CRYOBIOLOGY
13, 214-217
(1976)
Transformation of Amino Acid Composition in Bacterial of Lactobacillus bulgaricus during Freeze-Drying STOJANKA Dairy
Institute
MATERIALS
AND
METHODS
Samples for quantitative and qualitative determinations of amino acids in cell suspensions of Lactobacillus bulgaricus (from the National Institute for Research in Dairying, Reading) were prepared in the following way. L. bulgaricus was inoculated in MRS broth, incubated at 37°C for 18 hr, and centrifuged at 4000 rpm. Bacterial cells were washed with Ringer’s solution and centrifuged again in order to remove all traces of the nutrient medium. Cell suspenReceived
December
13, 1974.
MITIC
of Yugoslavia,
Previous studies on the effect bacterial freeze-drying have shown that this technique can be evaluated satisfactorily by means of quantitative parameters for determining survival cell counts (2-5, 8, 9). MitiC (10) bases her explanation of biochemical phenomena manifested in bacterial death during freeze-drying upon deamination of free and some bound amino acids contained in the protein complex of lyophilized cell suspensions of Streptococcus diacetiluctis. In ,order to establish a relationship and confirm the degradation of proteins and polypeptides, new experiments were carried out with suspensions of Lactobacillus bulgaricus cells in which amino acid ‘cornposition was quantitatively and qualitatively analyzed both before and after freeze-drying.
Beograd,
Yugoslavia
sions were transferred into 10% saccharose solution. Cell suspension aliquots of 1 ml were distributed in special ampoules, frozen at -45°C and freeze-dried in a centrifugal freeze-drier (Model 30 P, Edwards High Vacuum Ltd. ) . Primary drying was carried out under a vacuum of 0.05-0.07 Torr for 16 hr. Secondary drying lasted 6 hr and was perfmormed in a separate manifold system with phosphorus pentoxide under a vacuum of 0.001 Ton-. Ampoules were then sealed under vacuum and an Edwards’ high frequency vacuum tester was used for detecting vacuum both in ampoules on the manifold and in sealed ampoules. RESULTS
In order to simplify the experimental conditions, cell suspensions of L. bulgaricus were freeze-dried in distilled water containing 1070 saccharose. We did not add any amino acid protector during freezedrying, therefore only 1% of the cells survived the freeze-drying procedure. The viable cell count amounted to 5.7 X 10’ beand 5.7 x lo6 after fore freeze-drying freeze-drying. Quantitation of identified amino acids in the total protein complex of the cells is presented in Table 1. Experimental data concerning basic acids show that lysine and histidine occur in lower concentrations than arginine. Percentage of histidine in the total cell protein is the lowest when compared with other amino acids examined. 214
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any farm reserved.
Cells
TRANSFORMATION TABLE
1
Quantitative Amino Acid Composit,ion L. bulguricus Cells (PM/ml)
of
Amino acid
Before freeaedrying
After freezedrying II
Lysine Histidine Arginine Asparagine acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine NH,
1,336 0.636 3.240 6.692 2.816 2.504 6.552 1.652 5.728 7.784 3.528 1.228 2.844 3.928 1.364 1.572 8.348
0.936 0.600 2.868 5.128 2.160 1.864 4.952 1.292 4.196 5.692 2.544 0.964 2.196 3.076 0.880 1.260 12.112
Asparagine acid and glutamic are present in very high amounts. Concentrations of amino acids in lyophilized cells were abo’ut 1.600 pM lower than initial concentrations in nonlyophilized cells. Data in Table 1 further show that alanine is the dominant amino acid, both in the group glycine, alanine, and serine and in the examined protein complex of L. bulgur&s cells. After freeze-drying, alanine concentration was approximately 2.092 PM lower than the initial concentration, indicating a high degree of deamination. Glycine concentration was also lower after freeze-drying ( 1.523 PM). In this amino acid group, serine showed the smallest observable decrease in concentration. Valine, isoleucine, and leucine belong to a group of amino acids present in medium amounts in the total protein of examined cells. Their concentrations ranged from 2.844 to 3.928 PM before freezing to 0.852-0.964 ,uM after freezing. The group of amino acids present in smallest amount includes proline, phenylalanine, and tyrosine. Their concentrations
IN BACTERIA
215
range from 1.364 to 1.652 PM before freezedrying, with a decrease of 0.360-0.484 PM during freeze-drying. Data on threonine and methionine content show that threonine concentration is very close to that of isoleucine, while methionine concentration is higher than histidine. After freeze-drying, threonine and methionine concentrations were reduced by 0.656 and 0.242 ,JLM, respectively. Variation in treatment of cell suspensions also caused changes in ammonia content. After freeze-drying, ammonia content was 3.712 PM higher than in control samples. DISCUSSION
In this paper, we used poor medium without amino acid protectors, because protecting the bacteria during freeze-drying could affect the quantitative and qualitative spectra of amino acids and make them different from those in the control samples before freeze-drying. However, in our previous work (1967) we used a more optimal medium (skimmed milk with 8% lactose and 0.2% pancreas extract) so that 32.5% of the L. bulgaricus cells survived freeze-drying (8.0 x lo* beand 2.6 x lo8 after fore freeze-drying, freeze-drying). On that occasion, it was found that the number of cells surviving freeze-drying depended essentially on the type of bacteria and the composition of medium. Analytical data show that a decrease in concentration of free and some bound amino acids of the protein complex is a general phenomenon occurring in all lyophilized samples as compared with control samples. Keeping in mind the high concentrations of these acids and their important role in biochemical deamination processes, we can conclude that these molecules are a significant part of the formation of cell protein in the examined L. bulguricus strain. Degree of deamination varies among amino acids, since these acids have
216
STOJANKA
different hydrophilic, structural, physical, and chemical properties as well as variations in frequencies and sequences in protein macromolecules and in the free amino acid pool. Increase in ammonia concentration observed in lyophilized cell suspensions contributes to the explanation of the general deamination of all amino acids. In her earlier work (1973) the author observed that deamination of all amino acids was also a general phenomenon in lyophilized cells of Streptococcus diacetilactis. Through qualitative identification, the presence of similar basic, neutral, and acidic amino acids was discovered in L. bulgaricus cells; however, quantitative analyses showed differences in amino acid concentration, i.e., glutamic acid dominated in S. diacetiluctis and alanine in L. hulgaricus. The greatest quantitative differences were observed with lysine which belonged to the dominant amino acid group in S. diacetilactis but to the third group in L. bulgaricus. This common characteristic has confirmed our assumption that some biochemical transformations of amino acids, as discovered by analyzing the amino acid composition before and after freeze-drying, might cause bacterial death during freezedrying. In the available literature we could not find any confirmation of our results, since, so far as we know, there is no work in which death of lyophilized cells would be ascribed to the deamination
13, 214-217
(1976)
Transformation of Amino Acid Composition in Bacterial of Lactobacillus bulgaricus during Freeze-Drying STOJANKA Dairy
Institute
MATERIALS
AND
METHODS
Samples for quantitative and qualitative determinations of amino acids in cell suspensions of Lactobacillus bulgaricus (from the National Institute for Research in Dairying, Reading) were prepared in the following way. L. bulgaricus was inoculated in MRS broth, incubated at 37°C for 18 hr, and centrifuged at 4000 rpm. Bacterial cells were washed with Ringer’s solution and centrifuged again in order to remove all traces of the nutrient medium. Cell suspenReceived
December
13, 1974.
MITIC
of Yugoslavia,
Previous studies on the effect bacterial freeze-drying have shown that this technique can be evaluated satisfactorily by means of quantitative parameters for determining survival cell counts (2-5, 8, 9). MitiC (10) bases her explanation of biochemical phenomena manifested in bacterial death during freeze-drying upon deamination of free and some bound amino acids contained in the protein complex of lyophilized cell suspensions of Streptococcus diacetiluctis. In ,order to establish a relationship and confirm the degradation of proteins and polypeptides, new experiments were carried out with suspensions of Lactobacillus bulgaricus cells in which amino acid ‘cornposition was quantitatively and qualitatively analyzed both before and after freeze-drying.
Beograd,
Yugoslavia
sions were transferred into 10% saccharose solution. Cell suspension aliquots of 1 ml were distributed in special ampoules, frozen at -45°C and freeze-dried in a centrifugal freeze-drier (Model 30 P, Edwards High Vacuum Ltd. ) . Primary drying was carried out under a vacuum of 0.05-0.07 Torr for 16 hr. Secondary drying lasted 6 hr and was perfmormed in a separate manifold system with phosphorus pentoxide under a vacuum of 0.001 Ton-. Ampoules were then sealed under vacuum and an Edwards’ high frequency vacuum tester was used for detecting vacuum both in ampoules on the manifold and in sealed ampoules. RESULTS
In order to simplify the experimental conditions, cell suspensions of L. bulgaricus were freeze-dried in distilled water containing 1070 saccharose. We did not add any amino acid protector during freezedrying, therefore only 1% of the cells survived the freeze-drying procedure. The viable cell count amounted to 5.7 X 10’ beand 5.7 x lo6 after fore freeze-drying freeze-drying. Quantitation of identified amino acids in the total protein complex of the cells is presented in Table 1. Experimental data concerning basic acids show that lysine and histidine occur in lower concentrations than arginine. Percentage of histidine in the total cell protein is the lowest when compared with other amino acids examined. 214
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any farm reserved.
Cells
TRANSFORMATION TABLE
1
Quantitative Amino Acid Composit,ion L. bulguricus Cells (PM/ml)
of
Amino acid
Before freeaedrying
After freezedrying II
Lysine Histidine Arginine Asparagine acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine NH,
1,336 0.636 3.240 6.692 2.816 2.504 6.552 1.652 5.728 7.784 3.528 1.228 2.844 3.928 1.364 1.572 8.348
0.936 0.600 2.868 5.128 2.160 1.864 4.952 1.292 4.196 5.692 2.544 0.964 2.196 3.076 0.880 1.260 12.112
Asparagine acid and glutamic are present in very high amounts. Concentrations of amino acids in lyophilized cells were abo’ut 1.600 pM lower than initial concentrations in nonlyophilized cells. Data in Table 1 further show that alanine is the dominant amino acid, both in the group glycine, alanine, and serine and in the examined protein complex of L. bulgur&s cells. After freeze-drying, alanine concentration was approximately 2.092 PM lower than the initial concentration, indicating a high degree of deamination. Glycine concentration was also lower after freeze-drying ( 1.523 PM). In this amino acid group, serine showed the smallest observable decrease in concentration. Valine, isoleucine, and leucine belong to a group of amino acids present in medium amounts in the total protein of examined cells. Their concentrations ranged from 2.844 to 3.928 PM before freezing to 0.852-0.964 ,uM after freezing. The group of amino acids present in smallest amount includes proline, phenylalanine, and tyrosine. Their concentrations
IN BACTERIA
215
range from 1.364 to 1.652 PM before freezedrying, with a decrease of 0.360-0.484 PM during freeze-drying. Data on threonine and methionine content show that threonine concentration is very close to that of isoleucine, while methionine concentration is higher than histidine. After freeze-drying, threonine and methionine concentrations were reduced by 0.656 and 0.242 ,JLM, respectively. Variation in treatment of cell suspensions also caused changes in ammonia content. After freeze-drying, ammonia content was 3.712 PM higher than in control samples. DISCUSSION
In this paper, we used poor medium without amino acid protectors, because protecting the bacteria during freeze-drying could affect the quantitative and qualitative spectra of amino acids and make them different from those in the control samples before freeze-drying. However, in our previous work (1967) we used a more optimal medium (skimmed milk with 8% lactose and 0.2% pancreas extract) so that 32.5% of the L. bulgaricus cells survived freeze-drying (8.0 x lo* beand 2.6 x lo8 after fore freeze-drying, freeze-drying). On that occasion, it was found that the number of cells surviving freeze-drying depended essentially on the type of bacteria and the composition of medium. Analytical data show that a decrease in concentration of free and some bound amino acids of the protein complex is a general phenomenon occurring in all lyophilized samples as compared with control samples. Keeping in mind the high concentrations of these acids and their important role in biochemical deamination processes, we can conclude that these molecules are a significant part of the formation of cell protein in the examined L. bulguricus strain. Degree of deamination varies among amino acids, since these acids have
216
STOJANKA
different hydrophilic, structural, physical, and chemical properties as well as variations in frequencies and sequences in protein macromolecules and in the free amino acid pool. Increase in ammonia concentration observed in lyophilized cell suspensions contributes to the explanation of the general deamination of all amino acids. In her earlier work (1973) the author observed that deamination of all amino acids was also a general phenomenon in lyophilized cells of Streptococcus diacetilactis. Through qualitative identification, the presence of similar basic, neutral, and acidic amino acids was discovered in L. bulgaricus cells; however, quantitative analyses showed differences in amino acid concentration, i.e., glutamic acid dominated in S. diacetiluctis and alanine in L. hulgaricus. The greatest quantitative differences were observed with lysine which belonged to the dominant amino acid group in S. diacetilactis but to the third group in L. bulgaricus. This common characteristic has confirmed our assumption that some biochemical transformations of amino acids, as discovered by analyzing the amino acid composition before and after freeze-drying, might cause bacterial death during freezedrying. In the available literature we could not find any confirmation of our results, since, so far as we know, there is no work in which death of lyophilized cells would be ascribed to the deamination
