Zealand
Shao-Quan Liu 1 993
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NAME AND ADDRESS DATE
i
ABSTRACT
L-Arg i n i n e is a m aj o r a m i n o acid fou nd i n g rapes and wine w h ich is degraded by some wine lactic acid bacteria (LAB). The m echanism of this degradation and its oenological implications were examined i n tbis research.
lt was found that wine LAB able to degrade arginine do so by means of the arg i n i n e d e i m i nase pathway, demonstrated by meas u ring the e nzyme activities in ce l l-free ext racts : argi n i n e de i m i n a s e , o rn it h i n e trans
carbamylase and carbamate kinase. These enzymes were present in most h et e rofe rme ntative lactobaci l l i and leuconostocs, but we re absent i n homofermentative lactobacilli and pediococci.
The prese nce of arginine i ncreased the activities of arg i n i n e d e i m inase pathway enzymes in heterofermenters, but failed to induce these enzymes in homofermenters even under conditions of low glucose concentration ( 1 g/L).
Glucose did not repress arginine utilisation but fructose appeared to do so, as fructose and arginine were metabolised sequentially, with arginine being metabolised mainly after utilisation of the fructose.
D etailed studies o n Leuconostoc oenos OENO, Lactobacillus b uchneri CUC-3 and Lactobacillus brevis 250 showed that arginine was converted stoichiometrically to ammonia and ornithine as the major end-products and that arginine catabolism could supply energy (ATP) to support growth. lt was also demonstrated that citrulline was excreted during arginine catabolism by both the lactobacilli and the leuconostoc. Some of the excreted citru lline was reassimilated and catabolised after arginine depletion by the lactobacilli, but not by the leuconostoc.
The implication of citrulline excretion for the wine industry was explored by studying the formation of the carcinogen ethyl carbamate (urethane) in a synthetic wine and a white wine, since citrulline is a known precursor of ethyl carbamate. Duri ng growth of Le. oenos OENO and Lb. buchneri CUC-3 in the synthetic wine and wine, significant amounts of ethyl carbamate were
found i n the two wine types upon heat treatment of samples. The formation of ethyl carbamate correlated well with arginine degradation and citrulline excretion. Citrulline excretion during arginine degradation is of concern to the winemaker, si nce the reaction of citru lli ne and ethanol to form ethyl carbamate has been shown by other workers to occur even at normal wine storage temperatures. Winemakers, therefore, should avoid usi ng arginine
degrading LAB starter cultures for inducing malolactic fermentation ( MLF). In addition, spo ntaneous MLF i n wine by u ndefined LAB strai ns should be discouraged, as this may lead to formation of ethyl carbamate precursors.
Ammonia detection with Nessler's reagent provides a simple, rapid test to assess arginine degradation by wine LAB in a complex medium, but is useful o n ly for strai ns showing strong ammonia fo rmation . The more sensitive enzymatic determination of ammonia is required for strains showing weak ammonia formation.
i i i
ACKNOWLEDGEM ENTS
Fi rst of a l l , I si ncerely thank my chief supervisor O r G .J. Pilon e for the gu i d a n c e , s u p p ort and t i m e du ri n g the cou rse of t h i s study a n d for encouraging me to e nter the competition for the New Zealand
Microbi ological Society P h . D Student Prize, which I won , at the a nnual conference of NZMS held at Massey U niversity, May, 1993.
I wou ld also like to thank my eo-supe rvisors Associate P rofessors G . G . Pritchard and M.J. Hard man for their advice and supervision.
The D epartment of Microbiology and Genetics p rovided the facilities and Graduate Assisstantship which I gratefully acknowledge.
The American Vineyard Fou ndation provided a grant to cover the cost of ethyl carbamate analysis, which was carried out at a reduced rate by ETS Laboratory, St. Helena, California, USA. Both are greatly appreciated.
I am indebted to my parents and brothers for their encouragement, support and patie nce during my schooli ng, u n iversity eudcation and postgraduate training.
And last, but not least, a big THAN KYOU to my wife, Marlene, for the special consu ltancy about e nzyme assays ; and for h e r understanding, care and patience for awaiti ng me to complete my PhD programme. The arrival of Andi brought lots of fun and 'pain', a thankyou to you, too.
Table of Contents
Page Abstract
Acknowledgements Table of Contents List of Tables
iii iv ix
List of Figures xi
Chapter 1 Introduction to the Thesis Chapter 2 Literature Review
1 4 2.1 The pathways of arginine catabolism by
micororganisms 4
2.2 Arginine catabolism in wine lactic acid bacteria 6 2.3 Arginine catabolism in non-wine lactic acid
bacteria 7
2.3.1 Occurrence of the arginine deiminase
pathway enzymes 7
2.3.2 Regulation of the arginine deiminase pathway enzymes
2.3.3 Energetics of arginine catabolism and molar growth yield
2.3.4 Arginine transport and genetics of the arginine deiminase pathway system 2.4 Formation of ethyl carbamate in wine Chapter 3 Verification of Assay Systems for Arginine
Dei minase Pathway Enzymes in Selected Wine
7 8
10 11
Lactic Acid Bacteria 14
3.1 I ntroduction 14
3.2 -Materials and Methods 14
3.2.1 Lactic acid bacterial strai ns 14 3.2.2 Medium preparation, culturing and
harvesting of wine LAB 15
3.2.3 Preparation of cell-free extracts 16
3.2.4 Analytical methods 16
3.2.4.1 Determination of protein concentration 16 3.2.4.2 Determination of arginine concentration 16
3.2.4.3 Determination of citrulli n e
concentration 1 7
3.2.4.4 Determination of ornithine concentration 1 8 3.2.4.5 Analysis of ammonia, u rea, L-malic acid,
glucose and fructose 1 8
3.2.4.6 Measurement of growth 1 8
3.2.4. 7 Measurement of pH 1 9
3.2.5 Assay for arginine deiminase 1 9 3.2.6 Assay for ornithine transcarbamylase 20 3.2.7 Assay for carbamate kinase 2 1
3.3 Results and Discussion 23
3.3. 1 Enzyme concentration and reaction rate 23 3.3.2 Substrate concentration and e nzyme
activities
3.3.3 pH and activity of AD I, OTC and CK 3.3.4 Storage stability of cell-free extracts
and whole cells, and effects of freezing
25 3 1
on enzyme activities 36
Chapter 4 Occurrence of Arginine Deiminase Pathway Enzymes in Wine Lactic Acid Bacteria
4.1 I ntroduction
4.2 Materials and Methods
4.2. 1 Lactic acid bacterial strai ns
4.2.2 Culturing and harvesting of wine LAB 4.2.3 Measurement of enzyme activity 4.2.4 Other analysis
4.3 Results
4.3. 1 Determination of optimal harvest time 4.3.2 Occurrence of arginine deiminase pathway
enzymes in wine LAB
4.3.3 Distinction of activities of acetate ki nase and carbamate ki nases
4.4 Discussion
Chapter 5 Arginine Catabolism and Metabolite Formation in Selected Wine Lactic Bacteria
5.1 Introduction
5.2 Materials and Methods
38 38 38 38 38 40 4 1 4 1 4 1 46
48 49
53 53 53
V
5.2. 1 Lactic acid bacterial strai ns 53 5.2.2 Medium composition and preparation 53 5.2.3 Cultu ring procedures and sampling regime 54
5.2.4 Analysis 56
5.3 Results 56
5.3. 1 G rowth and metabolism of heterofermentative
wine LAB in the absence of fructose 56
5.3. 1 .1 Le. oenos 56
5.3. 1 .2 Lb. brevis 250 and Lb. buchneri CUC-3 60 5.3. 1 .3 Stoichiometry of arginine catabolism 64 5.3.2 Growth and metabolism of heterofermentative
wine LAB in the presence of g lucose and
fructose 6 5
5.3.3 Growth and metabolism of homofermentative
wine LAB 65
5.4 Discussion
Chapter 6 Energetics of Arginine and Citrulline Catabolism
69 in Selected Wine Lactic Acid bacteria 76
6.1 Introduction 76
6.2 Materials and Methods 76
6.2. 1 Lactic acid bacterial strai ns 76 6.2.2 Medium composition and preparation 76
6.2.3 Cultu ring procedures 77
6.2.4 Determination of cellular dry weight 78 6.2.5 Determination of molar g rowth yields
and Y ATP 79
6.2.6 Analysis 79
6.3 Results 79
6.3. 1 Growth in presence of arginine at different
initial pH values 79
6.3.2 Determination of growth-limiting glucose concentration and molar growth yield of glucose grown cultures
6.3.3 Growth at different concentrations of arginine and citrulline
6.3.4 Molar growth yields of arginine and citrulline
82 84 87
vii
6.4 Discussion 9 1
C hapter 7 Ethyl Carbamate Formation in Relation to Arginine
Catabolism by Selected Wine Lactic Bacteria 94
7. 1 I ntroduction 94
7.2 Materials and Methods 94
7.2 . 1 Lactic acid bacterial strai ns 94 7.2.2 Preparation of synthetic wine and wine 94 7.2.3 Cultu ring procedures, growth conditions and
sampling regime 95
7.2.4 Growth measurement and analyses 96
7.3 Results 96
7.3. 1 Growth, L-malic acid deg radation and pH
changes 96
7.3.2 Arginine catabolism and citrulline
production 97
7.3.3 Ethyl carbamate formation 97
7.3.4 Control experiments 1 02
7.3.5 Percentage yields of ethyl carbamate from
citrulline 1 03
7.4 Discussion 1 04
Chapter 8 Correlation of ammonia and citrulline formation with the Occurrence of Arginine Deiminase Pathway
Enzymes in wine lactic acid bacteria 1 09
8.1 Introduction 1 09
8.2 Materials and Methods 1 09
8.2. 1 Lactic acid bacterial strai ns 1 09
8.2.2 HFA media 1 1 0
8.2.3 Culturing procedures 1 1 0
8.2.4 Analyses 1 1 0
8.3 Results 1 1 0
8.4 Discussion 1 1 4
Chapter 9 Regulation of Enzyme Synthesis in the Arginine Deiminase Pathway of Selected Wi ne Lactic Acid
Bacteria 1 1 9
9 . 1 I ntroduction 1 1 9
9.2 Materials and Methods 1 1 9
8.2. 1 Lactic acid bacterial strai ns 1 1 9
8.2.2 Medium composition and preparation 1 1 9
8.2.3 Culturing procedures 1 20
8.2.4 Measurement of enzyme activities 1 21 8.2.5 Determination of cell mass, pH and arginine 1 21
9.3 Results 1 21
9.3. 1 Activities of arginine deiminase pathway enzymes from wine LAB strains grown with or without arginine
9.3.2 Time-course induction of the arginine deiminase pathway enzymes
9.4 Discussion C hapter 1 0 General Discussion
1 0. 1 The pathway of arginine catabolism in wine lactic acid bacteria
1 0.2 Excretion of citrulline during arginine catabolism by wi ne lactic acid bacteria
1 0.3 Relationship between the utilisation of arginine and citrulline and the possible uptake system
1 21 1 24 1 29 1 31 1 31 1 33
of arginine and citrulline 1 34
1 0.4 Oenological implications of the present studies 1 35
1 0.5 Suggestions for future work 1 37
C hapter 1 1 Summary 1 38
Literature Cited 1 41
List of Publications 1 50
ix
List of Tables
Table Page
3. 1 Relationship betwee n enzyme concentrations and
reaction rates 24
3.2 Storage stability of cell-free extracts 36
3.3 effect of overnight freezing of whole cells (-70°C) on
activities of ADI, OCT and Ck from Le. o en os MCW 37 3.4 Storage stability of whole cells at -70°C 37 4. 1 Wine lactic acid bacteria and thei r origi nal sources 39 4.2 Occurrence of arginine deiminase pathway e nzymes in wine
lactic acid bacteria 47
4.3 Relative specific activities of arginine deiminase
pathway enzymes in wine lactic acid bacteria 48 4.4 A comparison of activities of carbamate kinases and
acetate ki nases from wine LAB 49
5. 1 Composition of the synthetic medium 55
5.2 Stoichiometry of arginine catabolism 64
6 . 1 Molar growth yields o f arginine and citrulline with or
without correcting cell yield from glucose consumption 90 7. 1 Growth, metabolism and ethyl carbamate formation of
Le. o enos ML 34 in synthetic wine and wine 1 02 7.2 Percentage yields of ethyl carbamate from citrulline
in synthetic wine and wine 1 04
8 . 1 Correlation of ammonia formation from arginine in H FA broth with the occurrence of arginine deiminase
pathway enzymes 1 1 1
8.2 Correlation of citrulline excretion with arginine degradation by wine lactic acid bacteria cultured in
HFA broth 1 1 4
9 . 1 Specific activities of arginine deiminase pathway
enzymes from Lb. buehneri CUC-3 cultured i n a synthetic
medium with or without L-arginine 1 23
9.2 Specific activities of arginine deiminase pathway enzymes from Le. oenos OENO cultured in a synthetic medium with
or without L-arginine 1 23
9.3 Specific activities of arginine deiminase pathway e nzymes from Lb. brevis 250 cultured in a synthetic m edium with
or without L-arginine 1 24
xi
List of Fig u res
Figure Page
2 . 1 The arginine deiminase pathway 5
3. 1 Relationship between arginine concentrations and activities of ADI in extracts of four wine LAB.
Assays were carried out using the conditions described in Section 3.2.5, except that arginine
concentration was varied as specified. Reported values are based on duplicate measurements from one extract.
Specific activities are i n U/mg. 26
3.2 Relationship between ornithine concentrations and activities of OTC in extracts of four wine LAB. Reported values are based on duplicate measurements from one extract. Assays were conducted at pH 7.6 for strains MCW and OENO; pH 8.0 for strain 250 and pH 8.5 for
strain CUC-3. Specific activities are in U/mg. 28 3.3 Relationship between carbamyi-P concentrations and
activities of CK in extracts of four wine
LAB. Duplicate measurements were made from one extract.
Assay pH levels were the same as i n Fig. 3.2.
Specific activities are in U/mg. 29
3.4 Relationship between substrate concentrations and activities of CK in extracts of four wine LAB. Single measurements were made from one extract. C hanges i n substrate concentration or coupling enzyme level were made relative to those used in the standard (std) assay (Section 3.2. 7). Thus, 2[ ] or 1 /2[ ] means that a
substrate concentration of twice or half that i n the standard assay. Assays were conducted at pH 7.6 for strain MCW and pH 7.9 for others. Specific activities
are in U/mg. 30
3.5 Relationship between pH and activities of ADI in extracts of three wine LAB. Assay conditions as described i n
Section 3.2.5. Specific activities are in U/mg. 32 3.6 Relationship between pH and activities of OTC in extracts
of four wine LAB. Assay conditions as described in
Section 3.2.6. Specific activities are in U/mg. 34 3.7 Relationship between pH and activities of CK in extracts
of four wine LAB. Assay conditions are as described in
Section 3.2.7. Specific activities are in U/mg. 35 4.1 Time-course of activities of arginine deiminase
pathway enzymes during incubation (30°C) of Le. oenos
Er1 a in TJBAG broth. Specific activities are i n U/mg. 42 4.2 Time-course of activities of arginine deiminase
pathway enzymes during incubation (30°C) of Le. oenos
MCW i n TJ BAG broth . Specific activities are in U/mg. 43 4.3 Time-course of activities of arginine deiminase
pathway enzymes during incubation (30°C) of Le. oenos OENO in TJ BAG broth. Specific activities are in U/mg.
4.4 Time-course of activities of arginine deiminase 44 pathway enzymes during incubation (30°C) of Lb. buehneri
CUC-3 in T JBAG broth. Specific activities are in U/mg. 45 5. 1 G rowth (30°C) and metabolism of Le. oenos OENO in a
synthetic medium containing 5 g/L each g lucose and
arginine. 58
5.2 G rowth (30°C) and metabolism of L e. oenos OENO in a synthetic medium containing 1 g/L g lucose and 5 g/L
arginine. 59
5.3 G rowth (30°C) and metabolism of Lb. brevis 250 in a synthetic medium containing 1 g/L g lucose and 5 g/L
arginine. 6 1
5.4 G rowth (30°C) and metabolism of Lb. buehneri CUC-3 in a synthetic medium contai ning 1 g/L glucose and 5 g/L
arginine. 62
5.5 G rowth (30°C) and metabolism of Lb. buehneri CUC-3 in a synthetic medium containing 5 g/L each glucose and
arginine. 63
5.6 G rowth (30°C) and metabolism of Le. oenos OENO and Lb.
buehneri CUC-3 in a synthetic wine containing glucose
and arginine (2.5 g/L each). 66
5.7 G rowth (30°C) and metabolism of Lb. brevis 250 in a synthetic medium containing glucose and arginine
xi i i
(5 g/L each). c 67
5.8 G rowth (30°C) and metabolism of Lb. delbruekii " CUC-1 and Pd. sp CUC-4 i n a synthetic medium containing
1 g/L glucose and 5 g/L argini ne. 68
5.9 The urea cycle (modified after Stryer, 1 98 1 ). 70 6 . 1 G rowth o f Lb. brevis 250, Lb. buehneri CUC-3 and Le.
oenos OENO at different initial pH values i n synthetic
media containing glucose with or without arginine. 80 6.2 Cell yield of Lb. brevis 250 and Le. oenos OENO in
synthetic media containing glucose at i ncreasing
concentrations (initial pH 5.5, 30°C). 83
6.3 G rowth (30°C) of Lb. bre vis 250 and Lb. buehneri CUC-3 i n synthetic media containing glucose at 1 3.3 mM and
at various concentrations of arginine (initial pH 3.5). 85 6.4 G rowth (30°C) of Lb. buehneri CUC-3 and Le. oenos OENO
i n synthetic media containing glucose at 1 3.3 mM and
at various concentrations of citrulline (initial pH 3.5). 86 6.5 Cell yield from arginine consumption in Lb. brevis 250,
Lb. buehneri CUC-3 and Le. oenos OENO. 88
6.6 Cell yield from citrulline consumption i n Lb. buehneri
CUC-3. 89
7. 1 Arginine and L-malic acid catabolism and metabolite formation duri ng incubation (22°C) of Le. oenos OENO i n synthetic wine. Samples for measuring ethyl carbamate taken at the i ndicated times were determined with and without subsequent heating (71 °C/48 hrs). In graph C, the bars at 437 hours indicate the range of duplicate
determinations. 98
7.2 Arginine and L-malic acid catabolism and metabolite formation during incubation (22°C) of Le. oenos OENO in a white wine. Samples for measuring ethyl carbamate
taken at the i ndicated times were treated as in Fig. 7.1 . 99 7.3 Arginine and L-malic acid catabolism and metabolite
formation duri ng incubation (22°C) of Lb. buehneri CUC-3 i n synthetic wine. Samples for measu ring ethyl carbamate taken at the i ndicated times were treated as in Fig. 7.1 .
In graph C, the bars at 1 74 hours indicate the range of
duplicate determinations. 1 00 7.4 Arginine and L-malic acid catabolism and metabolite
formation during incubation (22°C) of Lb. buchneri CUC-3 in a white wine. Samples for measuring ethyl carbamate taken at the indicated times were treated as in Fig. 7.1 . The dashed plot of the u nheated samples i n g raph C indicates concentrations of ethyl carbamate below
detectable values. 1 01
9 . 1 G rowth, arginine catabolism, p H and activities of the arginine deiminase pathway enzymes duri ng incubation (30°C) of Lb. brevis 250 in a complex mediu m. Arrow indicates time of arginine addition. Enzyme activities at each point are the averages of duplicate measurements
in one extract of a single culture. 1 26
9.2 Growth, arginine catabolism, pH and activities of the arginine deiminase pathway enzymes during incubation (30°C) of Lb. buchneri CUC-3 in a complex medium. Arrow indicates time of arginine addition. Enzyme activities at each poi nt are the averages of duplicate measurements i n
one extract o f a single cultu re. 1 27
9.3 Growth, arginine catabolism, pH and activities of the arginine deiminase pathway enzymes duri ng incubation (30°C) of Le. oenos OENO in a complex medium. Arrow indicates time of arginine addition. Enzyme activities at each point are the averages of duplicate measurements
in two separate extracts of a single cultu res. 1 28
