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Amino acid limited growth of starter cultures in milk
FEMS Microbiology Ecology 45 (1987) 191-198 Published by Elsevier
191
FEC 00120
Amino acid limited growth of starter cultures in milk J. H u g e n h o l t z , M. D i j k s t r a a n d H. V e l d k a m p Department of Microbiolog)" Unit~ersiO' of Groningen, 9751 NN Haren (The Netherlands)
Received 22 September 1986 Revision received 20 February 1987 Accepted 2 April 1987 Key words: Amino acids; Limited growth; Starter cultures; Milk
1. S U M M A R Y The specific growth rates of several Streptococcus cremoris strains were 10-40% lower in milk than in other growth media. The growth rates in milk increased when an amino acid mixture or casein was added, whereas, when milk was diluted, the specific growth rate of the streptococci decreased. This decrease could be overcome by bringing the casein concentration in the diluted milk back to the normal value (3%). This indicates that casein-hydrolysis proceeded at a rate too low for the streptococci to reach their potential maxim u m specific growth rates in milk so that growth in milk is essentially amino acid-limited. This was subsequently demonstrated for S. cremoris by continuous cultivation in media with low casein concentrations. At a low dilution rate casein hydrolysis was fast enough to supply the cells with enough amino acids and lactose was growth-limiting, whereas at higher dilution rates amino acids became growth-limiting. In cultures exponentially growing in milk the concentration of free amino acids was measured to determine which amino
Correspondence to: Dr. H. Veldkamp, Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren (The Netherlands).
acid(s) was(were) absent and could possibly limit growth. A number of essential amino acids (leucine, methionine, glutamate and in some cases phenylalanine) were not detected and addition of these, together, stimulated the growth of S. cremoris in milk. The amino acids leucine and phenylalanine appeared to play a particularly important role in this stimulation. These two are, supposedly, the first amino acids that become limiting during growth in milk. The effect of competition for casein and amino acids by different organisms was studied in continuous cultures. At different dilution rates different strains became dominant in these mixed cultures, suggesting that differences in apparent affinity constants (Ks) for casein, leucine and glutamate existed between the strains.
2. I N T R O D U C T I O N Starter cultures currently in use in the Dutch cheese industry consist of many different strains of, mainly, S. cremoris [1]. These strains can vary strongly in the properties important during cheese manufacture such as proteolytic activity [2,3], acidification rate [4] and bacteriophage resistance [5]. All strains contribute to the production of a cheese with unique qualities in texture and flavour.
0168-6496/87/$03.50 © 1987 Federation of European Microbiological Societies
192 The use of mixed starters makes quality control of the cheese difficult since small shifts in the composition of the cultures can lead to a cheese with changed properties. These changes in the population have been reported regularly, They can be the result of bacteriocin-production by some strains [6], a difference in m a x i m u m specific growth rates [4,7], bacteriophage infections [8] or substrate or mineral deficiencies (e.g., Mn2+), as was found in Leuconostoc strains [9]. Milk contains all necessary growth factors for streptococci [10] and the energy source (lactose) and main carbon source (casein) are present at very high concentrations (_+4.5% and 2.5%, respectively). Yet previous reports have shown that maximum specific growth rates of S. cremoris measured in milk are lower than in other media [4] and growth stimulation has often been reported upon the addition of amino acids to milk [11-13]. It thus seems that growth of starter bacteria in milk does not proceed at potential m a x i m u m rates. The possibility that this might be due to an amino acid limitation was investigated by changing the composition of milk and comparing the growth rates of several S. cremoris strains in these media. It was found that the casein concentration did indeed determine the specific growth rate in milk and that a limited rate of casein hydrolysis led to limitation of growth by some amino acids, including leucine and phenylalanine. The significance of this limitation for the composition of mixed cultures has been studied in continuous cultures.
3. M A T E R I A L S A N D M E T H O D S 3.1. Bacterial strains S. cremoris strains HP, Es, ME1 and AM 1 were obtained from the Netherlands Institute for Dairy Research (NIZO, Ede, The Netherlands). The strains were routinely stored at - 2 0 ° C in 10% ( w / v ) reconstituted skimmed milk. 3.2. Milk-based media All media were prepared from skimmed milk powder. The casein concentration in milk was increased to 4.0% by the addition of extra casein and decreased to about 1.7% by centrifuging the
skimmed milk for 30 min at 12000 x g and discarding the pellet which consisted mainly of casein micelles. Concentrated milk (2 x and 1.5 × ) and diluted milk ( 1 . 3 3 X , 2 × , 4 x , 8 x and 1 0 x ) were prepared by dissolving the skimmed milk powder in appropriate amounts of demineralized water. To bring the casein concentration back to the normal value in the 10 x diluted milk, _+2.5% casein was added. All media were heat-sterilized for 5 min at 120 ° C. 3. 3. Synthetic casein medium This was based on the synthetic (RFP) medium described by Rogosa et al. [14] in which amino acids were replaced by casein in the required concentration. 3. 4. Medium for amino acid-limited growth A leucine- or glutamate-limited growth medium was obtained by reducing the amount of either leucine or glutamate in the R F P medium to 12.5 rag/1 [15] and 40.0 mg/1 [16], respectively. 3.5. Determination of tXm,x values Maximum specific growth rate (ff max ) values in milk were determined as described previously [4,12] by diluting samples tenfold in an EDTAborate buffer and measuring the absorbance of the clarified milk cultures at intervals of 30 min. ~max values in RFP-casein medium were determined in screw-capped tubes which fit in a Vitatron UC 200 Spectrophotometer (Vitatron Scientific Instruments, Dieren, The Netherlands). In all cases growth rates were determined at 30 ° C and absorbance was measured at 660 nm. 3.6. Continuous cultivation A chemostat was used as described previously [4,15-17]. RFP-casein (0.4% casein) medium and leucine- or glutamate-limited R F P - m e d i u m was used with 1% lactose in all experiments. 3. 7. Amino acid determination Milk cultures were centrifuged for 15 min at 6000 x g. An equal volume of 28% perchloric acid solution was added to the supernatant. This mixture was incubated on ice for 30 min followed by centrifugation at 6000 x g for 10 rain. An equal
193
volume of a solution of 2 N K O H and 2 N K H C O 3 was added to the supernatant cautiously. The precipitate formed was removed by centrifugation at 5000 × g for 5 min. The supernatant was diluted 5-fold in 40 m M NazCO3, p H 9.5. The amino acids in the resulting samples were dansylated by mixing the samples 2 : 1 with a 1.5 m g / m l dansylchloride solution in acetonitrile. The samples from the continuous cultures growing in RFP-casein medium were diluted 20fold in 40 m M N a 2 C O 3, p H 9.5 and mixed with the same dansylchloride solution. The dansylation reaction proceeded at 3 7 ° C for 2 h and was stopped by the addition of 4.0 m M methylamine. The dansylated amino acids were separated by reversed-phase high-performance liquid chrom a t o g r a p h y ( R F - H P L C ) on a C18 column (Waters, ~Bondapak C18; 3.9 m m x 30 cm) as described previously [17], using for elution a 0 to 55% ( v / v ) acetonitrile gradient in 35 rain. Amino acid concentrations of several /~M could be measured accurately by this method. Only the acidic amino acids glutamate and aspartate did not give very clear responses. This was probably due to poor dansylation of these amino acids. 3.8. Determination o f lactose
The concentration of lactose in the continuous cultures was measured as described previously, using an anthron reagent [18]. 3.9. Detection o f S. cremoris strains in m i x e d cultures
The composition of mixed cultures of different cremoris strains was analysed by the immunofluorescence method as described recently [19]. In case strain HP was used in the mixtures additional plating on milk agar was performed as described previously [4]. S.
4. R E S U L T S 4.1. Casein limitation in m i l k cultures
The specific growth rates in milk can be increased by the addition of mixtures of amino acids [11-13]. These data suggest a limitation of amino acids for streptococci during growth in milk. We
have measured the specific growth rates of several strains of S. cremoris in milk. For the strains E 8 (~ = 0.65-0.70), ML 1 (/x = 0.59-0.64), HP (~t = 0.55-0.60) and AM 1 (bt = 0.45-0.50) the specific growth rates in milk were 10% (Es) to 40% (AM1) lower than in amino acid-containing media such as MRS medium or R F P medium ([4]; see also Table 2). The main amino acid source in milk is casein, which is present in a concentration of approx. 2.5%. We have investigated whether this concentration is high enough for S. cremoris to reach its potential m a x i m u m specific growth rate in milk. Casein was added as a concentrated solution to milk to increase the concentration to 4.0%. This led to a 10-20% increase in the specific growth rates of several S. cremoris strains (Table 1, 'extra casein milk'). These /~m~× values in modified milk were similar to those measured in RFPmedium, especially for the strains E8 and HP. If the casein concentration was selectively decreased by centrifugation (Table 1, 'low casein milk'), the specific growth rates of the strains decreased concomitantly. It appeared from these data that the casein concentration determined the specific growth rate of S. cremoris in milk. This was confirmed by growth experiments in diluted milk. The growth rates of the strains decreased as the milk was diluted further, but growth of the
Table 1 Relative m a x i m u m specific g r o w t h rates (in %) of S. cremoris strains HP, E 8, M L 1 a n d A M 1 in milk-based media G r o w t h rates in ' n o r m a l ' milk are presented as 100%. The absolute values are presented in the text. S. cremoris strain
milk milk+casein milk - casein 2 × c o n c e n t r a t e d milk 1.5 x c o n c e n t r a t e d milk 3 / 4 x c o n c e n t r a t e d milk 1 / 2 x c o n c e n t r a t e d milk 1 / 4 x c o n c e n t r a t e d milk 1 / 8 x c o n c e n t r a t e d milk 1 / 1 0 x c o n c e n t r a t e d milk + casein n.d. = not d e t e r m i n e d
HP
E8
ML 1 AM 1
100 118 90 91 116 92 84 70 60 92
100 111 92 99 111 94 88 76 63 95
100 108 95 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
100 112 93 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
194
streptococci proceeded exponentially so that specific growth rates could still be calculated under these conditions (Table 1). By supplementing the diluted milk with pure casein the specific growth rate increased accordingly, even when ten fold diluted milk was used. The other essential nutrients apparently were present in large excess,
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4.2. Casein-limitation in R F P casein medium
In this medium the casein concentration determined the specific growth rates of the streptococci up to a concentration of 1.6% (Table 2). At this concentration the highest m a x i m u m specific growth rates of the S. cremoris strains were reached. It should be noticed that the four strains reacted differently to the variation in casein concentration. The data in Table 2 suggest that strain H P has the lowest apparent affinity-constant (K~) for casein since its specific growth rate at 0.2% casein was 46% of ~m~× and strain ML~ has the highest K~ with its ~ at 0.2% casein of only 30%
z e~ t
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~
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A limited rate of casein hydrolysis will lead to an amino acid limitation for the streptococci during growth. This could be demonstrated by cultivating S. cremoris in continuous culture on R F P casein with 0.4% casein. At low dilution rates the hydrolysis rate of casein was sufficiently high and all essential amino acids were detected in the culture fluid, indicating that there is no amino acid limitation under these conditions (Fig. 1A). At higher dilution rates the casein hydrolysis by the streptococci is not rapid enough, resulting in an amino acid limitation. This could be shown by
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o
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Table 2
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Specific growth rates (in h -1) in batch culture of S. cremoris strains HP, Es, ML~ and AM 1 in RFP-caseha medium with varying casein concentrations Casein concentration
S. cremoris strain
HP
E8
ML 1
AM~
0.2% 0.4% 0.8% t.2% 1.6%
0.32 0.43 0.52 0.65 0.69
0.32 0.44 0.59 0.71 0.78
0.22 0.35 0.66 0.73 0.73
0.30 0.45 0.69 0.74 0.74
ML Fig. 1. HPLC pattern of the free amino acids in steady-state culture of S. cremoris HP growing in a chemostat on RFPcasein medium (0.4% casein) at a dilution rate of 0.1 h ~ (A) and 0.4 h -1 (B). The numbers represent the elution time of the HPLC column in min. The same volume of culture fluid was used in A and B.
195
analysing the amino acids in the culture fluid (Fig. 1B). The number and amount of amino acids was lower than at low dilution rates and many of the essential amino acids (leucine, glutamate, lysine, histidine, alanine, methionine and arginine) were missing and could have become growth limiting. 4.3. Amino acid limitation in milk cultures
High specific growth rates are necessary for high acid production in milk, but casein concentrations are suboptimal, as was shown in Table 1. Under these conditions an amino acid limitation is to be expected. This was investigated by taking samples from growing cultures and determining the free amino acids in these samples (Fig. 2). Some of the essential amino acids were found, such as proline, lysine, histidine, tyrosine, arginine, valine and alanine, whereas others were conspicuously missing or present at very low concentrations (leucine, phenylalanine, methionine and glutamate). These amino acids are possibly growth-limiting in milk cultures. However, in the stationary phase of growth all amino acids present
,J
in casein were found in the medium (not shown). The streptococci had, apparently, continued to hydrolyse casein after their growth had stopped. The presence or absence of the acidic amino acids glutamate and aspartate could not be established since these amino acids were poorly dansylated using the described method, and furthermore they both eluted very close to the (large) Dns-OH peak. It was assumed that glutamate, which is essential for growth of streptococci, could become growthlimiting since it is accumulated in large amounts by these organisms [17]. When adding to milk a mixture of amino acids which most likely become growth-limiting, the specific growth rate increased by approx. 10%, whereas no stimulation was found when all other amino acids were added (Table 3). An attempt was made to establish which specific amino acids were growth-limiting in milk. Each of the stimulatory amino acids was added separately and some in different combinations. The effect of the additions on the growth rate of two strains of S. cremoris, HP and Es, was determined (not shown). These effects were occasionally different for the two strains which can be contributed to differences in proteolytic systems [3]. Only when leucine or phenylalanine were included, stimulation in specific growth rates was found (Table 3). 4.4. Competition in continuous cultures
When growing mixed starter cultures under
Table 3 Relative specific growth rates (in %) of S. cremoris strains HP and E s in milk enriched with different amino acid-mixtures T
The growtla rates in milk are presented as 100%. Amino acid mixture I: leu, ser, met, val, glu, phe, ile. Amino acid mixture II: arg, thr, gly, pro, tyr, asp, lys, trp, his, ala. All amino acids were added in the concentration present in RFP-medium [14]. S. cremoris strain
¢
'i
t'
r
i
Fig. 2. HPLC pattern of the amino acids produced during exponential growth of S. cremoris HP in milk. The detected amino acids are named in the Figure.
milk milk + milk + milk + milk + milk + milk +
0.2% bactotrypton amino acid mixture I amino acid mixture II leucine phenylalanine leu/phe
HP
Es
100 105 111 97 104 100 105
100 110 108 97 105 107 105
196
amino acid limitation,competition for these substrates will occur. Population changes resulting from competition were studied with a chemostat [20]. Since milk is not a suitable medium for continuous cultivation we have used the RFPcasein medium as described above, with 0.4% casein. S. cremoris strains HP and E 8 were grown in separate continuous cultures and when steady states were reached in both cultures they were mixed in a 1 : 1 ratio. In one of these cultivation was continued at a dilution rate of 0.1 and in the other the dilution rate was slowly increased to 0.3 h-1. In both cases S. cremoris H P outcompeted S. cremoris E s (Table 4). However, there was a big difference between the two experiments. At the low dilution rate a high cell density was reached (A660n m = about 2.0) and the residual lactose concentration was low (83 #M). Under these conditions enough casein could be hydrolysed by the streptococci for their supply of essential amino acids (Fig. 1A) resulting in a lactose-limited culture. Addition of extra lactose to these cultures led to an increase of the cell density (not shown). As was already shown in a previous publication [4] S. cremoris H P has a lower apparent affinity constant for lactose than S. cremoris E 8 and gradually became dominant in the culture. At the higher dilution rate the cell density in the culture was significantly lower ( A 6 6 0 n m = about 1.5) and the residual lactose concentration was 1290 ~tM. In this case lactose was definitely present in excess and the rate of casein hydrolysis had now become growth-limiting. The data presented in Table 2 already suggested that S. cremoris strain HP had a lower apparent affinity constant for casein than strain E s and it was indeed found that strain HP became dominant at the higher dilution rate dur-
Table 4 Competition in continuous culture between S. cremoris strains E 8 and HP in RFP-medium with 0.4% casein Dilution rate (h -1 )
Strain becoming dominant
Bacterial cell density (A 660nm)
Concentration of residual lactose (/IM)
0.1 0.3
HP HP
2.0 1.5
83 1290
ing the competition for casein and its hydrolysis products. Strain E 8 could not obtain a selective advantage in this medium since the specific growth rates of both strains on 0.4% casein were almost identical (Table 2). Competition between different strains of S. cremoris were also studied in leucine- and glutamate-limited chemostats. Under leucine limitation crossing /~-S curves were reported for the strains E s and ML 1 with E 8 having the highest /~max and ML1 the lowest K~ for. leucine [19]. When strain HP was used instead of strain ML 1 in the mixtures similar results were obtained (not shown). If glutamate was made growth-limiting in R F P medium, again S. cremoris HP outcompeted S. cremoris E 8 at a low dilution rate of 0.05 h -~. At a higher dilution rate ( D = 0.2 h 1) strain E s became dominant (Fig. 3). Thus, in all experiments competition for growth-limiting substrates gave rise to population changes due to differences in growth parameters of S. cremoris strains.
5. D I S C U S S I O N The
maximum
specific growth
rate
of
S.
cremoris in milk has been reported to be signifi-
cantly lower than that in other growth media [4,12]. This has been confirmed in our experiments. This relatively low specific growth rate could be increased by the addition of amino acids to milk [11-13]. This suggests an amino acid limitation in milk, despite the high concentration (3%) of casein present. We have tested this possibility by performing different growth experiments in milk with and without various additions. If amino acids would be growth-limiting during cultivation of streptococci in milk, this would be a consequence of casein hydrolysis by the bacteria proceeding at a rate too low to sustain m a x i m u m specific growth rates. This was indeed observed in the growth experiments in milk and RFP-casein medium where the casein concentration was varied. In milk 3% casein did not allow potential maxim u m specific growth rates (Table 1), whereas growth rates could be increased by 10 to 20% by the addition of extra casein. The experiments in which diluted milk was used clearly demonstrated
197
that casein is the growth rate-determining substrate in milk. Even when milk was diluted 10-fold and extra casein was added normal growth rates were observed (Table 1). In RFP-casein medium the casein concentration necessary for m a x i m u m specific growth rates (1.2-1.6%) was lower than in milk (Table 2). Milk contains high concentrations of Ca 2+ and most casein is present in the form of CaZ+-casein micelles. In RFP-casein, on the other hand, the Ca 2 +-concentration is much lower and it can be assumed that more soluble casein is present. Possibly this free casein is the actual substrate for the proteolytic system of the streptococci. A limited rate of hydrolysis of casein will lead to an amino acid-limitation for the streptococci. This was demonstrated with S. cremoris strain H P in continuous cultures with RFP-casein medium containing a low casein concentration. At a low dilution rate the casein-hydrolysis was sufficiently fast to supply all the essential amino acids for growth and lactose became the growth-limiting substrate. However, at a higher dilution rate the amino acids were not produced fast enough and became growth-limiting (Fig. 1). In an attempt to identify these amino acids, leucine and phenylalanine appeared to be of particular importance. The practical implications of these results are obvious. The growth rate and, concomitantly, the acidification rate of S. cremoris can be increased by additions to milk of: (1) an amino acid mixture, provided that leucine and phenylalanine are included and that glutamate is not added in inhibitory concentrations [12]; (2) extra casein, and (3) extra milk powder. All these additions result in an increase of 10 to 20% of the specific growth rate of S. cremoris. An alternative way of increasing the specific growth rates of the streptococci in milk is to increase the rate of proteolysis. This could be achieved by genetically manipulating the bacteria leading to overproduction of the proteases or by the addition of purified streptococcal proteases to the milk cultures. Both approaches will soon be possible since the genes coding for the proteases are currently being cloned in streptococci [21] and large amounts of proteases can now be purified (E.J. Smid, Dept. of Microbiology, University of Groningen, pers. commun.)
The observations on the amino acid/caseinlimitation in milk also have important implications for the population dynamics of mixed starter cultures. During propagation of these starters the composition of the mixtures will change due to differences in maximum specific growth rates [4,7]. These population changes were also observed in continuous cultures and besides differences in m a x i m u m specific growth rates, different apparent affinity constants for substrates have been found. Crossing ~-S curves under lactose limitation were reported for S. cremoris strains HP and Es, showing dominancy of strain HP at low dilution rates, whereas at high dilution rates strain E 8 came to the fore [4]. Lactose limitation, however, will only occur in the initial stages of the ripening process and definitely not during propagation of the starters in milk. As was described here, other substrates ( c a s e i n / a m i n o acids) are growth limiting in milk cultures. It was therefore relevant to study the role of these growth-limiting substrates in the population dynamics of mixed starter cultures. Differences in K S for the amino acid leucine in S. cremoris strains were described recently [18]. The crossing ~-S curves observed for leucine have also been found here for glutamate (Fig. 3).
100-
50-
[]
I
5
!
10
vol. changes(Dxt) Fig. 3. Competition of S. cremoris E 8 and S. cremoris H P in continuous culture under glutamate limitation at a dilution rate of 0.05 h - 1 (D) and at a dilution rate of 0.20 h 1 (m).
198 W e also r e p o r t h e r e d i f f e r e n c e s in K s for casein. S. c r e m o r i s H P o u t c o m p e t e d S. c r e m o r i s E 8 in continuous culture on RFP-casein medium cont a i n i n g 0.4% casein. T h e o u t c o m e of the e x p e r i m e n t was n o t d e p e n d e n t o n the d i l u t i o n rate. A t l o w d i l u t i o n rates t h e strains c o m p e t e d for l a c t o s e a n d at h i g h e r d i l u t i o n rates for casein. In b o t h c a s e s s t r a i n H P w a s m o r e c o m p e t i t i v e . If h i g h e r c o n c e n t r a t i o n s of c a s e i n w o u l d h a v e b e e n u s e d in the m e d i u m S. c r e m o r i s Es, w i t h its higher/x,,,~ x in R F P - m e d i u m t h a n s t r a i n H P ( T a b l e 2), w o u l d be e x p e c t e d to b e c o m e d o m i n a n t at d i l u t i o n rates a p p r o a c h i n g ~m,x" It c a n b e c o n c l u d e d t h a t (1) the r a t e o f c a s e i n h y d r o l y s i s d e t e r m i n e s the g r o w t h r a t e of S. c r e m o r i s in m i l k ; (2) the c a s e i n - l i m i t a t i o n l e a d s to a l i m i t a t i o n of a m i n o acids, e s p e c i a l l y l e u c i n e a n d phenylalanine; (3) t h e c o m p e t i t i o n for t h e g r o w t h - l i m i t i n g c a s e i n a n d a m i n o a c i d s has a p r o n o u n c e d e f f e c t o n the c o m p o s i t i o n o f m i x e d s t a r t e r cultures.
REFERENCES [1} Limsowtin, G.K.Y., Heap, H.A. and Lawrence, R.C. (1977) N.Z.J. Dairy Sci, Technol. 12, 101-106. [2] Law, B.A. and Kolstad, J. (1983) Antonie van Leeuwenhoek 49, 225-245. [3] Hugenholtz, J., Exterkate, F.A. and Konings, W.N. (1984) Appl. Environ. Microbiol. 48, 1105-1110. [4] Hugenholtz, J. and Veldkamp, H. (1985) FEMS Microbiol. Ecol. 31, 57-62. [5] Teuber, M. and Lembke, J. (1983) Antonie van Leeuwenhoek 49, 283-295. [6] Collins, E.B. (1961) Appl. Microbiol. 9, 200-209. [7] Lee, D.A. and Collins, E.B. (1976) J. Dairy Sci. 59, 405 409.
[8] Lawrence, R.C., Heap, H.A., Limsowtin, G.K.Y. and Jarvis, A.W. (1978) Cheddar cheese starters: current knowledge and practices of phage characteristics and strain selection. J. Dairy Sci. 61, 1181-1191. [9] De Man, J.C. and Galesloot, T.E. (1962) Neth. Milk Dairy J. 16, 1-10. [101 Banks, W., Dalgleish, D.G. and Rook, J.A.F. (1985) Milk and milk processing. In Robinson, R.K. (Ed.), Dairy Microbiology, I, Elsevier, Amsterdam, pp. 1-34. [11] Selby Smith, J., Hillier, A.J., Lees, G.J. and Jago, G.R. (1975) J. Dairy Res. 42, 123-138. [12] Otto, R. (1981) An Ecophysiological Study of Starter Streptococci. Ph.D. Thesis, University of Groningen. [13] Reiter, B. (1973) Some thoughts on cheese starters. J. Soc. Dairy Techn. 26, 3-15. [14] Rogosa, M., Franklin, and Perry, (1961) Correlation of the vitamin requirements with cultural and biochemical characters of Lactobacillus spp. J. Gen. Microbiol. 25, 473-482. [15] Otto, R. (1984) Uncoupling of growth and acid production in Streptococcus cremoris. Arch. Microbiol. 140, 225 -230. [16] Otto, R. (1985) The relation between phosphate potential and growth rate of Streptococcus cremoris. Arch. Microbiol. 142, 97-100. [17] Poolman, B., Smid, E.J., Veldkamp, H. and Konings, W.N. (1987) Bioenergetic consequences of lactose starvation for continuously cultured Streptococcus cremoris. J. Bacteriol., 160, 1460-1468. [18] Fairbairn, N.J. (1953) A modified anthrone reagent. Chem. Ind. (London) 86. [19] Hugenholtz, J., Veldkamp, H. and Konings, W.N. (1986) Detection of specific strains and variants of Streptococcus cremoris in mixed culture by immunofluorescence. Appl. Env. Microbiol. 53. 149-155. [201 Veldkamp, H. (1977) Ecological studies with the chemostat. Adv. Microbiol. Ecol. 1, 59-94. [21] Kok, J., Van Dijl, J.M., Van der Vossen, J.M.B.M. and G. Venema (1985) Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis. Appl. Env. Microbiol. 50, 94-101.
191
FEC 00120
Amino acid limited growth of starter cultures in milk J. H u g e n h o l t z , M. D i j k s t r a a n d H. V e l d k a m p Department of Microbiolog)" Unit~ersiO' of Groningen, 9751 NN Haren (The Netherlands)
Received 22 September 1986 Revision received 20 February 1987 Accepted 2 April 1987 Key words: Amino acids; Limited growth; Starter cultures; Milk
1. S U M M A R Y The specific growth rates of several Streptococcus cremoris strains were 10-40% lower in milk than in other growth media. The growth rates in milk increased when an amino acid mixture or casein was added, whereas, when milk was diluted, the specific growth rate of the streptococci decreased. This decrease could be overcome by bringing the casein concentration in the diluted milk back to the normal value (3%). This indicates that casein-hydrolysis proceeded at a rate too low for the streptococci to reach their potential maxim u m specific growth rates in milk so that growth in milk is essentially amino acid-limited. This was subsequently demonstrated for S. cremoris by continuous cultivation in media with low casein concentrations. At a low dilution rate casein hydrolysis was fast enough to supply the cells with enough amino acids and lactose was growth-limiting, whereas at higher dilution rates amino acids became growth-limiting. In cultures exponentially growing in milk the concentration of free amino acids was measured to determine which amino
Correspondence to: Dr. H. Veldkamp, Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren (The Netherlands).
acid(s) was(were) absent and could possibly limit growth. A number of essential amino acids (leucine, methionine, glutamate and in some cases phenylalanine) were not detected and addition of these, together, stimulated the growth of S. cremoris in milk. The amino acids leucine and phenylalanine appeared to play a particularly important role in this stimulation. These two are, supposedly, the first amino acids that become limiting during growth in milk. The effect of competition for casein and amino acids by different organisms was studied in continuous cultures. At different dilution rates different strains became dominant in these mixed cultures, suggesting that differences in apparent affinity constants (Ks) for casein, leucine and glutamate existed between the strains.
2. I N T R O D U C T I O N Starter cultures currently in use in the Dutch cheese industry consist of many different strains of, mainly, S. cremoris [1]. These strains can vary strongly in the properties important during cheese manufacture such as proteolytic activity [2,3], acidification rate [4] and bacteriophage resistance [5]. All strains contribute to the production of a cheese with unique qualities in texture and flavour.
0168-6496/87/$03.50 © 1987 Federation of European Microbiological Societies
192 The use of mixed starters makes quality control of the cheese difficult since small shifts in the composition of the cultures can lead to a cheese with changed properties. These changes in the population have been reported regularly, They can be the result of bacteriocin-production by some strains [6], a difference in m a x i m u m specific growth rates [4,7], bacteriophage infections [8] or substrate or mineral deficiencies (e.g., Mn2+), as was found in Leuconostoc strains [9]. Milk contains all necessary growth factors for streptococci [10] and the energy source (lactose) and main carbon source (casein) are present at very high concentrations (_+4.5% and 2.5%, respectively). Yet previous reports have shown that maximum specific growth rates of S. cremoris measured in milk are lower than in other media [4] and growth stimulation has often been reported upon the addition of amino acids to milk [11-13]. It thus seems that growth of starter bacteria in milk does not proceed at potential m a x i m u m rates. The possibility that this might be due to an amino acid limitation was investigated by changing the composition of milk and comparing the growth rates of several S. cremoris strains in these media. It was found that the casein concentration did indeed determine the specific growth rate in milk and that a limited rate of casein hydrolysis led to limitation of growth by some amino acids, including leucine and phenylalanine. The significance of this limitation for the composition of mixed cultures has been studied in continuous cultures.
3. M A T E R I A L S A N D M E T H O D S 3.1. Bacterial strains S. cremoris strains HP, Es, ME1 and AM 1 were obtained from the Netherlands Institute for Dairy Research (NIZO, Ede, The Netherlands). The strains were routinely stored at - 2 0 ° C in 10% ( w / v ) reconstituted skimmed milk. 3.2. Milk-based media All media were prepared from skimmed milk powder. The casein concentration in milk was increased to 4.0% by the addition of extra casein and decreased to about 1.7% by centrifuging the
skimmed milk for 30 min at 12000 x g and discarding the pellet which consisted mainly of casein micelles. Concentrated milk (2 x and 1.5 × ) and diluted milk ( 1 . 3 3 X , 2 × , 4 x , 8 x and 1 0 x ) were prepared by dissolving the skimmed milk powder in appropriate amounts of demineralized water. To bring the casein concentration back to the normal value in the 10 x diluted milk, _+2.5% casein was added. All media were heat-sterilized for 5 min at 120 ° C. 3. 3. Synthetic casein medium This was based on the synthetic (RFP) medium described by Rogosa et al. [14] in which amino acids were replaced by casein in the required concentration. 3. 4. Medium for amino acid-limited growth A leucine- or glutamate-limited growth medium was obtained by reducing the amount of either leucine or glutamate in the R F P medium to 12.5 rag/1 [15] and 40.0 mg/1 [16], respectively. 3.5. Determination of tXm,x values Maximum specific growth rate (ff max ) values in milk were determined as described previously [4,12] by diluting samples tenfold in an EDTAborate buffer and measuring the absorbance of the clarified milk cultures at intervals of 30 min. ~max values in RFP-casein medium were determined in screw-capped tubes which fit in a Vitatron UC 200 Spectrophotometer (Vitatron Scientific Instruments, Dieren, The Netherlands). In all cases growth rates were determined at 30 ° C and absorbance was measured at 660 nm. 3.6. Continuous cultivation A chemostat was used as described previously [4,15-17]. RFP-casein (0.4% casein) medium and leucine- or glutamate-limited R F P - m e d i u m was used with 1% lactose in all experiments. 3. 7. Amino acid determination Milk cultures were centrifuged for 15 min at 6000 x g. An equal volume of 28% perchloric acid solution was added to the supernatant. This mixture was incubated on ice for 30 min followed by centrifugation at 6000 x g for 10 rain. An equal
193
volume of a solution of 2 N K O H and 2 N K H C O 3 was added to the supernatant cautiously. The precipitate formed was removed by centrifugation at 5000 × g for 5 min. The supernatant was diluted 5-fold in 40 m M NazCO3, p H 9.5. The amino acids in the resulting samples were dansylated by mixing the samples 2 : 1 with a 1.5 m g / m l dansylchloride solution in acetonitrile. The samples from the continuous cultures growing in RFP-casein medium were diluted 20fold in 40 m M N a 2 C O 3, p H 9.5 and mixed with the same dansylchloride solution. The dansylation reaction proceeded at 3 7 ° C for 2 h and was stopped by the addition of 4.0 m M methylamine. The dansylated amino acids were separated by reversed-phase high-performance liquid chrom a t o g r a p h y ( R F - H P L C ) on a C18 column (Waters, ~Bondapak C18; 3.9 m m x 30 cm) as described previously [17], using for elution a 0 to 55% ( v / v ) acetonitrile gradient in 35 rain. Amino acid concentrations of several /~M could be measured accurately by this method. Only the acidic amino acids glutamate and aspartate did not give very clear responses. This was probably due to poor dansylation of these amino acids. 3.8. Determination o f lactose
The concentration of lactose in the continuous cultures was measured as described previously, using an anthron reagent [18]. 3.9. Detection o f S. cremoris strains in m i x e d cultures
The composition of mixed cultures of different cremoris strains was analysed by the immunofluorescence method as described recently [19]. In case strain HP was used in the mixtures additional plating on milk agar was performed as described previously [4]. S.
4. R E S U L T S 4.1. Casein limitation in m i l k cultures
The specific growth rates in milk can be increased by the addition of mixtures of amino acids [11-13]. These data suggest a limitation of amino acids for streptococci during growth in milk. We
have measured the specific growth rates of several strains of S. cremoris in milk. For the strains E 8 (~ = 0.65-0.70), ML 1 (/x = 0.59-0.64), HP (~t = 0.55-0.60) and AM 1 (bt = 0.45-0.50) the specific growth rates in milk were 10% (Es) to 40% (AM1) lower than in amino acid-containing media such as MRS medium or R F P medium ([4]; see also Table 2). The main amino acid source in milk is casein, which is present in a concentration of approx. 2.5%. We have investigated whether this concentration is high enough for S. cremoris to reach its potential m a x i m u m specific growth rate in milk. Casein was added as a concentrated solution to milk to increase the concentration to 4.0%. This led to a 10-20% increase in the specific growth rates of several S. cremoris strains (Table 1, 'extra casein milk'). These /~m~× values in modified milk were similar to those measured in RFPmedium, especially for the strains E8 and HP. If the casein concentration was selectively decreased by centrifugation (Table 1, 'low casein milk'), the specific growth rates of the strains decreased concomitantly. It appeared from these data that the casein concentration determined the specific growth rate of S. cremoris in milk. This was confirmed by growth experiments in diluted milk. The growth rates of the strains decreased as the milk was diluted further, but growth of the
Table 1 Relative m a x i m u m specific g r o w t h rates (in %) of S. cremoris strains HP, E 8, M L 1 a n d A M 1 in milk-based media G r o w t h rates in ' n o r m a l ' milk are presented as 100%. The absolute values are presented in the text. S. cremoris strain
milk milk+casein milk - casein 2 × c o n c e n t r a t e d milk 1.5 x c o n c e n t r a t e d milk 3 / 4 x c o n c e n t r a t e d milk 1 / 2 x c o n c e n t r a t e d milk 1 / 4 x c o n c e n t r a t e d milk 1 / 8 x c o n c e n t r a t e d milk 1 / 1 0 x c o n c e n t r a t e d milk + casein n.d. = not d e t e r m i n e d
HP
E8
ML 1 AM 1
100 118 90 91 116 92 84 70 60 92
100 111 92 99 111 94 88 76 63 95
100 108 95 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
100 112 93 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
194
streptococci proceeded exponentially so that specific growth rates could still be calculated under these conditions (Table 1). By supplementing the diluted milk with pure casein the specific growth rate increased accordingly, even when ten fold diluted milk was used. The other essential nutrients apparently were present in large excess,
@
,o,
~2
4.2. Casein-limitation in R F P casein medium
In this medium the casein concentration determined the specific growth rates of the streptococci up to a concentration of 1.6% (Table 2). At this concentration the highest m a x i m u m specific growth rates of the S. cremoris strains were reached. It should be noticed that the four strains reacted differently to the variation in casein concentration. The data in Table 2 suggest that strain H P has the lowest apparent affinity-constant (K~) for casein since its specific growth rate at 0.2% casein was 46% of ~m~× and strain ML~ has the highest K~ with its ~ at 0.2% casein of only 30%
z e~ t
a
÷ ~-
~
%
,,,,
L
:
~'£ m a x "
A limited rate of casein hydrolysis will lead to an amino acid limitation for the streptococci during growth. This could be demonstrated by cultivating S. cremoris in continuous culture on R F P casein with 0.4% casein. At low dilution rates the hydrolysis rate of casein was sufficiently high and all essential amino acids were detected in the culture fluid, indicating that there is no amino acid limitation under these conditions (Fig. 1A). At higher dilution rates the casein hydrolysis by the streptococci is not rapid enough, resulting in an amino acid limitation. This could be shown by
®
o
z~
z n
5~
f:
Table 2
¢~._.
Specific growth rates (in h -1) in batch culture of S. cremoris strains HP, Es, ML~ and AM 1 in RFP-caseha medium with varying casein concentrations Casein concentration
S. cremoris strain
HP
E8
ML 1
AM~
0.2% 0.4% 0.8% t.2% 1.6%
0.32 0.43 0.52 0.65 0.69
0.32 0.44 0.59 0.71 0.78
0.22 0.35 0.66 0.73 0.73
0.30 0.45 0.69 0.74 0.74
ML Fig. 1. HPLC pattern of the free amino acids in steady-state culture of S. cremoris HP growing in a chemostat on RFPcasein medium (0.4% casein) at a dilution rate of 0.1 h ~ (A) and 0.4 h -1 (B). The numbers represent the elution time of the HPLC column in min. The same volume of culture fluid was used in A and B.
195
analysing the amino acids in the culture fluid (Fig. 1B). The number and amount of amino acids was lower than at low dilution rates and many of the essential amino acids (leucine, glutamate, lysine, histidine, alanine, methionine and arginine) were missing and could have become growth limiting. 4.3. Amino acid limitation in milk cultures
High specific growth rates are necessary for high acid production in milk, but casein concentrations are suboptimal, as was shown in Table 1. Under these conditions an amino acid limitation is to be expected. This was investigated by taking samples from growing cultures and determining the free amino acids in these samples (Fig. 2). Some of the essential amino acids were found, such as proline, lysine, histidine, tyrosine, arginine, valine and alanine, whereas others were conspicuously missing or present at very low concentrations (leucine, phenylalanine, methionine and glutamate). These amino acids are possibly growth-limiting in milk cultures. However, in the stationary phase of growth all amino acids present
,J
in casein were found in the medium (not shown). The streptococci had, apparently, continued to hydrolyse casein after their growth had stopped. The presence or absence of the acidic amino acids glutamate and aspartate could not be established since these amino acids were poorly dansylated using the described method, and furthermore they both eluted very close to the (large) Dns-OH peak. It was assumed that glutamate, which is essential for growth of streptococci, could become growthlimiting since it is accumulated in large amounts by these organisms [17]. When adding to milk a mixture of amino acids which most likely become growth-limiting, the specific growth rate increased by approx. 10%, whereas no stimulation was found when all other amino acids were added (Table 3). An attempt was made to establish which specific amino acids were growth-limiting in milk. Each of the stimulatory amino acids was added separately and some in different combinations. The effect of the additions on the growth rate of two strains of S. cremoris, HP and Es, was determined (not shown). These effects were occasionally different for the two strains which can be contributed to differences in proteolytic systems [3]. Only when leucine or phenylalanine were included, stimulation in specific growth rates was found (Table 3). 4.4. Competition in continuous cultures
When growing mixed starter cultures under
Table 3 Relative specific growth rates (in %) of S. cremoris strains HP and E s in milk enriched with different amino acid-mixtures T
The growtla rates in milk are presented as 100%. Amino acid mixture I: leu, ser, met, val, glu, phe, ile. Amino acid mixture II: arg, thr, gly, pro, tyr, asp, lys, trp, his, ala. All amino acids were added in the concentration present in RFP-medium [14]. S. cremoris strain
¢
'i
t'
r
i
Fig. 2. HPLC pattern of the amino acids produced during exponential growth of S. cremoris HP in milk. The detected amino acids are named in the Figure.
milk milk + milk + milk + milk + milk + milk +
0.2% bactotrypton amino acid mixture I amino acid mixture II leucine phenylalanine leu/phe
HP
Es
100 105 111 97 104 100 105
100 110 108 97 105 107 105
196
amino acid limitation,competition for these substrates will occur. Population changes resulting from competition were studied with a chemostat [20]. Since milk is not a suitable medium for continuous cultivation we have used the RFPcasein medium as described above, with 0.4% casein. S. cremoris strains HP and E 8 were grown in separate continuous cultures and when steady states were reached in both cultures they were mixed in a 1 : 1 ratio. In one of these cultivation was continued at a dilution rate of 0.1 and in the other the dilution rate was slowly increased to 0.3 h-1. In both cases S. cremoris H P outcompeted S. cremoris E s (Table 4). However, there was a big difference between the two experiments. At the low dilution rate a high cell density was reached (A660n m = about 2.0) and the residual lactose concentration was low (83 #M). Under these conditions enough casein could be hydrolysed by the streptococci for their supply of essential amino acids (Fig. 1A) resulting in a lactose-limited culture. Addition of extra lactose to these cultures led to an increase of the cell density (not shown). As was already shown in a previous publication [4] S. cremoris H P has a lower apparent affinity constant for lactose than S. cremoris E 8 and gradually became dominant in the culture. At the higher dilution rate the cell density in the culture was significantly lower ( A 6 6 0 n m = about 1.5) and the residual lactose concentration was 1290 ~tM. In this case lactose was definitely present in excess and the rate of casein hydrolysis had now become growth-limiting. The data presented in Table 2 already suggested that S. cremoris strain HP had a lower apparent affinity constant for casein than strain E s and it was indeed found that strain HP became dominant at the higher dilution rate dur-
Table 4 Competition in continuous culture between S. cremoris strains E 8 and HP in RFP-medium with 0.4% casein Dilution rate (h -1 )
Strain becoming dominant
Bacterial cell density (A 660nm)
Concentration of residual lactose (/IM)
0.1 0.3
HP HP
2.0 1.5
83 1290
ing the competition for casein and its hydrolysis products. Strain E 8 could not obtain a selective advantage in this medium since the specific growth rates of both strains on 0.4% casein were almost identical (Table 2). Competition between different strains of S. cremoris were also studied in leucine- and glutamate-limited chemostats. Under leucine limitation crossing /~-S curves were reported for the strains E s and ML 1 with E 8 having the highest /~max and ML1 the lowest K~ for. leucine [19]. When strain HP was used instead of strain ML 1 in the mixtures similar results were obtained (not shown). If glutamate was made growth-limiting in R F P medium, again S. cremoris HP outcompeted S. cremoris E 8 at a low dilution rate of 0.05 h -~. At a higher dilution rate ( D = 0.2 h 1) strain E s became dominant (Fig. 3). Thus, in all experiments competition for growth-limiting substrates gave rise to population changes due to differences in growth parameters of S. cremoris strains.
5. D I S C U S S I O N The
maximum
specific growth
rate
of
S.
cremoris in milk has been reported to be signifi-
cantly lower than that in other growth media [4,12]. This has been confirmed in our experiments. This relatively low specific growth rate could be increased by the addition of amino acids to milk [11-13]. This suggests an amino acid limitation in milk, despite the high concentration (3%) of casein present. We have tested this possibility by performing different growth experiments in milk with and without various additions. If amino acids would be growth-limiting during cultivation of streptococci in milk, this would be a consequence of casein hydrolysis by the bacteria proceeding at a rate too low to sustain m a x i m u m specific growth rates. This was indeed observed in the growth experiments in milk and RFP-casein medium where the casein concentration was varied. In milk 3% casein did not allow potential maxim u m specific growth rates (Table 1), whereas growth rates could be increased by 10 to 20% by the addition of extra casein. The experiments in which diluted milk was used clearly demonstrated
197
that casein is the growth rate-determining substrate in milk. Even when milk was diluted 10-fold and extra casein was added normal growth rates were observed (Table 1). In RFP-casein medium the casein concentration necessary for m a x i m u m specific growth rates (1.2-1.6%) was lower than in milk (Table 2). Milk contains high concentrations of Ca 2+ and most casein is present in the form of CaZ+-casein micelles. In RFP-casein, on the other hand, the Ca 2 +-concentration is much lower and it can be assumed that more soluble casein is present. Possibly this free casein is the actual substrate for the proteolytic system of the streptococci. A limited rate of hydrolysis of casein will lead to an amino acid-limitation for the streptococci. This was demonstrated with S. cremoris strain H P in continuous cultures with RFP-casein medium containing a low casein concentration. At a low dilution rate the casein-hydrolysis was sufficiently fast to supply all the essential amino acids for growth and lactose became the growth-limiting substrate. However, at a higher dilution rate the amino acids were not produced fast enough and became growth-limiting (Fig. 1). In an attempt to identify these amino acids, leucine and phenylalanine appeared to be of particular importance. The practical implications of these results are obvious. The growth rate and, concomitantly, the acidification rate of S. cremoris can be increased by additions to milk of: (1) an amino acid mixture, provided that leucine and phenylalanine are included and that glutamate is not added in inhibitory concentrations [12]; (2) extra casein, and (3) extra milk powder. All these additions result in an increase of 10 to 20% of the specific growth rate of S. cremoris. An alternative way of increasing the specific growth rates of the streptococci in milk is to increase the rate of proteolysis. This could be achieved by genetically manipulating the bacteria leading to overproduction of the proteases or by the addition of purified streptococcal proteases to the milk cultures. Both approaches will soon be possible since the genes coding for the proteases are currently being cloned in streptococci [21] and large amounts of proteases can now be purified (E.J. Smid, Dept. of Microbiology, University of Groningen, pers. commun.)
The observations on the amino acid/caseinlimitation in milk also have important implications for the population dynamics of mixed starter cultures. During propagation of these starters the composition of the mixtures will change due to differences in maximum specific growth rates [4,7]. These population changes were also observed in continuous cultures and besides differences in m a x i m u m specific growth rates, different apparent affinity constants for substrates have been found. Crossing ~-S curves under lactose limitation were reported for S. cremoris strains HP and Es, showing dominancy of strain HP at low dilution rates, whereas at high dilution rates strain E 8 came to the fore [4]. Lactose limitation, however, will only occur in the initial stages of the ripening process and definitely not during propagation of the starters in milk. As was described here, other substrates ( c a s e i n / a m i n o acids) are growth limiting in milk cultures. It was therefore relevant to study the role of these growth-limiting substrates in the population dynamics of mixed starter cultures. Differences in K S for the amino acid leucine in S. cremoris strains were described recently [18]. The crossing ~-S curves observed for leucine have also been found here for glutamate (Fig. 3).
100-
50-
[]
I
5
!
10
vol. changes(Dxt) Fig. 3. Competition of S. cremoris E 8 and S. cremoris H P in continuous culture under glutamate limitation at a dilution rate of 0.05 h - 1 (D) and at a dilution rate of 0.20 h 1 (m).
198 W e also r e p o r t h e r e d i f f e r e n c e s in K s for casein. S. c r e m o r i s H P o u t c o m p e t e d S. c r e m o r i s E 8 in continuous culture on RFP-casein medium cont a i n i n g 0.4% casein. T h e o u t c o m e of the e x p e r i m e n t was n o t d e p e n d e n t o n the d i l u t i o n rate. A t l o w d i l u t i o n rates t h e strains c o m p e t e d for l a c t o s e a n d at h i g h e r d i l u t i o n rates for casein. In b o t h c a s e s s t r a i n H P w a s m o r e c o m p e t i t i v e . If h i g h e r c o n c e n t r a t i o n s of c a s e i n w o u l d h a v e b e e n u s e d in the m e d i u m S. c r e m o r i s Es, w i t h its higher/x,,,~ x in R F P - m e d i u m t h a n s t r a i n H P ( T a b l e 2), w o u l d be e x p e c t e d to b e c o m e d o m i n a n t at d i l u t i o n rates a p p r o a c h i n g ~m,x" It c a n b e c o n c l u d e d t h a t (1) the r a t e o f c a s e i n h y d r o l y s i s d e t e r m i n e s the g r o w t h r a t e of S. c r e m o r i s in m i l k ; (2) the c a s e i n - l i m i t a t i o n l e a d s to a l i m i t a t i o n of a m i n o acids, e s p e c i a l l y l e u c i n e a n d phenylalanine; (3) t h e c o m p e t i t i o n for t h e g r o w t h - l i m i t i n g c a s e i n a n d a m i n o a c i d s has a p r o n o u n c e d e f f e c t o n the c o m p o s i t i o n o f m i x e d s t a r t e r cultures.
REFERENCES [1} Limsowtin, G.K.Y., Heap, H.A. and Lawrence, R.C. (1977) N.Z.J. Dairy Sci, Technol. 12, 101-106. [2] Law, B.A. and Kolstad, J. (1983) Antonie van Leeuwenhoek 49, 225-245. [3] Hugenholtz, J., Exterkate, F.A. and Konings, W.N. (1984) Appl. Environ. Microbiol. 48, 1105-1110. [4] Hugenholtz, J. and Veldkamp, H. (1985) FEMS Microbiol. Ecol. 31, 57-62. [5] Teuber, M. and Lembke, J. (1983) Antonie van Leeuwenhoek 49, 283-295. [6] Collins, E.B. (1961) Appl. Microbiol. 9, 200-209. [7] Lee, D.A. and Collins, E.B. (1976) J. Dairy Sci. 59, 405 409.
[8] Lawrence, R.C., Heap, H.A., Limsowtin, G.K.Y. and Jarvis, A.W. (1978) Cheddar cheese starters: current knowledge and practices of phage characteristics and strain selection. J. Dairy Sci. 61, 1181-1191. [9] De Man, J.C. and Galesloot, T.E. (1962) Neth. Milk Dairy J. 16, 1-10. [101 Banks, W., Dalgleish, D.G. and Rook, J.A.F. (1985) Milk and milk processing. In Robinson, R.K. (Ed.), Dairy Microbiology, I, Elsevier, Amsterdam, pp. 1-34. [11] Selby Smith, J., Hillier, A.J., Lees, G.J. and Jago, G.R. (1975) J. Dairy Res. 42, 123-138. [12] Otto, R. (1981) An Ecophysiological Study of Starter Streptococci. Ph.D. Thesis, University of Groningen. [13] Reiter, B. (1973) Some thoughts on cheese starters. J. Soc. Dairy Techn. 26, 3-15. [14] Rogosa, M., Franklin, and Perry, (1961) Correlation of the vitamin requirements with cultural and biochemical characters of Lactobacillus spp. J. Gen. Microbiol. 25, 473-482. [15] Otto, R. (1984) Uncoupling of growth and acid production in Streptococcus cremoris. Arch. Microbiol. 140, 225 -230. [16] Otto, R. (1985) The relation between phosphate potential and growth rate of Streptococcus cremoris. Arch. Microbiol. 142, 97-100. [17] Poolman, B., Smid, E.J., Veldkamp, H. and Konings, W.N. (1987) Bioenergetic consequences of lactose starvation for continuously cultured Streptococcus cremoris. J. Bacteriol., 160, 1460-1468. [18] Fairbairn, N.J. (1953) A modified anthrone reagent. Chem. Ind. (London) 86. [19] Hugenholtz, J., Veldkamp, H. and Konings, W.N. (1986) Detection of specific strains and variants of Streptococcus cremoris in mixed culture by immunofluorescence. Appl. Env. Microbiol. 53. 149-155. [201 Veldkamp, H. (1977) Ecological studies with the chemostat. Adv. Microbiol. Ecol. 1, 59-94. [21] Kok, J., Van Dijl, J.M., Van der Vossen, J.M.B.M. and G. Venema (1985) Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis. Appl. Env. Microbiol. 50, 94-101.