World Journal
of Microbiology
& Biotechnology
Detection esterases N.E. Karam*
11, 559463
of polygalacturonases in lactic acid bacteria
and pectin
and A. Belarbi
Of 80 strains of lactic acid bacteria tested, only Lactobacihs cusei strains I-INK10 and Ll-8, Luctobucilfus phntutmm Lc5 and Luctococcus hctis NNOl produced polygalacturonases (EC 3.2.1.) and/or pectin-esterases (EC 3.1.X.). Crude extracellular extracts of strain LX-8 were able to clarify pectin. Key words:
Lactic acid bacteria, pectins, pectin-esterases,
polygalacturonases.
Increasing research efforts are being devoted to the study of lactic acid bacteria because of their economic importance and the crucial role they play in human and livestock health (Fuller 1989) and in the fermentation of animal (Romero & McKay 1985) and plant products (Dellaglio et al. 1984). The industrial processing of plants generally requires a pectin-degradation step. Pectins are methylated polysaccharides, constitute one of the main building blocks of plant cell walls and cause difficulties during extraction, filtration and clarification of fruit juices. Endogenous pectinases are often insufficiently active and full pectin degradation requires the exogenous addition of pectinases (Pilnik & Rombouts 1985). Pectinases include depolymerizing and demethoxylating enzymes. Depolymerizing enzymes include polygalacturonase (PG) (EC 3.2.1), which cleaves the a-1,4 glycosidic bonds between two non-methylated galacturonic acid residues, and pectin-lyase (PL) (EC 4.2.2), which catalyses a pelimination reaction between two methylated residues. Deesterifying enzymes include pectin-esterase (PE) (EC 3.1.1), which catalyses the demethoxylation of methylated pectin, producing methanol and pectin. The pectin produced can be hydrolysed by PG. Pectinolytic activities have been found in Kltryveromyces frugilis (Bamby et al. 1990), Saccburomyces cerevisiae (McKay 1988;
Gainvors
ef al.
1994),
Buciffus
1990) and in the phytopathogens Pseudomonas
marginalis
(Nasuno
subfibs Erwinia
& Starr
1966a,
(Nasser
et al.
and b). Commer-
curofovoru
The authors are with the Laboratoire de Microbiologic G&&ale et Mokulaire. Facultb des Sciences, Universitb de Reims, B.P. 347, 51062 Reims Cedex. France: fax: 26 05 3279. *Corresponding author.
@ 1995 Rapid Science
Publishers
cial pectinases are mainly from Aspergillus niger and include the three pectinase activities. Little work has been done to assess the pectinolytic capacity of lactic acid bacteria other than Lucfoobucihs plunfuram (Sakellaris et al. 1988), Luctobucihs breuis (Starrier & Stoyla 1968) and Leuconosfoc mesenferoides (luven et al. 1985). An extracellular PG from Lacfob. plunfurclm has been characterized (Sakellaris et al. 1989). The present study was on the pectinolytic activities of different strains of lactic acid bacteria, isolated from raw milk in Algeria.
Materials
and Methods
Bacterial Strains The following strains were used: citrate-utilizing Lucfococcw fucfis subsp. fucfis strains 89, B9, FS5, FS6, NS5, NS6, NS7, NS25, NS30J and NS44; non-citrate utilizing Lacfococcus fucfis ssp. fucfis BS, BSI, B82, B83, H4, NNOI, NNII, TFIS and V8; Lucfococclts hcfis ssp. cremoris HI& NN105, NSI, NSlB, NSIC, NS4, NS63 and NS63B; Lucfococctts sp. FS3, FS9, FSII, VI7 and SI20.2; Lucfobucillus phfurum B22, cha34, cha343, Lc5, TF5, TFS, V4, V82, VI9 and V21; Lucfobucillus cusei HNKIO, FL20, LI-8, Lcl, Lc3, Lc7, Lc9 and Lc12; Lucfobuciffw sufivurius B23, cha401, cha404, TFHI and VII; Lucfobuciffw buchneri cha309Y; Lucfobucilhs sp. 67, 210, TF3, TF13, cha310, cha402 and cha405; Leuconostoc fuctis chalO2, cha201 and cha403; Letlconostoc dextrunicum chalO3 and cha406; and Leuconostoc sp. SIOZ, V3, V15, cha35, cha408, cha410, cha411, cha412, cha4122 and cha4123. Ail were isolated from Algerian raw milk (Karam & Karam 1994). Three strains from the American Type Culture Collection (ATCC) were also used: Luctobucik plunturum ATCC 8014, Luctobucih sake ATCC 15521 and Luctococcus luctis ATCC 11454.
N. E. h-am
and A. Belarbi
Growth Media MRS (De Man et al. 1960) and Ml7 (Terzaghi & Sandine 1975) with or without sugar, were supplemented with 0.1 to I% (w/v) polygalacturonic acid from orange (Sigma), 0.1 to 2.5% (w/v) apple pectin (70% to 75% methylated; Fluka Biochemicals) or 0.1% to 2.5% (w/v) lemon pectin (63% to 66% methylated; Fluka Biochemicals) and adjusted to pH 6 before sterilization (lZl”C, 20 min). Agarose (0.5%) was used instead of agar to obtain solid media when pectin or polygalacturonic acid were used. A chemically defined minimum medium, called M-medium, based upon the medium of Bocquet (1988), was also used. This medium contained @g/l): o-biotin, 2; folic acid, 2; p-aminobenzoic acid, ZOO; riboflavin, 200; niacin, 400; o-pantothenic acid (calcium salt), 400; pyridoxine hydrochloride, 400; thiamine hydrochloride, 400; boric acid, 500; Z&O,.7 H,O, 500; Mn SO,.H,O, 500; Cu SO,.5 H,O, 200; KI, 200; and sodium molybdate, 200; and (g/l): adenine, 0.02; guanine, 0.02; cytosine, 0.02; L-phenylalanine, 0.2; L-serine, 0.2; L-threonine, 0.2; L-valine, 0.2; L-alanine, 0.2; L-aspartic acid, 0.2; r.-glutamine, 0.2; L-leucine, 0.2; L-arginine hydrochloride, 0.4; L-cysteine, 0.4; glycine, 0.4; L-proline, 0.4; L-tyrosine, 0.4; KH,PO,, I g K,HPO,, 5; NH,Cl, 5; (NH&SO,, 5; and sodium acetate.3 H,O, 12. Distilled water was added to a final volume of 950 ml and the mixture was stirred until complete solubilization. The solution was adjusted to pH 6.5 and sterilized through a 0.22~/*m pore cellulose acetate membrane (Millipore). The energy source [O.l% (v/v) glucose (MG), polygalacturonic acid (pH 6.5) (MP), apple (MA) or lemon pectin (ML)] was added just before use. A single colony was used to inoculate solid media. Liquid media were inoculated with I% (v/v) of a preculture grown in the same medium. Cultures were grown at 30°C on solid or liquid medium without shaking. Incubation varied from 24 to 48 h for MRS and MI7 cultures to 8 to 10 days for M-media cultures. Growth was routinely estimated by measuring A,,,. Spread-plate counting procedures were also used with MG, MP, MA and ML. Counts were made in triplicate. Plates were incubated aerobically at 30°C for 48 h. Pectinolytic enzymes were measured by the increase in reducing groups according to a modification (Milner & Avigad 1967) of the Nelson (1944) method. Cells were removed by centrifugation (5 min at 10,000 x g) and A,,, measured in triplicate using 300 ,~l supernatant. A calibration curve was made from a stock solution of 0.01 M galacturonic acid in 0.05% (w/v) benzoic acid solution. Vistlaliurfion of Pectinolytic Acfivify on Solid Media MRS or Ml7 agars, containing sugar and supplemented with 0.1 to 1% (w/w) apple or lemon pectin as described by McKay (1988), were inoculated with either bacterial cultures or 50 to 100~1 culture supematants (20 min at 11,500 x g and 4°C) of liquid media. In a few experiments, 2 ml supematant were treated with 3 vol. cold acetone according to Gainvors et al. (1994) and centrifuged (10 min at 11,500 x g and 4°C). The precipitated protein pellet was resuspended in 100 ,~l 0.1 M acetate buffer, pH 4.0, and 50 to 100 ,ul were deposited on solid medium. Plates were incubated at 30°C for 3 to 5 days before staining with 0.02% (w/v) Ruthenium Red solution. Excess stain was removed with distilled water. A dark-red halo surrounding the colony indicated PG activity. Ulfrafilfrafion One litre of supematant (20 min at 11,500 x g; and 4°C) from a 3-day culture grown in MRS or Ml7 containing sugar, with or without 0.1% (w/v) pectin, was subjected to tangential ultrafihration using a 100-kDa cut-off polysulphone membrane. The perme-
560
World ]oaml ofA4icrobiology 6 Biotechnology. Vof I?, 1995
ate (800 ml) was subjected to a second ultrafiltration kDa cut-off polysulphone membrane. This retentate called the ‘30 to 100 kDa’ cut.
using a 30. (50 ml) was
Zymograms Samples obtained by tangential ultrafiltration and precipitated protein pellets obtained as described above were analysed by electrophoresis under non-denaturating conditions on 10% polyacrylamide gels containing 0.1 to 0.5% (w/v) pectins as described by Cruikshank & Wade (1980). Migration was at 4°C and 150 V. Gels were incubated in 200 ml 0.1 M malic acid for 1 h, followed by incubation for 18 h at 30°C in 200 ml 0.1 M acetate buffer, pH 4.0, before staining with 0.02% (w/v) Ruthenium Red and washing with distilled water. Under these conditions, PG activity was indicated by clear zones and PE activity by dark-red zones. PG and PE colorations were stable for many days. PL activity, in contrast, appeared as light yellow zones which faded within a few min of incubation (Cruikshank & Wade 1980). Isoelectric Focusing Fractions obtained by tangential ultrafiltration were subjected to isoelectric focusing on polyacrylamide gels in the presence of 0.5% ampholines, pH 3.0 to 10.0 (Servalyt Precotes 3-10; Serva). Migration was carried out at 4°C and 3 W for 6 h. The gel was then cut into two parts. One, containing PI-standards (Pharmacia), was stained with Coomassie Blue according to Sambrook et al. (1989) and the other was covered with a 0.7% agarose containing 0.5 (w/v) apple pectin and incubated at 30°C for 18 to 24 h in 25 ml of 0.1 M acetate buffer, pH 4.0. The isoelectric focusing gel was subsequently stained and washed as described for the
zymogram. Clarification Assays Clarification was examined in a turbid solution called C-medium, containing 1% (w/v) apple pectin and 0.04% (w/v) BSA, adjusted to pH 4.0 with 0.01 M HCI. This C-medium, 5 ml, was inoculated with 50~1 of the ‘30 to 100 kDa’ cut from strain Ll-8 or strain NN105. The preparations were well mixed and incubated at room temperature for 24 h.
Results
and Discussion
Polygalacfuronase Lactic
ana’ Pectin-esferase
Acfivifies
in Strains
of
Bacteria
The reducing groups liberated during PG hydrolysis by the different strains could not be determined when bacteria were grown on MRS or MI7 medium with or without sugar and supplemented with 0.1% to 1% PG or 0.1% to 2.5% pectin. This was probably due to the presence of substances in these media which interfered with the detection process. MG, MP, MA and ML media were therefore used.
All
strains
grew
in MG,
reaching
10’
cells/ml
after
1
of incubation at 3O”C, whereas only four (Ll-8, HNKIO, Lc5 and NNOI) of the 80 strains tested grew on MP, reaching lo4 cells/ml. Figure 1 shows the number of reducing groups released by PG activity from the strains in MP after 1 week of cultivation. These four strains must be able to utilize polygalacturonic acid as sole energy source, as previously described for kzctobacillus brevis (Stamer & week
Polygalacfuronases and pectin esferases in lacfic acid bacteria showed that only strains Ll-8 and Lc5 were able to grow on this medium, reaching lo4 cells/ml at the end of incubation. As these two strains released reducing groups (i.e. products of PG activity) from highly methylated pectin (Figure I) they must have PE activity, which acts on and permits subsequent degradation of methylated pectin. The other strains tested (HNKIO, NNOl and NN105) were unable to grow on MA medium. Similar results were also obtained in ML.
HNKlO
Ll-8
NNOI
Strain
Figure 1. Reducing groups liberated by pectinolytic activity. ) medium for 1 week at 3O”C, Growth was in MP (ig:. ) or MA ( reaching lo4 cellsjml. Reducing groups were measured in 300~fit1 volumes of culture supernatants. Values are given for 1 ml
1
PE E
2
3
PC
Figure 2. Line drawing of pectic zymogram from lactic acid bacteria. Clear zones of the gel indicate PG activity whereas dark zones indicate PE activity. l--Strain Ll-8; 2-strain NN105; S-strain HNKlO.
Stoyla 1968), Leuconostoc mesenferoides (Juven et al. 1985) and Succharomyces cerevisiae (McKay 1990). Strain NN105, which did not grow on MP, was used as a pectinasenegative control. MA was used to check for PE activity. Experiments
Visualization of PG Activity on Solid Medium To determine whether the observed PG activity was extracellular, MRS or Ml7 agars containing the respective sugar (glucose or lactose) and 1% w/v polygalacturonic acid were used. Red halos were detected around colonies from all strains which were shown to have PG activity, indicating the presence of extracellular depolymerizing enzymes. No halo was found with the pectinase-negative strain NN105. Supernatants from cultures grown in liquid MRS or Ml7 containing sugar and supplemented with 1% (w/v) polygalacturonic acid did not generate any halos, probably because the concentration of enzyme present was too low. After concentrating the supematant by ultrafiltration, halos were detected in the ‘30 to 100 kDa’ cut from HNKlO but not in that from strain NN105. Zymogram of Pecfinases Figure 2 shows the zymogram of pectinase activity from the ‘30 to 100 kDa’ cuts of the cultures of strains HNKlO, Ll-8 and NN105. Three clear bands of PG activity are observed on the figure for strain HNKIO. No PG activity band could be detected for the pectinase-negative strain NN105. Strain Ll-8 showed faint PG activity and five dark-red bands of variable intensities. These coloured bands correspond to PE activity as described by Cruikshank & Wade (1980). The occurrence of these enzymes may explain the ability of the bacteria to utilize esterified pectins. Isoelectric Focusing and Further Defection of PG and PE Enzymes The appearance of several clear bands of PG activity on PAGE zymograms could indicate the existence of several PG enzymes with different electrophoretic properties. To check if this were so, 20 ,~l of the ‘30 to 100 kDa’ cut was analysed by isoelectric focusing (Figure 3). The cut from strain HNKlO produced two clear bands, corresponding to two PG activities, at p1 4.0 and 9.3 and one coloured band, corresponding to a PE enzyme, at p1 7.5. Strain Ll-8 gave four clear bands, corresponding to four PG activities, at pI 4.7, 5.0, 7.3 and 8.5, and two coloured bands, corresponding to two PE activities, at pI 4.5 and 8.7. Similar results have been described for Rhizoctonia sp. by Cruikshank (1990).
N.E.
Karam
and A. Belarbi
PI 8.7.
8.5-
4
PI
C
7.3 -
9.34
7.55.0 -
cl
4.7 -
sp. by Cruikshank (1990), who used zymograms as taxonomic criteria. Lactic acid bacteria contain PG, PE and perhaps PL enzymes like other bacteria such as Er. carofovora, Ps. marginalis and Ba. subtilis (Nasuno & Starr 1966a, b; Nasser ef al. 1990). It is not surprising that such bacteria have developed the enzymatic machinery that allows them to use plant pectins because plant materials are their main habitat. The present isolates were from milk and it would be of interest to check if isolates from plant material contained the same enzymes.
Acknowledgements The authors wish to thank Drs L. Legendre, for the translation, and A. Bensoltane, for critically reading the manuscript.
4.5 -
References
Flgure 3. Line drawing of result of isoelectrofocusing of PG and PE activities from (A) strain Ll-8 and (8) HNKlO. Arrows indicate PG activity (pale, faint zones of the gel) or PE activity (dark zone).
Clarification
with
fhe Pectinolytic
Enzymes
When C-medium was inoculated with the ‘30 to 100 kDa’ cut from strain LI-8, fluffy material developed at room temperature and progressively settled to the bottom of the liquid and clarification occurred. No change in the turbidity of C-medium was detected using the pectinase-negative strain NN105 or when the medium was not inoculated. These results indicate that pectinolytic enzymes are required for the clarification of the pectin-containing medium. General
Discussion
From the results of the present in-vivo and in-vitro studies, it is evident that four strains of the lactic acid bacteria tested contained pectinolytic activities. This allowed them to grow with polygalacturonic acid as sole source of energy. Strains Ll-8 and Lc5 were also capable of multiplying with only apple pectin as sole source of energy. All four strains are capable of utilizing galacturonic acid, the pectin hydrolysis product. Enzymes with different p1 exhibited similar pectinolytic activity. According to the observations by Cruikshank & Wade (1980) and Cruikshank (1990), one can provisionally conclude that these represent isoenzymes, as described for Kluyveromyces fragilis by Bamby et al. (1990) and Rhizoctonia
562
World Jmmal of Micmbiolqy
6 Biotechnology. Vol 1I. 1995
Bamby, F.M., Morpeth, F.F. & Pyle, D.L. 1990 Endopolygalacturonase production from Kluyveromyces marxiantts. I. Resolution, purification, and partial characterisation of the enzyme. Enzyme Microbiology and Technology 12, 891-897. Bocquet, J. 1988 Biotechnologie, 3rd edn. Paris: Techniques et Documentation - Lavoisier. Cruikshank, R.H. 1990 Pectic zymograms as criteria in taxonomy of Rhizoctonia. Mycologycal Research 94, 93&940. Cruikshank, R.H. & Wade, G.C. 1980 Detection of pectic enzymes in pectin-acrylamide gels. Analytical Biochemistry 107, 177-181. De Man, J.C., Rogosa, M. & Sharpe, M.E. 1960 A medium for the cultivation of lactobacilli. ]ownal of Applied Bacferiology 23, 130-135. Dellaglio, F., Vescovo, M., Morelli, L. & Torriani, S. 1984 Lactic acid bacteria in ensiled high-moisture corn grain: physiological and genetic characterization. Systematics and Applied Microbiology 5,534-544. Fuller, R. 1989 Probiotics in man and animals. ]ownal of Applied Bacteriology 66, 365-378. Gainvors, A., Frezier, V., Lemaresquier, H., Lequart, C., Aigle, M. & Belarbi, A. 1994 Detection of polygalacturonase, pectin-lyase and pectin-esterase in a Saccharomyces cereoisiae strain. Yeast 10, 1311-1319. Juven, B.J., Lindner, P. & Weisslowicz, H. 1985 Pectin degradation in plant material by Leuconostoc mesenteroides. ]oumal of Applied Bacteriology 58, 533-538. Karam, N.E. & Karam, H. 1994 Isolement et CaractCrisation de batteries lactiques d’AlgCrie. In Alimentation, Gint%que ef Santk de I’Enfant, eds Touhami, M. & Desjeux, J.F. pp. 257-264. Paris: Editions L’Harmattan. McKay, A.M. 1988 A plate assay method for the detection of fungal polygalacturonase secretion. FEMS Letlers 56, 355-358. McKay, A.M. I990 Degradation of polygalacturonic acid by Saccharomyces cereuisiae. Letters in Applied Microbiology 11, 4144.
Milner, Y. & Avigad, G. 1967 A copper reagent for the determination of hexuronic acids and certain ketohexoses. Carbohydrate Research 4, 359-361. Nasser, W., Chalet, F. & Robert-Baudouy, J. 1990 Purification and characterization of extracellular pectate lyase from Bacillus subtilis. Biochimie 72, 689-695.
Polygalacturonases Nasuno, S. 81 Starr, M.P. 1966a Polygalacturonase of Psetrdomonus margin&. Phyfopafhology 56, 1414. Nasuno, S. 81 Starr, M.P. 1966b Polygalacturonase of Envinia curofovoru. ]ourna~ of Biological Chemistry 241, 5298. Nelson, N. 1944 A photometric adaptation of the Sogomyi method for the determination of glucose. journal of Biologicul Chemistry 153,375. Pilnik, W. & Rombouts, F.M. 1985 Polysaccharides and food processing. Carbohydrate Research 142,93-105. Romero, D.A. & McKay, L.L. 1985 Isolation and plasmid characterization of a Luctobacillw species involved in the manufacture of fermented sausage. ]orrrnu~ of Food Profecfion 48, 1028-1035. Sakellaris, G., Nikolaropoulos, S. 81 Evangelopoulos, A.E. 1988 Polygalacturonase biosynthesis by Lucfobucillus phnfurwn: effect of cultural conditions on enzyme production. Journal of Applied Bucferiology 65, 397-404.
and pectin
esteruses
in lactic
acid
bacteria
Sakellaris, G., Nikolaropouios, S. & Evangelopoulos, A.E. 1989 Purification and characterization of an extracellular polygalacturonase from Lucfobucillm plunfurzm strain BAll. Journal of Applied Bacteriology 67, 77-85. Sambrook, J., Fritsch, E.F. & Maniatis, T. 1989 Molectclur Cloning: d Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Stamer, J.R. & Stoyla, B.O. 1968 Fermentation of glucuronic acid by Lucfobuciks breuis. ]ownul of Applied Bacteriology 16, 536 537. Terzaghi, B.E. & Sandine, W.E. 1975 Improved medium for lactic streptococci and their bacteriophages. Applied Microbiology 29, 807-813.
(Received
in revised
form
4 April
1995;
accepted
14 April
19%)
of Microbiology
& Biotechnology
Detection esterases N.E. Karam*
11, 559463
of polygalacturonases in lactic acid bacteria
and pectin
and A. Belarbi
Of 80 strains of lactic acid bacteria tested, only Lactobacihs cusei strains I-INK10 and Ll-8, Luctobucilfus phntutmm Lc5 and Luctococcus hctis NNOl produced polygalacturonases (EC 3.2.1.) and/or pectin-esterases (EC 3.1.X.). Crude extracellular extracts of strain LX-8 were able to clarify pectin. Key words:
Lactic acid bacteria, pectins, pectin-esterases,
polygalacturonases.
Increasing research efforts are being devoted to the study of lactic acid bacteria because of their economic importance and the crucial role they play in human and livestock health (Fuller 1989) and in the fermentation of animal (Romero & McKay 1985) and plant products (Dellaglio et al. 1984). The industrial processing of plants generally requires a pectin-degradation step. Pectins are methylated polysaccharides, constitute one of the main building blocks of plant cell walls and cause difficulties during extraction, filtration and clarification of fruit juices. Endogenous pectinases are often insufficiently active and full pectin degradation requires the exogenous addition of pectinases (Pilnik & Rombouts 1985). Pectinases include depolymerizing and demethoxylating enzymes. Depolymerizing enzymes include polygalacturonase (PG) (EC 3.2.1), which cleaves the a-1,4 glycosidic bonds between two non-methylated galacturonic acid residues, and pectin-lyase (PL) (EC 4.2.2), which catalyses a pelimination reaction between two methylated residues. Deesterifying enzymes include pectin-esterase (PE) (EC 3.1.1), which catalyses the demethoxylation of methylated pectin, producing methanol and pectin. The pectin produced can be hydrolysed by PG. Pectinolytic activities have been found in Kltryveromyces frugilis (Bamby et al. 1990), Saccburomyces cerevisiae (McKay 1988;
Gainvors
ef al.
1994),
Buciffus
1990) and in the phytopathogens Pseudomonas
marginalis
(Nasuno
subfibs Erwinia
& Starr
1966a,
(Nasser
et al.
and b). Commer-
curofovoru
The authors are with the Laboratoire de Microbiologic G&&ale et Mokulaire. Facultb des Sciences, Universitb de Reims, B.P. 347, 51062 Reims Cedex. France: fax: 26 05 3279. *Corresponding author.
@ 1995 Rapid Science
Publishers
cial pectinases are mainly from Aspergillus niger and include the three pectinase activities. Little work has been done to assess the pectinolytic capacity of lactic acid bacteria other than Lucfoobucihs plunfuram (Sakellaris et al. 1988), Luctobucihs breuis (Starrier & Stoyla 1968) and Leuconosfoc mesenferoides (luven et al. 1985). An extracellular PG from Lacfob. plunfurclm has been characterized (Sakellaris et al. 1989). The present study was on the pectinolytic activities of different strains of lactic acid bacteria, isolated from raw milk in Algeria.
Materials
and Methods
Bacterial Strains The following strains were used: citrate-utilizing Lucfococcw fucfis subsp. fucfis strains 89, B9, FS5, FS6, NS5, NS6, NS7, NS25, NS30J and NS44; non-citrate utilizing Lacfococcus fucfis ssp. fucfis BS, BSI, B82, B83, H4, NNOI, NNII, TFIS and V8; Lucfococclts hcfis ssp. cremoris HI& NN105, NSI, NSlB, NSIC, NS4, NS63 and NS63B; Lucfococctts sp. FS3, FS9, FSII, VI7 and SI20.2; Lucfobucillus phfurum B22, cha34, cha343, Lc5, TF5, TFS, V4, V82, VI9 and V21; Lucfobucillus cusei HNKIO, FL20, LI-8, Lcl, Lc3, Lc7, Lc9 and Lc12; Lucfobuciffw sufivurius B23, cha401, cha404, TFHI and VII; Lucfobuciffw buchneri cha309Y; Lucfobucilhs sp. 67, 210, TF3, TF13, cha310, cha402 and cha405; Leuconostoc fuctis chalO2, cha201 and cha403; Letlconostoc dextrunicum chalO3 and cha406; and Leuconostoc sp. SIOZ, V3, V15, cha35, cha408, cha410, cha411, cha412, cha4122 and cha4123. Ail were isolated from Algerian raw milk (Karam & Karam 1994). Three strains from the American Type Culture Collection (ATCC) were also used: Luctobucik plunturum ATCC 8014, Luctobucih sake ATCC 15521 and Luctococcus luctis ATCC 11454.
N. E. h-am
and A. Belarbi
Growth Media MRS (De Man et al. 1960) and Ml7 (Terzaghi & Sandine 1975) with or without sugar, were supplemented with 0.1 to I% (w/v) polygalacturonic acid from orange (Sigma), 0.1 to 2.5% (w/v) apple pectin (70% to 75% methylated; Fluka Biochemicals) or 0.1% to 2.5% (w/v) lemon pectin (63% to 66% methylated; Fluka Biochemicals) and adjusted to pH 6 before sterilization (lZl”C, 20 min). Agarose (0.5%) was used instead of agar to obtain solid media when pectin or polygalacturonic acid were used. A chemically defined minimum medium, called M-medium, based upon the medium of Bocquet (1988), was also used. This medium contained @g/l): o-biotin, 2; folic acid, 2; p-aminobenzoic acid, ZOO; riboflavin, 200; niacin, 400; o-pantothenic acid (calcium salt), 400; pyridoxine hydrochloride, 400; thiamine hydrochloride, 400; boric acid, 500; Z&O,.7 H,O, 500; Mn SO,.H,O, 500; Cu SO,.5 H,O, 200; KI, 200; and sodium molybdate, 200; and (g/l): adenine, 0.02; guanine, 0.02; cytosine, 0.02; L-phenylalanine, 0.2; L-serine, 0.2; L-threonine, 0.2; L-valine, 0.2; L-alanine, 0.2; L-aspartic acid, 0.2; r.-glutamine, 0.2; L-leucine, 0.2; L-arginine hydrochloride, 0.4; L-cysteine, 0.4; glycine, 0.4; L-proline, 0.4; L-tyrosine, 0.4; KH,PO,, I g K,HPO,, 5; NH,Cl, 5; (NH&SO,, 5; and sodium acetate.3 H,O, 12. Distilled water was added to a final volume of 950 ml and the mixture was stirred until complete solubilization. The solution was adjusted to pH 6.5 and sterilized through a 0.22~/*m pore cellulose acetate membrane (Millipore). The energy source [O.l% (v/v) glucose (MG), polygalacturonic acid (pH 6.5) (MP), apple (MA) or lemon pectin (ML)] was added just before use. A single colony was used to inoculate solid media. Liquid media were inoculated with I% (v/v) of a preculture grown in the same medium. Cultures were grown at 30°C on solid or liquid medium without shaking. Incubation varied from 24 to 48 h for MRS and MI7 cultures to 8 to 10 days for M-media cultures. Growth was routinely estimated by measuring A,,,. Spread-plate counting procedures were also used with MG, MP, MA and ML. Counts were made in triplicate. Plates were incubated aerobically at 30°C for 48 h. Pectinolytic enzymes were measured by the increase in reducing groups according to a modification (Milner & Avigad 1967) of the Nelson (1944) method. Cells were removed by centrifugation (5 min at 10,000 x g) and A,,, measured in triplicate using 300 ,~l supernatant. A calibration curve was made from a stock solution of 0.01 M galacturonic acid in 0.05% (w/v) benzoic acid solution. Vistlaliurfion of Pectinolytic Acfivify on Solid Media MRS or Ml7 agars, containing sugar and supplemented with 0.1 to 1% (w/w) apple or lemon pectin as described by McKay (1988), were inoculated with either bacterial cultures or 50 to 100~1 culture supematants (20 min at 11,500 x g and 4°C) of liquid media. In a few experiments, 2 ml supematant were treated with 3 vol. cold acetone according to Gainvors et al. (1994) and centrifuged (10 min at 11,500 x g and 4°C). The precipitated protein pellet was resuspended in 100 ,~l 0.1 M acetate buffer, pH 4.0, and 50 to 100 ,ul were deposited on solid medium. Plates were incubated at 30°C for 3 to 5 days before staining with 0.02% (w/v) Ruthenium Red solution. Excess stain was removed with distilled water. A dark-red halo surrounding the colony indicated PG activity. Ulfrafilfrafion One litre of supematant (20 min at 11,500 x g; and 4°C) from a 3-day culture grown in MRS or Ml7 containing sugar, with or without 0.1% (w/v) pectin, was subjected to tangential ultrafihration using a 100-kDa cut-off polysulphone membrane. The perme-
560
World ]oaml ofA4icrobiology 6 Biotechnology. Vof I?, 1995
ate (800 ml) was subjected to a second ultrafiltration kDa cut-off polysulphone membrane. This retentate called the ‘30 to 100 kDa’ cut.
using a 30. (50 ml) was
Zymograms Samples obtained by tangential ultrafiltration and precipitated protein pellets obtained as described above were analysed by electrophoresis under non-denaturating conditions on 10% polyacrylamide gels containing 0.1 to 0.5% (w/v) pectins as described by Cruikshank & Wade (1980). Migration was at 4°C and 150 V. Gels were incubated in 200 ml 0.1 M malic acid for 1 h, followed by incubation for 18 h at 30°C in 200 ml 0.1 M acetate buffer, pH 4.0, before staining with 0.02% (w/v) Ruthenium Red and washing with distilled water. Under these conditions, PG activity was indicated by clear zones and PE activity by dark-red zones. PG and PE colorations were stable for many days. PL activity, in contrast, appeared as light yellow zones which faded within a few min of incubation (Cruikshank & Wade 1980). Isoelectric Focusing Fractions obtained by tangential ultrafiltration were subjected to isoelectric focusing on polyacrylamide gels in the presence of 0.5% ampholines, pH 3.0 to 10.0 (Servalyt Precotes 3-10; Serva). Migration was carried out at 4°C and 3 W for 6 h. The gel was then cut into two parts. One, containing PI-standards (Pharmacia), was stained with Coomassie Blue according to Sambrook et al. (1989) and the other was covered with a 0.7% agarose containing 0.5 (w/v) apple pectin and incubated at 30°C for 18 to 24 h in 25 ml of 0.1 M acetate buffer, pH 4.0. The isoelectric focusing gel was subsequently stained and washed as described for the
zymogram. Clarification Assays Clarification was examined in a turbid solution called C-medium, containing 1% (w/v) apple pectin and 0.04% (w/v) BSA, adjusted to pH 4.0 with 0.01 M HCI. This C-medium, 5 ml, was inoculated with 50~1 of the ‘30 to 100 kDa’ cut from strain Ll-8 or strain NN105. The preparations were well mixed and incubated at room temperature for 24 h.
Results
and Discussion
Polygalacfuronase Lactic
ana’ Pectin-esferase
Acfivifies
in Strains
of
Bacteria
The reducing groups liberated during PG hydrolysis by the different strains could not be determined when bacteria were grown on MRS or MI7 medium with or without sugar and supplemented with 0.1% to 1% PG or 0.1% to 2.5% pectin. This was probably due to the presence of substances in these media which interfered with the detection process. MG, MP, MA and ML media were therefore used.
All
strains
grew
in MG,
reaching
10’
cells/ml
after
1
of incubation at 3O”C, whereas only four (Ll-8, HNKIO, Lc5 and NNOI) of the 80 strains tested grew on MP, reaching lo4 cells/ml. Figure 1 shows the number of reducing groups released by PG activity from the strains in MP after 1 week of cultivation. These four strains must be able to utilize polygalacturonic acid as sole energy source, as previously described for kzctobacillus brevis (Stamer & week
Polygalacfuronases and pectin esferases in lacfic acid bacteria showed that only strains Ll-8 and Lc5 were able to grow on this medium, reaching lo4 cells/ml at the end of incubation. As these two strains released reducing groups (i.e. products of PG activity) from highly methylated pectin (Figure I) they must have PE activity, which acts on and permits subsequent degradation of methylated pectin. The other strains tested (HNKIO, NNOl and NN105) were unable to grow on MA medium. Similar results were also obtained in ML.
HNKlO
Ll-8
NNOI
Strain
Figure 1. Reducing groups liberated by pectinolytic activity. ) medium for 1 week at 3O”C, Growth was in MP (ig:. ) or MA ( reaching lo4 cellsjml. Reducing groups were measured in 300~fit1 volumes of culture supernatants. Values are given for 1 ml
1
PE E
2
3
PC
Figure 2. Line drawing of pectic zymogram from lactic acid bacteria. Clear zones of the gel indicate PG activity whereas dark zones indicate PE activity. l--Strain Ll-8; 2-strain NN105; S-strain HNKlO.
Stoyla 1968), Leuconostoc mesenferoides (Juven et al. 1985) and Succharomyces cerevisiae (McKay 1990). Strain NN105, which did not grow on MP, was used as a pectinasenegative control. MA was used to check for PE activity. Experiments
Visualization of PG Activity on Solid Medium To determine whether the observed PG activity was extracellular, MRS or Ml7 agars containing the respective sugar (glucose or lactose) and 1% w/v polygalacturonic acid were used. Red halos were detected around colonies from all strains which were shown to have PG activity, indicating the presence of extracellular depolymerizing enzymes. No halo was found with the pectinase-negative strain NN105. Supernatants from cultures grown in liquid MRS or Ml7 containing sugar and supplemented with 1% (w/v) polygalacturonic acid did not generate any halos, probably because the concentration of enzyme present was too low. After concentrating the supematant by ultrafiltration, halos were detected in the ‘30 to 100 kDa’ cut from HNKlO but not in that from strain NN105. Zymogram of Pecfinases Figure 2 shows the zymogram of pectinase activity from the ‘30 to 100 kDa’ cuts of the cultures of strains HNKlO, Ll-8 and NN105. Three clear bands of PG activity are observed on the figure for strain HNKIO. No PG activity band could be detected for the pectinase-negative strain NN105. Strain Ll-8 showed faint PG activity and five dark-red bands of variable intensities. These coloured bands correspond to PE activity as described by Cruikshank & Wade (1980). The occurrence of these enzymes may explain the ability of the bacteria to utilize esterified pectins. Isoelectric Focusing and Further Defection of PG and PE Enzymes The appearance of several clear bands of PG activity on PAGE zymograms could indicate the existence of several PG enzymes with different electrophoretic properties. To check if this were so, 20 ,~l of the ‘30 to 100 kDa’ cut was analysed by isoelectric focusing (Figure 3). The cut from strain HNKlO produced two clear bands, corresponding to two PG activities, at p1 4.0 and 9.3 and one coloured band, corresponding to a PE enzyme, at p1 7.5. Strain Ll-8 gave four clear bands, corresponding to four PG activities, at pI 4.7, 5.0, 7.3 and 8.5, and two coloured bands, corresponding to two PE activities, at pI 4.5 and 8.7. Similar results have been described for Rhizoctonia sp. by Cruikshank (1990).
N.E.
Karam
and A. Belarbi
PI 8.7.
8.5-
4
PI
C
7.3 -
9.34
7.55.0 -
cl
4.7 -
sp. by Cruikshank (1990), who used zymograms as taxonomic criteria. Lactic acid bacteria contain PG, PE and perhaps PL enzymes like other bacteria such as Er. carofovora, Ps. marginalis and Ba. subtilis (Nasuno & Starr 1966a, b; Nasser ef al. 1990). It is not surprising that such bacteria have developed the enzymatic machinery that allows them to use plant pectins because plant materials are their main habitat. The present isolates were from milk and it would be of interest to check if isolates from plant material contained the same enzymes.
Acknowledgements The authors wish to thank Drs L. Legendre, for the translation, and A. Bensoltane, for critically reading the manuscript.
4.5 -
References
Flgure 3. Line drawing of result of isoelectrofocusing of PG and PE activities from (A) strain Ll-8 and (8) HNKlO. Arrows indicate PG activity (pale, faint zones of the gel) or PE activity (dark zone).
Clarification
with
fhe Pectinolytic
Enzymes
When C-medium was inoculated with the ‘30 to 100 kDa’ cut from strain LI-8, fluffy material developed at room temperature and progressively settled to the bottom of the liquid and clarification occurred. No change in the turbidity of C-medium was detected using the pectinase-negative strain NN105 or when the medium was not inoculated. These results indicate that pectinolytic enzymes are required for the clarification of the pectin-containing medium. General
Discussion
From the results of the present in-vivo and in-vitro studies, it is evident that four strains of the lactic acid bacteria tested contained pectinolytic activities. This allowed them to grow with polygalacturonic acid as sole source of energy. Strains Ll-8 and Lc5 were also capable of multiplying with only apple pectin as sole source of energy. All four strains are capable of utilizing galacturonic acid, the pectin hydrolysis product. Enzymes with different p1 exhibited similar pectinolytic activity. According to the observations by Cruikshank & Wade (1980) and Cruikshank (1990), one can provisionally conclude that these represent isoenzymes, as described for Kluyveromyces fragilis by Bamby et al. (1990) and Rhizoctonia
562
World Jmmal of Micmbiolqy
6 Biotechnology. Vol 1I. 1995
Bamby, F.M., Morpeth, F.F. & Pyle, D.L. 1990 Endopolygalacturonase production from Kluyveromyces marxiantts. I. Resolution, purification, and partial characterisation of the enzyme. Enzyme Microbiology and Technology 12, 891-897. Bocquet, J. 1988 Biotechnologie, 3rd edn. Paris: Techniques et Documentation - Lavoisier. Cruikshank, R.H. 1990 Pectic zymograms as criteria in taxonomy of Rhizoctonia. Mycologycal Research 94, 93&940. Cruikshank, R.H. & Wade, G.C. 1980 Detection of pectic enzymes in pectin-acrylamide gels. Analytical Biochemistry 107, 177-181. De Man, J.C., Rogosa, M. & Sharpe, M.E. 1960 A medium for the cultivation of lactobacilli. ]ownal of Applied Bacferiology 23, 130-135. Dellaglio, F., Vescovo, M., Morelli, L. & Torriani, S. 1984 Lactic acid bacteria in ensiled high-moisture corn grain: physiological and genetic characterization. Systematics and Applied Microbiology 5,534-544. Fuller, R. 1989 Probiotics in man and animals. ]ownal of Applied Bacteriology 66, 365-378. Gainvors, A., Frezier, V., Lemaresquier, H., Lequart, C., Aigle, M. & Belarbi, A. 1994 Detection of polygalacturonase, pectin-lyase and pectin-esterase in a Saccharomyces cereoisiae strain. Yeast 10, 1311-1319. Juven, B.J., Lindner, P. & Weisslowicz, H. 1985 Pectin degradation in plant material by Leuconostoc mesenteroides. ]oumal of Applied Bacteriology 58, 533-538. Karam, N.E. & Karam, H. 1994 Isolement et CaractCrisation de batteries lactiques d’AlgCrie. In Alimentation, Gint%que ef Santk de I’Enfant, eds Touhami, M. & Desjeux, J.F. pp. 257-264. Paris: Editions L’Harmattan. McKay, A.M. 1988 A plate assay method for the detection of fungal polygalacturonase secretion. FEMS Letlers 56, 355-358. McKay, A.M. I990 Degradation of polygalacturonic acid by Saccharomyces cereuisiae. Letters in Applied Microbiology 11, 4144.
Milner, Y. & Avigad, G. 1967 A copper reagent for the determination of hexuronic acids and certain ketohexoses. Carbohydrate Research 4, 359-361. Nasser, W., Chalet, F. & Robert-Baudouy, J. 1990 Purification and characterization of extracellular pectate lyase from Bacillus subtilis. Biochimie 72, 689-695.
Polygalacturonases Nasuno, S. 81 Starr, M.P. 1966a Polygalacturonase of Psetrdomonus margin&. Phyfopafhology 56, 1414. Nasuno, S. 81 Starr, M.P. 1966b Polygalacturonase of Envinia curofovoru. ]ourna~ of Biological Chemistry 241, 5298. Nelson, N. 1944 A photometric adaptation of the Sogomyi method for the determination of glucose. journal of Biologicul Chemistry 153,375. Pilnik, W. & Rombouts, F.M. 1985 Polysaccharides and food processing. Carbohydrate Research 142,93-105. Romero, D.A. & McKay, L.L. 1985 Isolation and plasmid characterization of a Luctobacillw species involved in the manufacture of fermented sausage. ]orrrnu~ of Food Profecfion 48, 1028-1035. Sakellaris, G., Nikolaropoulos, S. 81 Evangelopoulos, A.E. 1988 Polygalacturonase biosynthesis by Lucfobucillus phnfurwn: effect of cultural conditions on enzyme production. Journal of Applied Bucferiology 65, 397-404.
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esteruses
in lactic
acid
bacteria
Sakellaris, G., Nikolaropouios, S. & Evangelopoulos, A.E. 1989 Purification and characterization of an extracellular polygalacturonase from Lucfobucillm plunfurzm strain BAll. Journal of Applied Bacteriology 67, 77-85. Sambrook, J., Fritsch, E.F. & Maniatis, T. 1989 Molectclur Cloning: d Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Stamer, J.R. & Stoyla, B.O. 1968 Fermentation of glucuronic acid by Lucfobuciks breuis. ]ownul of Applied Bacteriology 16, 536 537. Terzaghi, B.E. & Sandine, W.E. 1975 Improved medium for lactic streptococci and their bacteriophages. Applied Microbiology 29, 807-813.
(Received
in revised
form
4 April
1995;
accepted
14 April
19%)
