Journal of Dairy Research (1975), 42, 139-146
139
Citrate utilization in milk by Leuconostoc cremoris and Streptococcus diacetilactis B Y T. M. COGAN National Dairying Research Centre, The Agricultural Institute, Fermoy, Co. Cork, Irish Republic (Received 13 May 1974) STJMMABY. Citrate utilization and diacetyl, acetoin and acetaldehyde production by 2 strains each of Leuconostoc cremoris and Streptococcus diacetilactis in milk were studied. With the leuconostoc bacteria no growth and little citrate utilization occurred unless a stimulant (yeast extract) was present, when complete utilization of citrate without concomitant production of diacetyl or acetoin was obtained. The addition of Mn 2+ stimulated growth and citrate utilization in the presence of yeast extract. Addition of citric acid after 65-h growth resulted in diacetyl and acetoin production. Destruction of diacetyl and acetoin occurred when the citric acid level fell to c. 1000 and 600/ig/g in the case of Leuc. cremoris FR8-1 and CAFl, respectively. Only strain FR8-1 produced acetaldehyde. In contrast, Str. diacetilactis produced diacetyl, acetoin and acetaldehyde concomitant with citrate utilization.
Many workers have shown that citrate is the precursor compound for diacetyl and acetoin production by mixed-strain starter cultures growing in milk. The production of these compounds during growth by pure cultures of Streptococcus diacetilactis and Leuconostoc species has been less well studied. Lightbody (1962) compared diacetyl production by several strains oiStr. diacetilactis in cream. Maximum amounts were produced when the pH fell to c. 5-5, after which destruction occurred. Chuang & Collins (1968) using Str. lactis and Eschenbruch (1970) using Leuc. citrovorum and Leuc. mesenteroides showed that maximum amounts of acetoin were produced at the end of the exponential phase of growth in the case of cells grown on glucose and malate respectively after which destruction occurred. Lactobacillus casei produced maximum concentrations of diacetyl in milk during the stationary phase of growth (Keenan & Lindsay, 1968). We have shown (Walsh & Cogan, 1973) that diacetyl and acetoin production in milk by mixed cultures proceeds as long as citrate is present. Once citrate utilization is complete, destruction of both compounds occurs. Thus, analysis at one point during growth could lead to misinterpretation of results. In the present study, citrate utilization and diacetyl and acetoin production by pure cultures of Leuc. cremoris and Str. diacetilactis in milk were studied.
140
T. M. COGAN MATERIALS AND METHODS
Cultures. Leuc. cremoris FR8-1 was isolated from FR8 mixed-strain starter, obtained from Dr J. Stadhouders, NIZO, Ede, The Netherlands, and Leuc. cremoris CAFl (Leuc. citrovorum) and Str. diacetilactis DRCl were obtained from Mr R. A. Speckman, Department of Food Science and Technology, University of California, Davis, California, U.S.A. Str. diacetilactis DRC3 was obtained from M. J.-P. Accolas, INRA, Jouy-en-Josas, France. The leuconostoc cultures were identified as Leuc. cremoris by the methods recommended by Sharpe, Fryer & Smith (1966). Propagation. Str. diacetilactis cultures were routinely grown at 25 °C in litmus milk using 1 % inocula while the leuconostoc strains were grown in litmus milk containing 0-3% (w/v) yeast extract. Experimental procedure. Autoclaved (5 min, 121 °C) 10% skim-milk was inoculated with 1-2 % of the culture being studied. Separately sterilized yeast extract (0-3% w/v) was added to the leuconostoc cultures. The inoculated milk was thoroughly mixed and divided into approximately 50-ml vol. in sterile bottles and incubated. Single bottles were removed periodically for the various analyses. Where additions of citric acid were made after growth, the volumes (generally 1 1) of inoculated milk were accurately measured before incubation using a sterile graduated cylinder. After about 65 h, sufficient citric acid was added to give the desired final concentration, the pH measured and the control culture brought to the same pH with 5 N-HC1. Before each sampling (generally at 1-h intervals) the flasks and contents were shaken. All incubations were at 21 °C. Manganese, as MnS0 4 , was made up to a concentration of 1 mg Mn2+/ml and filtersterilized. Chemical analysis. The percentage of lactic acid developed was measured by titrating 10 g of culture to pH 8-3 with 0-11 N-NaOH using a Radiometer pH stat and correcting for the inherent acidity of the uninoculated milk. Occasionally acid production was measured by pH. Citrate was measured by the method of Marier & Boulet (1958) and acetaldehyde by the method of Lindsay & Day (1965) except that a sample of the first "10 ml of steam distillate from 20 g of culture was used. Diacetyl and acetoin were estimated by modifications of the Prill & Hammer and Westerfeld methods respectively (Walsh & Cogan, 1974a, b).
RESULTS
Leuconostoc cremoris
The addition of yeast extract (0-3 %, w/v, final concentration) to the skim-milk stimulated both growth and citrate utilization by strain CAFl (Fig. la). In the presence of yeast extract, citrate utilization was complete at a per cent developed lactic acid of 0*014 (pH 6-00). No detectable amounts of diacetyl or acetoin were produced at any stage during growth. The addition of Mn2+ (4 /tg/ml) as well as yeast extract resulted in further stimulation of growth and more rapid citrate utilization without detectable amounts of diacetyl or acetoin being produced (Fig. 16). Similar results were obtained for strain FR8-1. Incubation of strain CAFl for 65 h before addition of citric acid resulted in
Citrate utilization by starters
1000
141
-
500 -
U
001
16
Fig. 1. Growth of strain CAFl in 10% skim-milk, (a) Lactic acid production (O, • ) and citric acid utilization (A, A) in cultures with (•, • ) or without (O. A) 0-3% w/v yeast extract. (6) Lactic acid production (O, • ) and citric acid utilization (Q, • ) in cultures containing 0-3% w/v yeast extract with (•, • ) or without (O, A) 4 fig Mn2+/ml. No diacetyl or acetoin was produced in either case.
diacetyl and acetoin production (Fig. 2). Much greater amounts of acetoin than of diacetyl were produced. However, production of the 2 compounds paralleled each other. The addition of citric acid lowered the initial pH value from 5-1 to 4-7, 4-5 and 4-1 in the case of 7-0, 14-9 and 31-8 mM added citric acid, respectively. The largest amounts of acetoin and diacetyl were produced at the highest level of citric acid added while no production occurred in the absence of added citric acid. Maximum production of both acetoin and diacetyl occurred after 2 and 5-5 h incubation in the case of 7-0 and 14-9 mM added citric acid, respectively, after which destruction occurred when the citric acid level fell below 650 /£g/g. A similar response was obtained for strain FR8-1 except that destruction occurred when the citrate level fell to 1000/ig/g. Assuming that 2 moles of citric acid give rise to 1 mole of diacetyl + acetoin + 2,3-butylene glycol, little of the citric acid utilized by either culture was recovered as acetoin and diacetyl (Table 1). The addition of Mn2+ (4/tg/ml) and 16-7 mM citric acid to preincubated cultures of strain CAFl led to an increased rate of citrate utilization and rapid destruction of acetoin (Fig. 3). Maximum production occurred earlier in the culture receiving Mn2+. Destruction occurred in both cases when the citrate level had fallen to about 500 jug/g. Similar results were obtained for strain FR8-1.
142
T. M. COGAN Diacetyl
Acetoin
10000 5000
1000
500
100
10
50
5
10
10 S
U
0-5
5
I
10
15
10
0 Time, h
15
Fig. 2. Effect of citric acid (•, A, • ) on acetoin and diacetyl production (O, A, • ) in 10% skim-milk containing 0-3% yeast extract by strain CAFl. The culture was preincubated for 65 h at 21 °C before addition of citric acid. No diacetyl or acetoin production occurred in the absence of added citric acid. t , 7 0 mM; • , 14-9 mM; A, 31-8 mM citric acid.
Table 1. Maximum concentrations (HIM) of diacetyl and acetoin produced and citric acid utilized by Leuconostoc cremoris CAFl and FR8-1 Initial level* of citric acid added
Citric acid utilized
Diacetyl produced
Acetoin produced
Total diacetyl + acetoin
Expected yieldt
CAFl
70 14-9
3-3 9-6
002 006
0-57 2-62
0-59 2-68
1-7 4-8
FR8-1
6-9 15-2
2-2 7-7
001 006
013 105
014 Ml
11 3-9
Strain
* Cultures were preincubated for 65 h before addition of citric acid. f Total of diacetyl + acetoin + 2,3-butylene glycol.
Citrate utilization by starters
143
Time, h Fig. 3. Effect of 4 fig Mna+/ml ( • • ) on citric acid utilization (O. • ) and acetoin production (O, • ) in 10% skim-milk containing 0-3% yeast extract by strain CAF1. The culture was preincubated for 65 h at 21 °C before additions of citric acid and Mn2+.
The addition of 15-7 mM trisodium citrate to the skim-milk prior to inoculation raised the initial pH value from 6-5 to 6-9 and resulted in traces of acetoin (maximum 7-0 /ig/g), but no diacetyl production by strain CAFl. Little citrate had been utilized when the maximum amount of acetoin was produced. Under similar conditions, strain FR8-1 produced no acetoin or diacetyl. Strain CAFl produced no detectable amounts of acetaldehyde under any experimental condition whereas strain FR8-1 did. The amounts produced varied and did not parallel diacetyl or acetoin production. Acetaldehyde production by strain FR8-1 gradually increased during growth and generally reached a maximum of 65 /tg/g after 60 h incubation. The addition of citric acid after growth for 65 h had no effect on acetaldehyde production by FR8-1 which remained constant at about 65 fig/g throughout the experiment.
DAR 42
144
T. M. COGAN
1000 500
100
10
50
0-5
100
0-1
50
005
10
001
0-5
0-1
I
8
12 Time, h
16
20
Fig. 4. Diaoetyl (•), acetoin (D), acetaldehyde (•), and lactic acid (A) production and citric acid (O) utilization by Streptococcus diacetilactis DRC3 in 10% skim-milk.
Streptoccus diacetilactis ' Production of diacetyl and acetoin and utilization of citrate by Str. diacetilactis DRC3 in skim-milk is shown in Fig. 4. Little diacetyl (c. 1 /tg/ml), but large amounts of acetoin (c. 250/jg/ml) were produced. Maximum amounts of the latter were produced only when citrate utilization was complete after 9 h incubation. Little if any destruction of acetoin occurred. The addition of citrate resulted in higher amounts of both diacetyl and acetoin and slightly greater amounts of lactic acid being produced. Similar results were obtained with strain DRCl except for slight destruction of acetoin, which coincided with the disappearance of citrate. Mn 2+ (4 /ig/ml) had no effect on citrate utilization or diacetyl and acetoin production by strains DRCl and DRC3.
Citrate utilization by starters
145
DISCUSSION
It is well documented that mixed-strain starter cultures containing Leuc. cremoris and Str. diacetilactis produce diacetyl and acetoin from citrate when grown in milk. However, in pure culture these bacteria behaved entirely differently - in the case of Leuc. cremoris, yeast extract was necessary for growth and citrate was utilized without concomitant production of diacetyl and acetoin, while the opposite was true of Str. diacetilactis. The reason for the unexpected results with the leuconostoc bacteria remains obscure, but we are at present endeavouring to elucidate it. The phenomenon is probably a general one since other leuconostoc strains tested behaved in a similar manner (unpublished results). However, not all leuconostoc species will utilize citrate (Dr E. I. Garvie, personal communication). These results are in agreement with those of van Beynum & Pette (1939), who showed that leuconostoc bacteria growing in neutral media containing citrate produced no aroma. Michaelian, Farmer & Hammer (1933) found that 29 out of 35 citric-acid-fermenting streptococci (leuconostocs) produced no aroma in milk after 7 d at 21 °C. These workers also found that cultures preincubated for 24 h in milk before acidification with one of several acids produced diacetyl and acetoin. Further studies (Michaelian, Hoecker & Hammer, 1938) showed that citric acid was a much better acidulant than either lactic or sulphuric acid for the production of diacetyl+ acetoin. However, their cultures were not examined for 48 h after acidifying, while the present data suggest that unless a large amount of citric acid is added, maximum acetoin production occurs within 2-5 h. In acidified cultures of leuconostoc much lower amounts of diacetyl and acetoin were produced than citrate utilized suggesting that, unless 2,3-butylene glycol is also produced, citrate is being utilized in the formation of other compounds in the cell. During this time it is unlikely that the cultures were actively growing because of the low pH. In the control flask where HC1 was the acidifying agent the pH did not change during the 14 h of the experiment. The pH in the cultures acidified with citrate increased as utilization proceeded. Thus, it is possible that diacetyl and acetoin are produced only when active growth is unfavourable. Acetoin is quantitatively a much more important compound - in fact recovery of diacetyl could conveniently be ignored in metabolic studies. Mn2+ stimulated growth and citrate utilization in milk by the leuconostoc cultures, as shown by de Man & Galesloot (1962). Mn2+ also stimulated citrate uptake and reduction of acetoin in acidified cultures under conditions where growth was unlikely to occur, suggesting that both the enzymes necessary for uptake and breakdown have a Mn2+ requirement. pH may also be important, since Harvey & Collins (1962) have shown that strain CAFl has a pH-dependent citrate permease. Production and destruction of both diacetyl and acetoin paralleled each other, which is surprising since Speckman & Collins (1968) have shown that 2 distinct biosynthetic pathways are involved. The reduced compound must be presumably 2,3-butylene glycol. The production of acetaldehyde by strain FR8-1 is surprising since Walsh & Cogan (1973) showed that the parent mixed culture produced little if any acetaldehyde. A possible explanation may be that the lactic streptococci present in the
146
T. M. COGAN
culture reduced the acetaldehyde as it was formed. In this connexion Bills & Day (1966) have noted that at least one strain each of Str. lactis and Str. cremoris can reduce added acetaldehyde in milk. The 2 strains of Str. diacetilactis showed similar production patterns for diacetyl, acetoin and acetaldehyde as D and BD cultures (Walsh & Cogan, 1973), suggesting that in a BD culture it is the Str. diacetilactis component and not Leuc. cremoris which is mainly responsible for flavour production. Presumably, in B type cultures, the Leuc. cremoris component, although stimulated by the lactic streptococci, still grows slowly. In these conditions sufficient citrate is present at the end of the customary growth period of 12-16 h to act as a precursor of diacetyl and acetoin production in the acid conditions present in the culture. This is borne out by the results of Walsh & Cogan (1973), who showed that in B type cultures citrate utilization does not start until after about 10 h of incubation at 21 °C. The author is indebted to Mr P. Thornbill and Mr F. Drinan for excellent technical assistance, to Dr S. Condon and Mr F. O'Connor for their help in preparation of the manuscript and to Dr E. I. Garvie for confirming that strain CAFl is Leuc. cremoris.
REFERENCES BILLS, D. D. & DAY, E. A. (1966). Journal of Dairy Science 49, 1473. CHUANG, L. F. & COLLINS, E. B. (1968). Journal of Bacteriology 95, 2083. DE MAN, J. C. & GALESLOOT, TH. E. (1962). Netherlands Milk and Dairy Journal 16, 1. ESCHENBRUCH, R. (1970). Vitis 9, 218. HARVEY, B. J. & COLLINS, E. B. (1962). Journal of Bacteriology 83, 1005. KEENAN, T. W. & LINDSAY, K. C. (1968). Journal of Dairy Science 51, 188. LIOHTBODY, L. G. (1962). Australian Journal of Dairy Technology 17, 36. LINDSAY, R. C. & DAY, E. A. (1965). Journal of Dairy Science 48, 665. MARIEB, J. R. & BOULET, M. (1958). Journal of Dairy Science 41, 1683. MICHAELIAN, M. B., FARMER, R. S. & HAMMER, B. W. (1933). Research Bulletin No. 155, Iowa State College of Agriculture and Mechanic Arts. MICHAELIAN, M. B., HOECKER, W. H. & HAMMER, B. W. (1938). Journal of Dairy Science 21, 213.
SHARPE, M. E., FRYER, T. F. & SMITH, D. G. (1966). Technical Series, Society for Applied Bacteriology 1,69. SPECKMAN, R. A. & COLLINS, E. B. (1968). Journal of Bacteriology 95, 174. VAN BEYNUM, J. & PETTE, J. W. (1939). Journal of Dairy Research 10, 250. WALSH, B. & COGAN, T. M. (1973). Applied Microbiology 26, 820. WALSH, B. & COGAN, T. M. (1974a). Journal of Dairy Research 41, 25. WALSH, B. & COGAN, T. M. (19746). Journal of Dairy Research 41, 31.
Printed in Great Britain
139
Citrate utilization in milk by Leuconostoc cremoris and Streptococcus diacetilactis B Y T. M. COGAN National Dairying Research Centre, The Agricultural Institute, Fermoy, Co. Cork, Irish Republic (Received 13 May 1974) STJMMABY. Citrate utilization and diacetyl, acetoin and acetaldehyde production by 2 strains each of Leuconostoc cremoris and Streptococcus diacetilactis in milk were studied. With the leuconostoc bacteria no growth and little citrate utilization occurred unless a stimulant (yeast extract) was present, when complete utilization of citrate without concomitant production of diacetyl or acetoin was obtained. The addition of Mn 2+ stimulated growth and citrate utilization in the presence of yeast extract. Addition of citric acid after 65-h growth resulted in diacetyl and acetoin production. Destruction of diacetyl and acetoin occurred when the citric acid level fell to c. 1000 and 600/ig/g in the case of Leuc. cremoris FR8-1 and CAFl, respectively. Only strain FR8-1 produced acetaldehyde. In contrast, Str. diacetilactis produced diacetyl, acetoin and acetaldehyde concomitant with citrate utilization.
Many workers have shown that citrate is the precursor compound for diacetyl and acetoin production by mixed-strain starter cultures growing in milk. The production of these compounds during growth by pure cultures of Streptococcus diacetilactis and Leuconostoc species has been less well studied. Lightbody (1962) compared diacetyl production by several strains oiStr. diacetilactis in cream. Maximum amounts were produced when the pH fell to c. 5-5, after which destruction occurred. Chuang & Collins (1968) using Str. lactis and Eschenbruch (1970) using Leuc. citrovorum and Leuc. mesenteroides showed that maximum amounts of acetoin were produced at the end of the exponential phase of growth in the case of cells grown on glucose and malate respectively after which destruction occurred. Lactobacillus casei produced maximum concentrations of diacetyl in milk during the stationary phase of growth (Keenan & Lindsay, 1968). We have shown (Walsh & Cogan, 1973) that diacetyl and acetoin production in milk by mixed cultures proceeds as long as citrate is present. Once citrate utilization is complete, destruction of both compounds occurs. Thus, analysis at one point during growth could lead to misinterpretation of results. In the present study, citrate utilization and diacetyl and acetoin production by pure cultures of Leuc. cremoris and Str. diacetilactis in milk were studied.
140
T. M. COGAN MATERIALS AND METHODS
Cultures. Leuc. cremoris FR8-1 was isolated from FR8 mixed-strain starter, obtained from Dr J. Stadhouders, NIZO, Ede, The Netherlands, and Leuc. cremoris CAFl (Leuc. citrovorum) and Str. diacetilactis DRCl were obtained from Mr R. A. Speckman, Department of Food Science and Technology, University of California, Davis, California, U.S.A. Str. diacetilactis DRC3 was obtained from M. J.-P. Accolas, INRA, Jouy-en-Josas, France. The leuconostoc cultures were identified as Leuc. cremoris by the methods recommended by Sharpe, Fryer & Smith (1966). Propagation. Str. diacetilactis cultures were routinely grown at 25 °C in litmus milk using 1 % inocula while the leuconostoc strains were grown in litmus milk containing 0-3% (w/v) yeast extract. Experimental procedure. Autoclaved (5 min, 121 °C) 10% skim-milk was inoculated with 1-2 % of the culture being studied. Separately sterilized yeast extract (0-3% w/v) was added to the leuconostoc cultures. The inoculated milk was thoroughly mixed and divided into approximately 50-ml vol. in sterile bottles and incubated. Single bottles were removed periodically for the various analyses. Where additions of citric acid were made after growth, the volumes (generally 1 1) of inoculated milk were accurately measured before incubation using a sterile graduated cylinder. After about 65 h, sufficient citric acid was added to give the desired final concentration, the pH measured and the control culture brought to the same pH with 5 N-HC1. Before each sampling (generally at 1-h intervals) the flasks and contents were shaken. All incubations were at 21 °C. Manganese, as MnS0 4 , was made up to a concentration of 1 mg Mn2+/ml and filtersterilized. Chemical analysis. The percentage of lactic acid developed was measured by titrating 10 g of culture to pH 8-3 with 0-11 N-NaOH using a Radiometer pH stat and correcting for the inherent acidity of the uninoculated milk. Occasionally acid production was measured by pH. Citrate was measured by the method of Marier & Boulet (1958) and acetaldehyde by the method of Lindsay & Day (1965) except that a sample of the first "10 ml of steam distillate from 20 g of culture was used. Diacetyl and acetoin were estimated by modifications of the Prill & Hammer and Westerfeld methods respectively (Walsh & Cogan, 1974a, b).
RESULTS
Leuconostoc cremoris
The addition of yeast extract (0-3 %, w/v, final concentration) to the skim-milk stimulated both growth and citrate utilization by strain CAFl (Fig. la). In the presence of yeast extract, citrate utilization was complete at a per cent developed lactic acid of 0*014 (pH 6-00). No detectable amounts of diacetyl or acetoin were produced at any stage during growth. The addition of Mn2+ (4 /tg/ml) as well as yeast extract resulted in further stimulation of growth and more rapid citrate utilization without detectable amounts of diacetyl or acetoin being produced (Fig. 16). Similar results were obtained for strain FR8-1. Incubation of strain CAFl for 65 h before addition of citric acid resulted in
Citrate utilization by starters
1000
141
-
500 -
U
001
16
Fig. 1. Growth of strain CAFl in 10% skim-milk, (a) Lactic acid production (O, • ) and citric acid utilization (A, A) in cultures with (•, • ) or without (O. A) 0-3% w/v yeast extract. (6) Lactic acid production (O, • ) and citric acid utilization (Q, • ) in cultures containing 0-3% w/v yeast extract with (•, • ) or without (O, A) 4 fig Mn2+/ml. No diacetyl or acetoin was produced in either case.
diacetyl and acetoin production (Fig. 2). Much greater amounts of acetoin than of diacetyl were produced. However, production of the 2 compounds paralleled each other. The addition of citric acid lowered the initial pH value from 5-1 to 4-7, 4-5 and 4-1 in the case of 7-0, 14-9 and 31-8 mM added citric acid, respectively. The largest amounts of acetoin and diacetyl were produced at the highest level of citric acid added while no production occurred in the absence of added citric acid. Maximum production of both acetoin and diacetyl occurred after 2 and 5-5 h incubation in the case of 7-0 and 14-9 mM added citric acid, respectively, after which destruction occurred when the citric acid level fell below 650 /£g/g. A similar response was obtained for strain FR8-1 except that destruction occurred when the citrate level fell to 1000/ig/g. Assuming that 2 moles of citric acid give rise to 1 mole of diacetyl + acetoin + 2,3-butylene glycol, little of the citric acid utilized by either culture was recovered as acetoin and diacetyl (Table 1). The addition of Mn2+ (4/tg/ml) and 16-7 mM citric acid to preincubated cultures of strain CAFl led to an increased rate of citrate utilization and rapid destruction of acetoin (Fig. 3). Maximum production occurred earlier in the culture receiving Mn2+. Destruction occurred in both cases when the citrate level had fallen to about 500 jug/g. Similar results were obtained for strain FR8-1.
142
T. M. COGAN Diacetyl
Acetoin
10000 5000
1000
500
100
10
50
5
10
10 S
U
0-5
5
I
10
15
10
0 Time, h
15
Fig. 2. Effect of citric acid (•, A, • ) on acetoin and diacetyl production (O, A, • ) in 10% skim-milk containing 0-3% yeast extract by strain CAFl. The culture was preincubated for 65 h at 21 °C before addition of citric acid. No diacetyl or acetoin production occurred in the absence of added citric acid. t , 7 0 mM; • , 14-9 mM; A, 31-8 mM citric acid.
Table 1. Maximum concentrations (HIM) of diacetyl and acetoin produced and citric acid utilized by Leuconostoc cremoris CAFl and FR8-1 Initial level* of citric acid added
Citric acid utilized
Diacetyl produced
Acetoin produced
Total diacetyl + acetoin
Expected yieldt
CAFl
70 14-9
3-3 9-6
002 006
0-57 2-62
0-59 2-68
1-7 4-8
FR8-1
6-9 15-2
2-2 7-7
001 006
013 105
014 Ml
11 3-9
Strain
* Cultures were preincubated for 65 h before addition of citric acid. f Total of diacetyl + acetoin + 2,3-butylene glycol.
Citrate utilization by starters
143
Time, h Fig. 3. Effect of 4 fig Mna+/ml ( • • ) on citric acid utilization (O. • ) and acetoin production (O, • ) in 10% skim-milk containing 0-3% yeast extract by strain CAF1. The culture was preincubated for 65 h at 21 °C before additions of citric acid and Mn2+.
The addition of 15-7 mM trisodium citrate to the skim-milk prior to inoculation raised the initial pH value from 6-5 to 6-9 and resulted in traces of acetoin (maximum 7-0 /ig/g), but no diacetyl production by strain CAFl. Little citrate had been utilized when the maximum amount of acetoin was produced. Under similar conditions, strain FR8-1 produced no acetoin or diacetyl. Strain CAFl produced no detectable amounts of acetaldehyde under any experimental condition whereas strain FR8-1 did. The amounts produced varied and did not parallel diacetyl or acetoin production. Acetaldehyde production by strain FR8-1 gradually increased during growth and generally reached a maximum of 65 /tg/g after 60 h incubation. The addition of citric acid after growth for 65 h had no effect on acetaldehyde production by FR8-1 which remained constant at about 65 fig/g throughout the experiment.
DAR 42
144
T. M. COGAN
1000 500
100
10
50
0-5
100
0-1
50
005
10
001
0-5
0-1
I
8
12 Time, h
16
20
Fig. 4. Diaoetyl (•), acetoin (D), acetaldehyde (•), and lactic acid (A) production and citric acid (O) utilization by Streptococcus diacetilactis DRC3 in 10% skim-milk.
Streptoccus diacetilactis ' Production of diacetyl and acetoin and utilization of citrate by Str. diacetilactis DRC3 in skim-milk is shown in Fig. 4. Little diacetyl (c. 1 /tg/ml), but large amounts of acetoin (c. 250/jg/ml) were produced. Maximum amounts of the latter were produced only when citrate utilization was complete after 9 h incubation. Little if any destruction of acetoin occurred. The addition of citrate resulted in higher amounts of both diacetyl and acetoin and slightly greater amounts of lactic acid being produced. Similar results were obtained with strain DRCl except for slight destruction of acetoin, which coincided with the disappearance of citrate. Mn 2+ (4 /ig/ml) had no effect on citrate utilization or diacetyl and acetoin production by strains DRCl and DRC3.
Citrate utilization by starters
145
DISCUSSION
It is well documented that mixed-strain starter cultures containing Leuc. cremoris and Str. diacetilactis produce diacetyl and acetoin from citrate when grown in milk. However, in pure culture these bacteria behaved entirely differently - in the case of Leuc. cremoris, yeast extract was necessary for growth and citrate was utilized without concomitant production of diacetyl and acetoin, while the opposite was true of Str. diacetilactis. The reason for the unexpected results with the leuconostoc bacteria remains obscure, but we are at present endeavouring to elucidate it. The phenomenon is probably a general one since other leuconostoc strains tested behaved in a similar manner (unpublished results). However, not all leuconostoc species will utilize citrate (Dr E. I. Garvie, personal communication). These results are in agreement with those of van Beynum & Pette (1939), who showed that leuconostoc bacteria growing in neutral media containing citrate produced no aroma. Michaelian, Farmer & Hammer (1933) found that 29 out of 35 citric-acid-fermenting streptococci (leuconostocs) produced no aroma in milk after 7 d at 21 °C. These workers also found that cultures preincubated for 24 h in milk before acidification with one of several acids produced diacetyl and acetoin. Further studies (Michaelian, Hoecker & Hammer, 1938) showed that citric acid was a much better acidulant than either lactic or sulphuric acid for the production of diacetyl+ acetoin. However, their cultures were not examined for 48 h after acidifying, while the present data suggest that unless a large amount of citric acid is added, maximum acetoin production occurs within 2-5 h. In acidified cultures of leuconostoc much lower amounts of diacetyl and acetoin were produced than citrate utilized suggesting that, unless 2,3-butylene glycol is also produced, citrate is being utilized in the formation of other compounds in the cell. During this time it is unlikely that the cultures were actively growing because of the low pH. In the control flask where HC1 was the acidifying agent the pH did not change during the 14 h of the experiment. The pH in the cultures acidified with citrate increased as utilization proceeded. Thus, it is possible that diacetyl and acetoin are produced only when active growth is unfavourable. Acetoin is quantitatively a much more important compound - in fact recovery of diacetyl could conveniently be ignored in metabolic studies. Mn2+ stimulated growth and citrate utilization in milk by the leuconostoc cultures, as shown by de Man & Galesloot (1962). Mn2+ also stimulated citrate uptake and reduction of acetoin in acidified cultures under conditions where growth was unlikely to occur, suggesting that both the enzymes necessary for uptake and breakdown have a Mn2+ requirement. pH may also be important, since Harvey & Collins (1962) have shown that strain CAFl has a pH-dependent citrate permease. Production and destruction of both diacetyl and acetoin paralleled each other, which is surprising since Speckman & Collins (1968) have shown that 2 distinct biosynthetic pathways are involved. The reduced compound must be presumably 2,3-butylene glycol. The production of acetaldehyde by strain FR8-1 is surprising since Walsh & Cogan (1973) showed that the parent mixed culture produced little if any acetaldehyde. A possible explanation may be that the lactic streptococci present in the
146
T. M. COGAN
culture reduced the acetaldehyde as it was formed. In this connexion Bills & Day (1966) have noted that at least one strain each of Str. lactis and Str. cremoris can reduce added acetaldehyde in milk. The 2 strains of Str. diacetilactis showed similar production patterns for diacetyl, acetoin and acetaldehyde as D and BD cultures (Walsh & Cogan, 1973), suggesting that in a BD culture it is the Str. diacetilactis component and not Leuc. cremoris which is mainly responsible for flavour production. Presumably, in B type cultures, the Leuc. cremoris component, although stimulated by the lactic streptococci, still grows slowly. In these conditions sufficient citrate is present at the end of the customary growth period of 12-16 h to act as a precursor of diacetyl and acetoin production in the acid conditions present in the culture. This is borne out by the results of Walsh & Cogan (1973), who showed that in B type cultures citrate utilization does not start until after about 10 h of incubation at 21 °C. The author is indebted to Mr P. Thornbill and Mr F. Drinan for excellent technical assistance, to Dr S. Condon and Mr F. O'Connor for their help in preparation of the manuscript and to Dr E. I. Garvie for confirming that strain CAFl is Leuc. cremoris.
REFERENCES BILLS, D. D. & DAY, E. A. (1966). Journal of Dairy Science 49, 1473. CHUANG, L. F. & COLLINS, E. B. (1968). Journal of Bacteriology 95, 2083. DE MAN, J. C. & GALESLOOT, TH. E. (1962). Netherlands Milk and Dairy Journal 16, 1. ESCHENBRUCH, R. (1970). Vitis 9, 218. HARVEY, B. J. & COLLINS, E. B. (1962). Journal of Bacteriology 83, 1005. KEENAN, T. W. & LINDSAY, K. C. (1968). Journal of Dairy Science 51, 188. LIOHTBODY, L. G. (1962). Australian Journal of Dairy Technology 17, 36. LINDSAY, R. C. & DAY, E. A. (1965). Journal of Dairy Science 48, 665. MARIEB, J. R. & BOULET, M. (1958). Journal of Dairy Science 41, 1683. MICHAELIAN, M. B., FARMER, R. S. & HAMMER, B. W. (1933). Research Bulletin No. 155, Iowa State College of Agriculture and Mechanic Arts. MICHAELIAN, M. B., HOECKER, W. H. & HAMMER, B. W. (1938). Journal of Dairy Science 21, 213.
SHARPE, M. E., FRYER, T. F. & SMITH, D. G. (1966). Technical Series, Society for Applied Bacteriology 1,69. SPECKMAN, R. A. & COLLINS, E. B. (1968). Journal of Bacteriology 95, 174. VAN BEYNUM, J. & PETTE, J. W. (1939). Journal of Dairy Research 10, 250. WALSH, B. & COGAN, T. M. (1973). Applied Microbiology 26, 820. WALSH, B. & COGAN, T. M. (1974a). Journal of Dairy Research 41, 25. WALSH, B. & COGAN, T. M. (19746). Journal of Dairy Research 41, 31.
Printed in Great Britain
