Proteolytic Characterization of Lactobacillus delbrueckii ssp ...

Proteolytic Characterization of Lactobacillus delbrueckii ssp. bulgaricus Strains by the o-Phthaldialdehyde Test and Amino Acid Analysis' CRAIG J. OBERG? BART c. WEIYER, LYNN v. MOYES,~ RODNEY J. BROWN, and GARY H. RICHARDSON Department of Nutrition and Food Sciences Utah State University Logan 843228700

An important metabolic trait of dairy lactic is their ability to degrade casein. This cultures The o-phthaldialdehydetest and amino proteinase system has been described for acid analysis were used to characterize mesophdic lactic organisms (19, 26, 27); howproteolysis of milk proteins during ever, few studies have been done with the growth of Lactobacillus delbrueckii ssp. 1actobaciJli species, particularly Lactobacillus bulgaricus. Thirty-four strains were indelbrueckii ssp. bulgaricus (1, 18). Several cubated in sterile 10% NDM for 12 h. physical parameters that affect proteinase and Extent of proteolysis as estimated by the peptidase activity in L. delbrueckii ssp. bulgari0-phthaldialdehyde test revealed a large have been identified (11, 15). Argyle et al. cus variance in total proteolysis. For seven (3) characterized a cell-bound proteinase in L. strains, trichloroacetic acid filtrates of indelbrueckii ssp. bulgaricus NCDO 1489. Using oculated 10% NDM were analyzed by the same organism, Chandan et al. ( 5 ) examclassical ion-exchange amino acid analyined its proteolytic ability on native micellar sis. Each strain had a distinct pattern of casein using the 2,4,6-trinitrobenzene sulfonic individual amino acid concentrations. acid assay (TNBS)and noted that Pcasein was Amino acid profiles provided information the most susceptiblefraction. Previously, Ohmabout the proteolytic activity of these iya and Sat0 (22) had made this same observastrains that was not available from the otion, noting that intracellular proteases from L. phthaldiddehyde test. Cluster analysis, based on amino acid profiles of each delbrueckii ssp. bulgaricus and Lactobacillus strain, differentiated the seven strains behelveticus hydrolyzed Pcasein more readily yond what was possible by visual comthan qlcasein. Ezzat et al. (10, 12) found cell parison of the amino acid analysis results. wall bound proteinase activity in L. delbrueckii (Key words: thermolactic cultures, protessp. bulgaricus by using "k2 methylated casein olysis, amino acid analysis, Lactobacillus as substrate. delbrueckii ssp. bulgaricus) Peptidase activity has been more closely examined in lactobacilli (14). Eggimann and Abbreviation key: OPA = o-phthaldialdehyde Bachmann (7) isolated and characterized an test, TNBS = triniaobenzene sulfonic acid asaminopeptidase from Lactobacillus delbrueckii say. ssp. lacfis. Peptidase activity in Lactobacillus casei has also been studied (8, 9). Meyer and INTRODUCTION Jordi (20) purified and characterized x-prolyldipeptidyl-amhopptidase from L. delbrueckii ssp. kzctis and compared it with enzymes found Receivcd August 28, 1989. in Streptococcus salivarius ssp. thermophilus. Accepted October 26, 1990. Atlan et al. (2) extracted cell wall proteins and 'Contribution Number 3873 o f the Utah Agricultural four aminopeptidases from L. delprofiled Experiment Station.Approved by the director. Mention of companies or products does not constitute.endorsement by brueckii ssp. bulgaricus CNRZ 397. Strains of Utah State University, Utah Agricultural Experiment Sta- L. delbrueckii ssp. bulgaricus have also been tion, or Weber State University over similar products not screened for aminopeptidase activity to evalumentioned. 2Departmmt of ~icrobiology,weber state university, ate their potential in accelerating cheese ripenO g d q UT 84408-2506. ing (15). ABSTRACT

1991 J Dairy Sci 74:398403

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PROTEINASE ACTIMTY IN THERMOLACTIC CULTURES

399

Because proteinases and peptidases play a incubation of separate tubes at W C for 12 h, critical role in cheese flavor and body develop samples were prepared according to the p r w e ment it is important to characterize these en- dure of Church et al. (6). A 2.5 ml sample from zyme activities in lactic cultures used for each tube was mixed with 1 ml distilled water cheese making (13). Wide variation in protee before 5 ml of .75 N trichloroacetic acid was lytic ability among L. delbrueckii ssp. bulgari- added while tube contents were vortexed. After cus strains has been seen by several research- setting at room temperature for 10 min the ers. Ayres et al. (4) found variance in amount acidified samples were filtered through a Whatof tyrosine released from strains screened for man (Clifton, NJ) Number 2 filter paper. Aliproteolytic ability. Singh et al. (24, 25) quots of the filtrate were kept frozen at -70'C screened 10 strains of L. delbrueckii ssp. bul- until analyzed. Controls were prepared simiguricus on casein agar, 4 strains exhibited a larly from uninoculated 10% sterile NDM. high degree of proteolysis and 6 exhibited much lower levels. Ohmiya and Sat0 (21, 22) 0-Phthaldlaldehyde Analysls studied intracellular proteams from L. delDuplicate aliquots from each TCA filtrate brueckii ssp. bulgaricus and L. helveticus and found a wide variability of proteolysis on were analyzed by OPA testing according to the whole casein and casein fractions among the method described by Church et al. (6) using a strains screened. Ezzat et al. (10) and Hickey et Beckman DU-8B W-Visible spectrophotomal. (16) also observed variability among strains eter (Beckman Instruments, Palo Alto, CA) of lactobacilli when screening for proteolytic with matched quartz cuvettes. The OPA reagent activity. was prepared by combining 25 ml of 100 mM Tests used to measure proteolytic ability in- sodium borate, 2.5 ml20% (wt/wt) SDS, 40 mg clude the TNBS test (5), the Hull test (17), the OPA dissolved in 1 ml methanol, and 100 pl j30-phthaldialdehyde test (OPA) (6), and classical mercaptoethanol, then adjusting the volume to ionexchange amino acid analysis (16, 28). 50 ml with H20. Fifty microliters of each Hickey et al. (16) used amino acid analysis to thawed TCA filtrate was mixed with 1 ml of determine whether species of lactobacilli re OPA reagent in a 1.5 ml quartz cuvette and the lease amino acids from sodium caseinate more absorbance was read at 340 nm. An average of rapidly than Lactococcus lactis ssp. cremoris or 10 readings for each aliquot was recorded. AbS. salivarius ssp. thermophilus. We report use sorbance of the OPA reagent with the 10% of classical ion-exchange amino acid analysis NDM control was subtracted from each readto characterize proteolysis among L. delbrueckii ing. ssp. bulguricus strains. Amlno Add Analysls MATERIALS AND METHODS

Duplicate aliquots from each TCA fiitrate were prepared for classical ion exchange amino Cultures acid analysis as outlined by Weimer et al. (28). Cultures of L. delbrueckii ssp. bulgaricus, Thawed aliquots (2.5 ml) of each TCA filtrate Luctococcus lactis ssp. lactis, L. lactis ssp. were lyophilized prior to addition of 1 ml 6N cremoris, L. helveticus, and S. salivarius ssp, HCL The acidified mixture was flushed with thennophilus were obtained from the Depart- nitrogen while in an ultrasonic water bath, ment of Microbiology culture bank at Weber flamesealed in a 5-ml ampule, and held in a State University and were maintained as previ- heat block at llo'C for 20 h. After hydrolysis, the sample was filtered through a . 2 - p pore ously described (29). size syringe filter. Ten microliters of filtrate was transferred to a test tube and dried by Sample Preparation blowing gently with nitrogen gas. Each dried After cultures had grown for 18 h in basal sample was hydrated with 250 p.1 of Beckman broth (30),a 1% inoculum of each culture was Na-S dilution buffer, filtered through a .2-pm transferred into separate tubes, each containing pore size syringe filter, and loaded into an 10 ml of 10% sterile NDM. Following further amino acid analyzer sample cartridge. FollowJournal of Dairy Science Vol. 74, No. 2, 1991

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OBERG ET At.

ing analysis in a Beckman System 6300 Amino Acid Analyzer, amino acid concentrations were calculated based on Beckman amino acid standards, which had been treated similarly to the samples. Cluster Analysis

TABLE 1. Proteolysis ranges for selected dairy strains Osisg the 0-phthaldialdehyde test. Strains

Culture

(=w

Lactococcus lactis ssp. lactis 5 Lactococcus lactis ssp. cremoris 13 Lactobacillus helveticus 9 Streptococcus salivarius ssp. rhennophilus 8 Lactobacillus delbrueckii ssp. bulgaricus 34

Incubation Range time @)

(~wd

24

.14-.17

24 12

.15-.29 .45-.7 1

Individual concentrations of fifteen amino acids for each of 7 strains of L. delbrueckii ssp. 12 .21-.39 bulgaricus were reduced to two dimensions by 12 .20-1.3 canonical discriminant analysis (23). This technique is a Combination of principal component 'Absorbance at 340 nm. analysis and canonical correlation. Three replicates of the nominal variable strain (n = 7, df = 6) and a matrix of amino acid concentrations (n = 15, df = 14) were analyzed to determine served with few exceptions. However, with a canonical variables. 24-h incubation time, individual strains of L. delbrueckii ssp. bulgaricus could not be distinguished because most cultures had absorbance RESULTS AND DISCUSSION readings >1. The extreme range of proteolysis noted for strains of L. delbrueckii ssp. bulgaria-Phthaldlaldehyde Analysls cus was not observed among samples of other Extent of proteolysis based upon OPA lactic genera and species examined by the same values was variable among the 34 strains of L. procedure (Table 1). delbrueckii ssp. bulgaricus examined (Figure 1). These measurements were made from dupli- Amlno Acid Analysls cate incubations with only one TCA filtrate per Seven of the 34 strains of L. delbrueckii ssp. incubation. Absorbance readings ranged from bulgaricus screened by the OPA method were .20 to 1.29 for cultures that were incubated in examined by amino acid analysis. Fifteen 10%NDM for 12 h. When incubation time was decreased to 10 h, the same ranking was ob- amino acids were selected to profile each strain (28). The profiles in Figure 2 show the relative concentrations of amino acids in NDM medium after incubation, divided by an uninoculated control. These measurements were made from triplicate incubations with one TCA filtrate per incubation. Amino acid analysis was run in duplicate for each filtrate. The average coefficient of variation for the seven strains measured in triplicate was 7.3%. Because relative concentrations of individual amino acids differed among strains, amino acid profiles also differed. Figure 2 adds detail to what is available from the ranking in Figure 1. For example, the prominence of the histidine peak in strains 133, 100, and 132 contrasts with the histidine Strain valley for the other four strains. Also noticeable Rgure 1. proteolytic activity of Lactobacillus del- is the valley for aspartic acid in strains 133 and bpueckii ssp. bulgaricus strai0s using the 0-phtbaldialde100 compared with the opposite in strains 117, hyde test. Cultures were incubated in duplicate for 12 h in 118, 111, and 114. Aspartic acid is lower than 10% NDM and measured by averaging 10 spectrophotometer readings. Points are means of two incubations and glycine or alanine in strains 133 and 100 but error bars are staadard errors of the means. higher in the other strains. Phenylalanine is Journal of Dairy Science Vol. 74, No. 2, 1991

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PROTEINASE AcrrvLTy IN THERMOLACI'IC CULTURES

lower than glycine in strains 133 and 100, but not in any of the other strains. More subtle differences, such as the glutamic acid peak in strain 117 and the proline peak in strain 100, can be seen also. Total amino acid concentration comelates well with values obtained by the OPA method (R2 = .95) (28). Therefore, the information relating to extent of proteolysis (Figure 1) could have been obtained fmm amino acid analysis by replacing OPA measurements with the sums of amino acid concentrations (28). Relative percentages of amino acids appeared similar among strains 117, 118, 111, and 114, which promoted more proteolysis. Greater variability in amino acid profiles was observed among strains 133, 100, and 132, which were least proteolytic.

sus second canoNcal variables is shown in Figure 3. Cluster analysis was run using concentrations of fifteen amino acids as quantitative variables. Amino acid analysis was run in duplicate for each of the three TCA filtrate aliquots (each from a separate incubation) of each culture, making a total of 42 amino acid analyses for the 21 aliquots of seven cultures. Means of analyses by aliquot produced three points for each of the seven cultures. Means of these three points were plotted in Figure 3. In all seven cases, standard errors of the means were so small that they were hidden by the plot symbols. The value of such plots is in identifying similarities and differences among samples, in this case among strains. Those points closest to each other are most similar and those furthest apart are least similar. Points found so close that they are indistinguishable are identical. Cluster Analysis Absolute distances between the 21 possible Canonical variables are linear combinations pairs of points in Figure 3 are listed in Table 2. that allow reduction of amino acid concentra- Attempts to assign specific meanings to vertical tions to two dimensions. A two-dimensional or horizontal positions in Figure 3 are not arrangement of the cultures plotted as first ver- justifiable. Both canonical variables in Figure 3 are made up of concentrations of all fifteen amino acids. Although it is easy to discern from Figure 2 that strains 111 and 118 are similar, cluster analysis showed that they are the most similar of the 21 possible pairs of two strains (Table 2). Strains 132 and 133 appear different in

Figure 2. Comparison of amino acid profiles in filtered NDM media following growth for 12 h of seven strains of Lactobacillus delbrueckii ssp. bulgaricus. Each amino acid concentration within a proffie was divided by the concentrationof the corresponding amino acid within a control (no culture). Readings represent means of three aliquots removed from the TCA filtrate, each analyzed in duplicate. Amino acids are phenylalanine (Phe), glycine (Gly). aspartic acid (Asp), alanine (Ala), arginine (Arg),histidine (His), isoleocine (Ile), glutamic acid (Glu), serine (Ser), lysine (Lys), leucine (Leu), proline (Pro), valine (Val), tyrosine W), and -nine W).

0

IIYI

'

1111

VI

I

5,,

lill

1511

Firs1 Canonical Variable

Figure 3. Clustering of seven strains of Lactobacillus delbrueckii ssp. bulgoricus based on amino acid profiles in filtered NDM media following growth. Each point represents the mean of three aliquots removed from the TCA fillrate, each analyzed in duplicate. Error bars showing standard errors of the means among aliquots are so small they are hidden by the symbols.

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OBERG ET AL.

TABLE! 2. Relative distances between pairs of straias of Lactobacillus delbrueckii ssp. buigan'cus based on amino acid profidea in filtered NDM media following growtb. Units match those in Figure 3. Strain

114

Strain ~~

114 118 111 133 Io0 117 132

~~

118

~

0 56 49 33 108

178 209

...

0 16 87 128 133 213

111 ~~

... ... ~~

0

76 135 148

224

Figure 2, but it would be difficult to conclude from Figure 2 that they are the least similar pair. Strain 133 is much more like 114 than 132 is, although Figure 2 could lead us to believe the opposite. Many other examples that show the importance of statistical analysis rather than just visual comparisons can be seen by comparing Figure 2 with Table 2 and Figure 3. It should be apparent also that statistical comparison without visual evaluation of the profiles is inadequate. Moving from Figure 2 to Figure 3 consolidates much information into a more easily comprehended form, but it does so at the loss of many degrees of freedom.

133 ~

...

... ...

0 125 211 229

100 ~

... ... ... ... ~

0 165 104

117

... ... ... ... ... 0 181

132 ~

...

~

...

...

... ... ...

0

amino acid analysis data is used to replace

OPA measurements (28).

ptoteolytic characterization of lactic acid bacteria by amino acid analysis revealed differences among amino acid profiles which reflected differences in proteinase, peptidase, and transport activities of lactic culture strains. A specific amino acid profile might correlate with a particular type of enzymic activity. Lactobaciiius deibrueckii ssp. bulgaricus is used in the manufacture of Mozzarella cheese where the physical properties of the cheese are critical to quality (4). The wide variation in total proteolysis among strains of this organism (Figure 1) has major implications for physical properties of Mozzarella cheese. Profiling culCONCLUSIONS tures by statistical analysis of amino acid analyThe three methods used in this paper repre- sis data as demonstrated in this paper could sent a succession of increasing ability to assess identify those strains that will give the most proteolysis associated with culture strains. The desirable characteristics in cultured products. OPA procedure (Figure 1) revealed a wide The techniques used here should be especially variation in amount of proteolysis. Amino acid helpful in screening large numbers of strains analysis profiles Figure 2) provided informa- for proteolytic characteristics. Use of individual tion about the kind of proteolysis associated proteins as substrates rather than NDM should with each strain. Statistical procedures Figure allow better definition of the proteolytic activ3 and Table 2) were necessary to extract ac- ity of strains than demonstrated in this paper. curately the similarities and differences among ACKNOWLEDGMENTS strains from the amino acid analysis data. After looking at Figure 3 and Table 2, hindWe thank the National Dairy Promotion and sight allowed us to see some of these relationResearch Board and the Western Daiq Foods ships by visual comparison of amino acid pro- Research Center for funding this research. files in Figure 2. It also made apparent the mistakes we would have made by relying on REFERENCES comparisons of amino acid analysis profiles without statistical analysis. The kinds of infor1 Accolas, I. P., and J. Auclair. 1983. Thermophilic lactic startm. Ir. J. Food Sci. Technol. 727. mation presented in figures 1,2, and 3 are all LAttan, D.,P. Laloi, and R. Portalicr. 1989. Isolation needed for a complete characterization of protea d charactfaizatim of aminopepti-deficient &IColysis among culture strains. Amino acid analyroboc111~ bJgoricus mutants. Appl. Enviroa Miaosis can provide all this information if a sum of biol. 55:1717.

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ACI'MTY IN THERMOLAcIlC CULTURES

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Journal of Dairy Science Vol. 74, No. 2, 1991