Proteolytic and other hydrolytic enzyme activities in non-starter lactic acid bacteria (NSLAB) isolated from cheddar cheese manufactured in the United Kingdom

PII : S0958-6946(97)00092-7

Int. Dairy Journal 7 (1997) 763—774 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00

Proteolytic and Other Hydrolytic Enzyme Activities in Non-starter Lactic acid Bacteria (NSLAB) Isolated from Cheddar Cheese Manufactured in the United Kingdom Alan G. Williams* and Jean M. Banks Hannah Research Institute, Ayr KA6 5HL, Scotland (Received 21 August 1997; revised version accepted 13 November 1997) ABSTRACT The populations of non-starter lactic acid bacteria (NSLAB) in a selection of 15 good-quality UK-manufactured Cheddar cheeses that had been matured for 6—9 months ranged from 105 to 107 bacteria g~1. Fifteen different species of lactic acid bacteria were identified using commercially-available identification systems. The species isolated most frequently were ¸actobacillus paracasei subsp. paracasei and ¸b. plantarum; 10 other species were isolated from two or more cheeses and three species were recovered from only a single cheese. There were marked differences in the NSLAB populations of the cheeses produced by different UK manufacturers, and differences were also apparent in the populations of two cheeses produced on different occasions at the same creamery. Forty-one isolates, selected to include all the species identified and the dominant strains present in cheeses produced at several different creameries, were screened for activities of 34 proteolytic, five glycoside hydrolase and five esterolytic enzymes. All the NSLAB possessed a wide range of hydrolytic enzymes and therefore had the potential to contribute at some stage to the development of cheese flavour during the maturation and ripening period. Inter-species and strain differences in enzyme profiles and levels of activity were apparent and were determinants for the non-random selection of NSLAB for use as adjunct cultures in subsequent cheese-making trials. The breakdown of diagnostic substrates was indicative of the presence of multiple proteinase, tripeptidase, dipeptidase (including prolinase- and prolidase-like), dipeptidyl peptidase, prolyl, proline, aspartyl, pyroglutamyl (pyrrolidonecarboxyl) and general aminopeptidase activities. ( 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION

fermentative strains that occur in the later stages of ripening have been associated with the occurrence of undesirable flavours in Cheddar (Tittsler et al., 1947; Dacre, 1953), whilst certain ¸actobacillus strains adversely affected flavour formation in both Gouda and Cheddar cheeses (Kleter, 1977; Puchades et al., 1989). NSLAB have also been implicated in the calcium D-lactate surface defect (Khalid and Marth, 1990). In other cheesemaking trials, it was suggested that thermophilic lactobacilli did not influence the development of Cheddar flavour (Tittsler et al., 1947, 1948; Lloyd et al., 1980). However, in an increasing number of recent trials, the addition of selected adjunct strains of ¸actobacillus spp. has positively influenced the quality of the cheese produced (McSweeney et al., 1994; Drake et al., 1996; Lane and Fox, 1996; Lynch et al., 1996; Muir et al., 1996). Likewise, the addition of viable or attenuated adjunct ¸actobacillus cultures has been advocated to accelerate the ripening of standard and reduced-fat Cheddar cheese (Tre´panier et al., 1991a, b, 1992; El Soda, 1993; Johnson et al., 1995). In addition, adjuncts of lactobacilli may suppress the growth of unwanted bacteria in the cheese (Martley and Crow, 1993). Although the exact role of NSLAB in the development of the flavour of Cheddar cheese is incompletely understood, it appears that the potential exists to manipulate the chemical and sensory characteristics of Cheddar cheese by the use of selected cultures of ¸actobacillus as

Cheese flavour develops as a consequence of the microbial breakdown and subsequent transformation of the degradation products of the protein, fat and carbohydrate components present initially in the curd. Lactic acid bacteria play a central role in the ripening process. The bacteria may have been added deliberately to the cheese as the starter culture or may have entered adventitiously from the milk and the immediate surroundings during cheese manufacture. The starter lactococcal population declines during the maturation of Cheddar cheese and the initially small population of adventitious nonstarter lactic acid bacteria (NSLAB) ultimately becomes the dominant bacterial population in the maturing cheese (Peterson and Marshall, 1990; Martley and Crow, 1993). The dominant non-starter bacteria in Cheddar cheese are homo- and heterofermentative species of lactobacilli (Sherwood, 1939; Franklin and Sharpe, 1963; Broome et al., 1990; Jordan and Cogan, 1993), although there are reports of the occurrence of pediococci, micrococci and ¸euconostoc spp. (Bhowmik and Marth, 1990; Peterson and Marshall, 1990). The effect of NSLAB on flavour formation has yet to be satisfactorily elucidated. Hetero*Corresponding author. Tel.: 44 1292 674081. E-mail: [email protected]. 763

764

A. G. Williams, J. M. Banks

adjuncts to the ¸actococcus starter. The criteria for adjunct selection are often not defined and frequently isolates from a good-quality cheese have been selected for evaluation. There is thus a need to identify biochemical characteristics of NSLAB that could potentially contribute to the overall maturation process. The lactobacilli affect proteolysis and lipolysis during ripening (McSweeney et al., 1993; Lane and Fox, 1996), but the proteolytic and lipolytic enzyme systems of the NSLAB are less well characterized than those of the starter lactococci (Bockelmann, 1995; Kunji et al., 1996). The objectives of this investigation were the identification of the predominant NSLAB present in Cheddar cheese produced by commercial manufacturers in the UK and a detailed survey of the activity and range of proteindegrading and other hydrolytic enzymes formed. It was intended that the activity profiles would be used subsequently to aid the selection of isolates for evaluation as adjunct cultures in cheese-making trials. MATERIALS AND METHODS Isolation of NSLAB Preliminary experiments confirmed that MRS (96 h, 30°C; de Man et al., 1960) was the most appropriate medium for the cultivation and enumeration of the population of lactic acid bacteria in Cheddar cheese, whereas the NSLAB lactobacilli present could be selectively quantified and isolated on LBS agar (96 h, 30°C; Rogosa et al., 1951). A selection of 15 good-quality Cheddar cheeses, that had been matured for 6—9 months, was obtained from seven commercial producers. Cheese suspensions were prepared in maximum recovery diluent (MRD), that had been prewarmed to 45°C, by homogenization for 5 min using a stomacher. Serial dilutions of the homogenate (10 g cheese, 90 mL MRD) were prepared in MRD and plates of both LBS and MRS agar were inoculated using a spiral plater. Duplicate plates were incubated under both aerobic and anaerobic conditions for 96 h at 30°C; the anaerobic series of plates were incubated in an anaerobic cabinet (Don Whitley Scientific Ltd, Shipley) under an oxygen-free atmosphere of N : CO : H (80 : 10 : 10%, 2 2 2 v/v). The total number of colonies developing on both media after aerobic and anaerobic incubation was enumerated and the number of each morphological colony type determined for the LBS cultures. A representative of each morphological type of colony was sub-cultured to purity on MRS at 30°C. Stock cultures were stored on glass beads at !70°C with glycerol (15%, v/v) as cryoprotectant (Feltham et al., 1978) and as combined slope/ stab cultures on MRS at 4°C. Culture media and MRD were purchased from Oxoid Ltd (Basingstoke, UK) and Fisher Scientific (Loughborough, UK). Identification of NSLAB isolates Isolates recovered from each type of colony were identified to the species level by morphological characteristics, Gram reaction and by biochemical reactions using the API 50CHL system (Biomerieux, Basingstoke, UK) or Biolog GP system (Biolog Inc., Hayward, CA, USA). The proteolytic capability of the isolates was assessed qualitatively following growth on MRS—caseinate me-

dium (48 h, 30°C). Although the lactococci grew well on the caseinate agar of Martley et al. (1970), the growth of some NSLAB was poor. In view of this, proteolysis was assessed on MRS medium modified by the inclusion of sodium caseinate (1%, w/v), tri-sodium citrate (15 mM) and 20 mM CaCl . 2 Preparation of cell lysates The NSLAB isolates included in the enzyme-screening experiments were selected so that the numerically dominant strains present in cheeses produced by all of the manufacturers, at as many creameries as possible, were monitored. At least one isolate of each of the ¸actobacillus species identified in the cheese survey was included in the screening procedure. The isolates were grown for 48 h at 30°C in MRS broth containing 2% (w/v) glucose. The cells were recovered by centrifugation (16,000g, 30 min, 4°C) and washed by resuspension in pre-cooled (4°C) 0.1 M 2-(N-morpholino)ethanesulphonic acid (MES) buffer (pH 7.0). After recovery by centrifugation, the washed cells were resuspended in 0.1 M MES buffer (pH 7.0) and disrupted by ultrasonic treatment at 4°C using an MSE Soniprep 150 ultrasonic disintegrator fitted with a small probe (tip diameter, 9.5 mm) at an amplitude level of 10 km for ten 30 s periods, with 30 s intervals. The supernatant fraction of the ultrasonicate after centrifugation (10,000 g, 15 min, 4°C) was not purified further and represented the crude cell lysate fraction. Aliquots (500 kL) of the lysate were stored at !20°C prior to assay. Enzyme assay procedures Proteolytic enzyme activities (endopeptidases, peptidases, aminopeptidases) were determined using synthetic p-nitroanilide substrates. The amino- and carboxytermini are both blocked in endopeptidase substrates, whereas aminopeptidase substrates had a free aminoterminus (Sarath et al., 1990). The assays were performed in 96-well plates. The reaction mixture (0.15 mL final volume) contained 0.1 M MES buffer (pH 7.0) and the appropriate p-nitroanilide derivative (0.5 mM); the reaction was started by addition of the enzyme preparation (0.02 mL). The p-nitroaniline released after incubation for a maximum period of 60 min at 30°C was determined spectrophotometrically at 405 nm using known standards. The sensitivity of p-nitroaniline detection was increased by diazotisation of the assay supernatant and colorimetric determination with N1-naphthylethylenediamine dihydrochloride (Appel, 1974). Specific activities are expressed as nmol p-nitroaniline released per mg protein in 1 min. Certain di-, tri- and aminopeptidase activities in the crude lysates were assayed using peptide substrates. The reaction mixture (0.5 mL) contained 0.1 M MES buffer (pH 7.0), peptide substrate (1 mM) and enzyme (0.03 mL); the reaction was started by addition of the enzyme preparation. The amino acid released after incubation at 30°C was determined colorimetrically using the modified Cd—ninhydrin method of Doi et al. (1981). Specific activities are given as nmol amino acid released per mg protein in 1 min. Glycoside hydrolase and esterase activities were determined by measuring the rate of p-nitrophenol release from the appropriate p-nitrophenyl derivative (1 mM)

765

Hydrolytic enzymes of NSLAB

after incubation at pH 7 in 0.1 M MES and 30°C for a maximum period of 60 min. After incubation, the p-nitrophenol released by the crude enzyme (0.02 mL) was determined spectrophotometrically at 420 nm; the colour was enhanced by the addition of 0.5 M glycine buffer (pH 9.0, 0.1 mL) to the incubation supernatant (0.15 mL). Specific activities are expressed as nmol p-nitrophenol released mg~1 protein min~1. Substrate-free incubations and controls using heatinactivated cell lysates (100°C, 10 min) were conducted for all assays to quantify non-specific colour formation and non-enzymic substrate breakdown during the reaction period. The suppliers of the substrates used were Sigma Chemical Co. Ltd (Poole, UK), Novabiochem (Nottingham, UK) and Bachem UK Ltd (Saffron Walden, UK). The protein content of the cell lysates was measured by the dye-binding method of Bradford (1976). RESULTS NSLAB populations present in Cheddar cheese Characterisation of NSLAB populations in creamerymanufactured cheese was undertaken using a selection of 15 good-quality Cheddar cheeses from seven commercial producers in the UK. The cheeses had been matured for 6—9 months at the time of microbiological analysis. Populations were monitored after growth on LBS and MRS media at 30°C under both aerobic and anaerobic conditions. Representative colonies of the various morphological forms present on both media after growth under aerobic and anaerobic conditions were selected for identification; as the starter culture grew on the MRS plates, only Gram-positive rods were selected for identification.

The NSLAB population in the cheeses examined ranged from 105 to 107 cfu g~1 after incubation for 96 h under either aerobic or anaerobic conditions on LBS agar (Table 1). The NSLAB populations in Cheddar cheese were dominated by mesophilic ¸actobacillus spp. Thirteen different species of ¸actobacillus and two ¼eissella were represented amongst the 143 isolates recovered from all growth conditions (Table 1). Using the BioMerieux API 50CHL system, it was possible to assign only 75 of the isolates to the species level; a further 45 isolates were identified to species using the Biolog GP system for lactic acid bacteria. Of the 23 unassigned isolates, 13 were identified as ¸actobacillus spp., but 10 could not be identified satisfactorily to the genus level using either of the commercially available identification systems. The unidentified Gram-positive isolates were recovered from cheeses D, I, J, K and L after both aerobic and anaerobic incubation; eight were recovered from MRS plates. The NSLAB that occurred at the highest frequency were ¸b. paracasei subsp. paracasei and ¸b. plantarum which were isolated from 13 and 10 of the cheeses examined, respectively. ¸actobacillus curvatus and ¸b. casei were recovered from four cheeses, six species (¸b. brevis, ¸b. helveticus, ¸b. fermentum, ¸b. bifermentans, ¸b. buchneri, ¸b. parabuchneri) occurred in at least three cheeses, ¸b. farciminis and ¸b. kefir were isolated twice and three species were isolated only once (Table 1). The number of different species identified in an individual cheese ranged from 2 to 7 (average 3.7$1.5). The predominant species in individual cheeses varied. ¸b. paracasei subsp. paracasei was the dominant NSLAB in seven of the cheeses, but in the remaining eight cheeses different NSLAB predominated. It was apparent that there were marked differences in the NSLAB populations of the cheeses produced by the different UK manufacturers. Differences

Table 1. NSLAB Populations in Cheddar Cheese Produced in the UK Producer

1 2

3 4 5 6 7

Cheese code

AC C! D! A O E F G N H M I J K L

NSLAB population" Anaerobic

Aerobic

4.4 0.2 0.7 2.8 40 15 22 1.0 0.8 13 39 26 13 33 30

1.8 0.2 0.4 2.4 7.9 0.5 16 1.3 0.4 8.4 18 9.7 12 9.7 34

NSLAB assigned#

¸actobacillus species identified$

80 100 90 87 67 100 89 83 80 60 70 60 40 50 78

pcas, pla, sp. cur, far, kef *, pcas, pla* cas, cur*, pcas, vir, sp. bre, cur, fer, pcas, pla, sp. cas, fer, pcas, pla, sp. pcas, pla far, hal, pcas, pla, sp. cas, fer, pcas, pla, sp. hel, pla*, sp. pcas, sp. bif, bre, buc, pbuc, pcas, sp. cas, hel, pcas, sp. bif *, hel, pcas, pla*, sp. bre, buc, cur, pbuc*, sp. bif *, buc, col, kef, pbuc, pcas, pla, sp.

(10)

(25) (40) (20) (8)

! Produced at same creamery. " cfu]106 g~1. # percentage of representative NSLAB isolates selected from an individual cheese that were assigned to the species level. Percentage of isolates that could not be assigned to genus given in parentheses. $ NSLAB species identified that were present at'105 cfu g~1 or *(105 cfu g~1 cheese are represented as: bif, ¸b. bifermentans; bre, ¸b. brevis; buc, ¸b. buchneri; cas, ¸b. casei; col, ¸b. collinoides; cur, ¸b. curvatus; far, ¸b. farciminis; fer, ¸b. fermentum; hal, ¼eissella (syn ¸b.) halotolerans; hel, ¸b. helveticus; kef, ¸b. kefir; pbuc, ¸b. parabuchneri; pcas, ¸b. paracasei subsp. paracasei; pla, ¸b. plantarum; vir, ¼eissella (syn ¸b.) viridescens; sp., unidentified ¸actobacillus sp.

766

A. G. Williams, J. M. Banks

were also apparent in the NSLAB populations of the two cheeses (C and D) produced in different production runs at the same creamery (Table 1). Hydrolytic enzyme activities of NSLAB isolated from Cheddar cheese Forty-one isolates representing 15 species of ¸actobacillus and ¼eisella identified in the NSLAB populations were screened for activities of 34 proteolytic enzymes (10 endopeptidases, 15 aminopeptidases, nine di-, tri- and dipeptidyl peptidases), five glycoside hydrolases and five esterases. The isolates studied were dominant in the NSLAB populations and were recovered from cheeses produced at all of the creameries identified as manufacture sites by the producers involved; the identity of the creamery responsible for the manufacture of the individual cheeses was revealed for only seven cheeses (A, C, D, AC, I, J and O). The enzyme activity profiles of the NSLAB isolates are summarized in the following sections. Bacterial cultivation, enzyme preparation and assay methods for the survey were optimised in a series of preliminary experiments. Proteinase Approximately half of the isolates selected (20 of 41) were weakly proteolytic on caseinate agar. However, endopeptidase activity was detectable in all of the isolates surveyed although there were pronounced inter- and intra-species (strain) variations in the range and level of activity (Table 2). Of the 41 isolates examined, only ¸b. brevis M2 had detectable activity against all 10 proteinase substrates. In some isolates, activity was low or undetectable on the substrates used and these NSLAB are not included in Table 2. For example, lysates of

¸b. fermentum AR11, ¸b. collinoides L8 and ¼. viridescens D5 had low activity in the range 0.02—0.06 nmol p-nitroaniline release mg~1 protein min~1 against only two of the substrates tested while detectable activity was present against only Abz Ala Ala Phe Phe in the lysate of ¸b. fermentum G9. Although ¸b. buchneri K10 and ¸b. parabuchneri strains L2 and M12 had a similarly restricted substrate range (Bz Tyr, Abz Ala Ala Phe Phe), the specific activities were marginally higher (0.13—0.35 nmol mg~1 protein min~1); activity was also present in the lysates of K10 and M12 against Suc Phe Pro Phe and Bz Pro Phe Arg, respectively. The proteinase profiles of isolates within an NSLAB species varied considerably. Thus, although seven of the 9 ¸b. paracasei subsp. paracasei isolates possessed activities against at least seven substrates (range 4—8), six different activity profiles were detected; only four of the isolates had the same proteinase profiles. Two of nine ¸b. plantarum strains had the same profile; these intra-species differences in proteinase profiles were apparent in other NSLAB species, although only 2—4 strains were examined. Likewise, there was considerable inter-strain variation in the specific activity of the detected enzymes (Table 2). The synthetic p-nitroanilide substrates are potentially diagnostic for different classes of proteinases (Dunn, 1990). The metalloprotease substrate Abz Ala Ala Phe Phe was degraded by some isolates of all of the NSLAB species examined; activity was undetectable in lysates of only three strains (Table 2). Activity against the cysteine protease substrate, Bz Pro Phe Arg, was less widely distributed whereas activities '1.0 nmol mg~1 min~1 were measured in some lysates with serine protease substrates (Table 2). However, other derivatives for serine proteases were not as effective as substrates. Activity against Suc Ala was detectable in eight of the 3

Table 2. Proteinase Activities! in NSLAB Isolated from Cheddar Cheese Strains tested

Cbz Ala

Cbz Lys

Bz Phe Val Arg"

Bz Tyr"

Suc Phe"

9

ND(1)—0.30 0.18 ND

ND%

ND(4)—0.21 0.055 ND(4)—0.93 0.087 ND(2)—1.91 0.52 0.20—0.27 0.23 ND—0.28 0.14 0.10—0.61 0.35 0.41—2.34 1.37 0.63 0.16 0.51

ND(3)—0.19 0.054 ND(6)—1.58 0.22 ND(1)—0.93 0.29 0.02—0.43 0.22 0.065—0.10 0.082 ND—0.62 0.31 0.19—0.32 0.25 0.05 0.36 0.070

0.04—1.01 ND(1)—0.16 0.56 0.089 ND ND(1)—1.38 0.35 ND ND(1)—0.60 0.21 ND 0.01—0.07 0.038 ND—2.78 0.17—0.69 1.39 0.43 0.04—2.16 0.31—0.72 1.10 0.51 ND—0.02 0.30—0.98 0.01 0.64 0.01 0.11 0.04 0.16 ND 0.22

¸b. paracasei subsp. paracasei ¸b. plantarum

9

¸b. curvatus

4

¸b. casei

2

¸b. helveticus

2

¸b. farciminis

2

¸b. kefir

2

ND—0.63 0.31 ND—1.11 0.55 ND

¸b. bifermentans ¸b. brevis ¼. halotolerans

1 1 1

ND 0.13 ND

ND(3)—0.15 0.038 ND

ND ND(3)—0.08 0.021 ND—0.03 0.017 ND ND—0.85 0.42 ND—0.07 0.035 0.030 0.13 0.030

Abz Ala Ala Bz Pro Phe Arg$ Phe Phe# ND(1)—0.18 0.088 ND(6)—0.20 0.028 ND(1)—1.41 0.50 0.13—0.17 0.15 0.38—0.83 0.60 0.01—0.07 0.04 ND—0.03 0.015 ND 0.46 0.22

! Mean specific activities (nmol p-nitroaniline formed mg~1 protein min~1 and range of activities for number of strains indicated. The blocking groups are Abz, aminobenzoyl; Bz, benzyl; Cbz, benzyloxycarbonyl; Suc, succinyl. The protease substrate specificities are " serine (chymotrypsin). # metalloprotease. $ cysteine protease. % ND, no detectable activity; the number of strains in which activity was not detected is indicated in parentheses where the number of strains examined was '2.

2

2

2

2

2

2

2

1 1 1 1 1

¸b. casei

¸b. helveticus

¸b. buchneri

¸b. parabuchneri

¸b. farciminis

¸b. fermentum

¸b. kefir

¸b. bifermentans ¸b. brevis ¸b. collinoides ¼. halotolerans ¼. viridescens

0.64—4.37 1.96 0.27—1.99 0.94 ND—2.39 0.90 0.01—1.05 0.53 2.25—5.72 3.98 ND—0.05 0.02 ND—0.42 0.21 1.72—2.68 2.20 0.53—1.17 0.85 0.01—1.94 0.92 0.64 0.27 0.14 2.00 2.48

Ala pNA

0.54—5.67 2.06 1.27—9.32 4.02 0.08—9.60 2.58 0.17—3.60 1.88 1.32—5.40 3.36 0.92—2.60 1.76 0.04—0.31 0.17 1.34—16.11 8.72 1.38—3.22 2.30 0.21—10.49 5.35 1.96 0.35 0.50 6.04 0.73

Arg pNA

ND(1)—2.1 0.93 ND(2)—18.1 4.7 ND(1)—1.2 0.64 ND—85.1 42.5 ND—1.4 0.70 2.1—4.1 3.1 2.4—4.0 3.2 2.5—83.3 42.9 2.6—5.2 3.9 0.9—4.7 2.8 11.0 7.5 11.3 0.14 ND

Asp pNA"

ND(1)—1.73 0.93 0.09—2.69 0.74 ND(2)—1.69 0.48 ND—0.08 0.04 1.42—8.29 4.85 0.16—0.35 0.25 0.31—0.64 0.47 0.4—2.34 1.37 0.35—1.06 0.70 0.11—4.43 2.27 0.43 0.60 0.02 0.75 0.27

Benzyl Cys pNA

0.05—2.63 1.34 0.03—0.13 0.08 ND—0.57 0.28 0.11 0.13 ND 0.42 0.09

ND(1)$—0.15 0.07 ND(6)—0.16 0.04 ND(1)—0.44 0.21 ND—0.11 0.05 0.45—0.51 0.48 0.03—0.26 0.14 ND

Gly pNA

0.03—6.04 1.91 0.60—2.58 1.49 0.14—5.62 2.13 0.15—3.13 1.64 0.05—5.30 2.90 0.21—0.31 0.26 0.08—1.64 0.86 4.14—13.67 8.90 1.27—2.02 1.64 0.09—6.60 3.34 1.21 0.32 0.45 5.16 0.10

Leu pNA

1.39—8.47 2.86 1.23—9.56 3.54 0.07—3.73 1.40 0.04—0.14 0.09 1.45—5.29 3.37 1.40—2.02 1.71 ND—0.46 0.23 1.53—15.64 8.58 2.37—3.10 2.73 0.24—11.15 5.69 2.14 3.32 0.32 5.09 0.72

Lys pNA

0.62—2.62 1.25 0.23—1.87 0.74 0.01—2.40 0.91 0.06—2.43 1.24 1.88—7.65 4.76 ND—0.10 0.05 ND—0.33 0.15 1.67—4.76 3.21 0.43—0.79 0.61 0.06—2.22 1.14 0.37 0.20 0.09 2.09 2.13

Met pNA

Aminopeptidase substrate

0.56—2.99 1.20 0.16—2.60 1.25 ND(1)—2.16 0.72 0.06—0.18 0.12 3.16—3.71 3.43 0.10—0.28 0.19 ND—0.45 0.22 0.79—11.53 6.16 0.79—1.59 1.19 0.31—4.46 2.38 0.36 1.38 0.02 3.96 0.34

Phe pNA

ND—0.05 0.02 0.56—0.96 0.76 0.02—0.03 0.025 ND—0.14 0.07 (0.01 1.11 (0.01 0.20 0.35

0.06—0.32 0.16 ND(7)—0.16 0.03 ND(2)—1.12 0.36 ND—0.20 0.10 0.57—0.75 0.66 ND

Pro pNA

ND(3)—0.06 0.03 ND(8)—0.14 0.015 ND(2)—0.48 0.12 ND—0.10 (0.01 0.15—0.31 0.23 0.09—0.25 0.17 ND—0.02 0.01 0.22—0.27 0.24 ND—0.01 (0.01 0.10—0.54 0.32 0.10 0.51 0.03 0.17 0.06

Pyroglut pNA

1.06—7.62 2.49 ND(1)—2.00 0.50 0.01—3.69 1.18 ND—0.64 0.32 1.84—3.47 2.65 ND—0.12 0.06 ND—0.04 0.02 0.44—4.90 2.67 0.38—0.39 0.38 0.01—1.05 0.53 0.04 0.16 0.06 0.81 0.69

Tyr pNA

0.32—2.23 1.32 ND(1)—2.02 0.58 ND(1)—1.28 0.58 0.01—0.28 0.14 1.53—3.72 2.62 ND—0.07 0.03 (0.01—0.01 (0.01 0.80—3.80 2.30 0.53—0.89 0.71 (0.01—2.72 1.36 0.28 0.13 0.03 1.78 0.65

Val pNA

ND—5.0 2.5 ND ND ND 8.1 ND

ND(1)—12.2 4.8 ND(1)—9.8 4.6 ND(1)—4.85 2.7 ND—3.8 1.9 ND—2.2 1.1 5.5—15.81 10.6 ND—11.0 5.5 ND—14.1 7.0 ND

Bradykinin (fragment 1—5)#

! Mean specific activities expressed as nmol p-nitroaniline released mg~1 protein min~1 or " in 15 h, or # nmol amino acid as arginine released mg~1 protein min~1 from bradykinin (1—5) and range of activities for the number of strains indicated. The aspartyl (PepA-like) and PepP activities of ¸b. curvatus and ¸b. plantarum/¸b. paracasei were determined with three and 4 strains, respectively. ND, no detectable activity; the number of strains having no detectable activity is indicated in parentheses (when more than two strains were examined).

ND—0.01 0.01 ND 0.05 ND (0.01 ND

ND—0.01 (0.01 ND

ND

ND—0.45 0.22 ND

ND(3)—0.05 0.01 ND

4

¸b. curvatus

Acetyl Ala

ND(4)—0.16 0.05 ND

Strains tested

¸b. paracasei 9 subsp. paracasei ¸b. plantarum 9

NSLAB

Table 3. Aminopeptidase Activities! in NSLAB Isolated from Cheddar Cheese

Hydrolytic enzymes of NSLAB 767

768

A. G. Williams, J. M. Banks

¸b. paracasei subsp. paracasei lysates and in one strain of each of ¸b. plantarum, ¸b. curvatus and ¸b. helveticus; the specific activity in ¸b. helveticus N6 was highest at 0.28 nmol mg~1 protein min~1. Specific activities '0.1 nmol mg~1 protein min~1 were measured in two strains of ¸b. plantarum and ¸b. helveticus and in single isolates of ¸b. paracasei subsp. paracasei, ¸b. buchneri and ¸b. farciminis using the serine protease substrate, Suc Phe Pro Phe. Activity on Cbz Arg was detected in only four isolates and ranged from 0.01—0.22 nmol mg~1 protein min~1 being highest in ¸b. brevis M2.

¸b. paracasei subsp. paracasei isolates measured with the dialanine and trialanine p-nitroanilide derivatives were 1.14 (0.67—1.48) and 1.06 (0.03—1.85) nmol p-nitroaniline formed mg~1 protein min~1. Dipeptidyl peptidase activities were also present in many of the NSLAB isolates and substrates (Gly Phe p-nitroanilide and Gly Pro p-nitroanilide), indicators of the presence of type I and type IV activities (McDonald and Barrett, 1986) were degraded (Table 4). There were again marked strain and inter-species variations in enzyme activity.

Aminopeptidase

Esterase

A wide range of aminopeptidase activities was detected in the NSLAB isolates using synthetic p-nitroanilide derivatives as substrates (Table 3). There were marked intra-species (strain) differences in the levels of activity and although high-activity strains were detected in which the specific activities of the individual aminopeptidases were consistently higher than the levels detected in other strains of the species (e.g. ¸b. curvatus C8, ¸b. farciminis F4, ¸b. helveticus N6, ¸b. kefir C13, ¸b. paracasei subsp. paracasei H6), in other NSLAB species the highest activities of all the aminopeptidases were not located in a single strain. For example, the highest levels of at least three different aminopeptidase activities occurred in three of the nine strains of ¸b. plantarum monitored. The aminopeptidase profiles of the different NSLAB species isolated from Cheddar cheese were similar; the interspecies differences in the level of aminopeptidase activity and the intra-species range of specific activity were also similar (Table 3). In many of the NSLAB isolates, the highest aminopeptidase activity was against substrates containing a basic amino acid (lysine, arginine), but activity was present in most isolates to facilitate the cleavage of the terminal amino acid irrespective of its type. Thus, the NSLAB also had the enzymic capability to release aliphatic, acidic, aromatic, heterocyclic and sulphur-containing amino acids (Table 3). Using the assay conditions described, the presence of prolyl aminopeptidase and proline aminopeptidase (aminopeptidase P-like) activities were detected in many isolates using the diagnostic substrates proline p-nitroanilide and the N-terminal pentapeptide of bradykinin (bradykinin fragment 1—5), respectively (Table 3). The diagnostic substrate for pyroglutamyl aminopeptidase (PCP-like) activity was also degraded by lysates of many of the NSLAB. Significant acylaminoacyl peptidase activity was detected in only six species of NSLAB (10 isolates). Carboxypetidase activity was not detectable in any of the lysates, that had been stored at !20°C, using N-(3-[2-furyl]acryloyl) derivatives of AlaLys, Gly-Gly, Gly-Leu or Phe-Phe.

Esterase activities were detected in most of the NSLAB isolates examined using synthetic p-nitrophenyl substrates (Table 5). Inter-species and strain variations in activity occurred. Specific activities were lower when the carbon chain length of the esterified fatty acid was increased. Highest activities were measured using esters of acetate, butyrate and caproate (C2—C6), although differences were apparent in substrate specificities. Thus, in most strains of ¸b. paracasei subsp. paracasei (8 of 9), ¸b. curvatus (3 of 4), ¸b. brevis and ¼. halotolerans, the specific activity was highest on the caproate ester, for ¸b. casei and ¸b. fermentum the preferred substrate was the butyrate ester, whereas higher activities were detected with the acetate ester in lysates of ¸b. plantarum (5 of 9), ¸b. buchneri, ¸b. parabuchneri, ¸b. farciminis, ¸b. bifermentans and ¸b. kefir. The NSLAB isolates that had higher esterase activities also tended to have higher peptidolytic activities as well.

Peptidase The presence of dipeptidase and tripeptidase activities in the lysates of all of the NSLAB examined was confirmed using dialanine and trialanine, respectively, as substrates (Table 4). The dipeptides, Pro-Leu and LeuPro, diagnostic substrates for prolinase and prolidase, were also degraded by most of the isolates. Dipeptidase and tripeptidase activities were also detected using synthetic p-nitroanilide derivatives. The mean specific activities of the di- and tripeptidase activities of the 9

Glycoside hydrolase The i-casein component of the casein molecule is glycosylated; these carbohydrate structures may impede proteolysis and the NSLAB lysates were monitored for glycoside hydrolase (glycosidase) activities that could cleave the carbohydrate moieties from the protein structure. b-Galactosidase activity was detectable in lysates of 37 NSLAB isolates and was particularly active in some of the strains (e.g. ¸b. kefir C13, L12, ¸b. parabuchneri M12, ¸b. curvatus D7, ¸b. plantarum G2) where activities 530 nmol mg~1 protein min~1 were detected (Table 6). a-Galactosidase activities were, however, lower and less widely distributed among the isolates; activity was detectable in only 19 of the isolates (Table 5). a- and b-Galactosaminidase activities were detected in only 13 and 14 of the lysates, respectively, and were generally low (Table 6), while N-acetyl-a-D-neuraminidase activity was present in single strains of ¸b. buchneri and ¸b. helveticus (specific activity (0.1 nmol p-nitrophenol released mg~1 protein min~1) and all nine isolates of ¸b. paracasei subsp. paracasei examined. In the latter species, the specific activity ranged from 0.08 to 2.0 (average 0.42) nmol mg~1 protein min~1. Strains of ¸b. paracasei subsp. paracasei may have a role in the cleavage of b-galactosamine and neuraminic acid moieties from the glycosylated casein molecule. DISCUSSION The predominant non-starter lactic acid bacteria present in the 15 UK-produced Cheddar cheeses that were

9

9

4

2

2

2

2

2

2

2

1 1 1 1 1

¸b. paracasei subsp. paracasei ¸b. plantarum

¸b. curvatus

¸b. casei

¸b. helveticus

¸b. buchneri

¸b. parabuchneri

¸b. farciminis

¸b. fermentum

¸b. kefir

¸b. bifermentans ¸b. brevis ¸b. collinoides ¼. halotolerans ¼. viridescens

36.3—211 120 5.0—228 136 55.8—72.9 64.2 68.8—244 156 100—102 101 35.7—51.4 43.6 44.8—101 72.8 20—118 69.0 70.1—106 88.0 59.4—246 153 142 79.6 47.2 29.5 169

Ala Ala Ala"

198—262 218 57.4—266 160 26.3—138 78.2 125—197 161 130—201 165 34.5—54.9 44.7 9.0—54.1 31.5 129—265 197 52.1—109 80.5 39.8—172 106 112 62.4 26.5 47.4 139

Ala Ala"

Tripeptidase

9.4—48.3 25.5 1.7—46.6 18.4 ND(1)—16.2 9.2 8.8—39.4 24.1 11.6—28.2 19.9 4.9—10.7 7.8 2.0—27.5 14.7 ND—82.8 41.4 2.6—5.6 4.1 8.7—23.7 16.2 27.6 9.3 10.6 15.4 28.8

Prolinase# 18.9—43.3 33.7 0.8—28.2 8.2 ND(1)—17.9 9.4 1.2—5.6 3.4 18.3—20.3 19.3 3.2—3.9 3.5 3.2—9.2 6.2 0.2—509 254 11.8—24.3 18.0 ND—0.7 0.35 ND 14.0 15.6 15.0 9.5

Prolidase#

Dipeptidase

0.01—1.11 0.29 ND(4)$—0.34 0.14 ND(1)—0.57 0.33 0.16—0.63 0.39 0.67—0.88 0.77 0.17—0.22 0.19 ND—0.32 0.16 0.62—1.24 0.93 0.21—0.25 0.23 0.71—1.11 0.91 0.61 0.21 ND 0.47 0.69

Gly Arg pNA

0.96—3.54 2.25 0.11—0.19 0.15 0.15—1.27 0.71 (0.01 0.02 ND 0.27 0.36

0.09—1.29 0.95 ND(2)—2.63 0.74 ND(2)—0.95 0.32 (0.01—0.02 0.010 0.67—4.39 2.53 0.02—0.08 0.052 ND

Gly Phe pNA 0.42—4.20 1.59 ND(4)—5.79 1.41 0.92—6.56 3.73 0.60—4.69 2.64 3.61—5.30 4.45 0.23—2.29 1.26 0.08—0.39 0.23 7.15—9.80 8.47 3.86—4.19 4.02 2.04—10.02 6.03 4.24 5.35 0.047 4.91 6.47

Ala Pro pNA

Dipeptidyl peptidase!

0.35—4.30 1.82 ND(3)—3.93 1.04 1.06—4.91 2.39 0.50—3.24 1.87 2.65—4.02 3.33 0.26—1.99 1.12 0.08—0.37 0.22 7.66—8.37 8.01 2.40—2.56 2.48 1.59—8.25 5.07 4.04 2.70 0.047 4.76 5.29

Arg Pro pNA

0.74—5.82 1.83 ND(4)—4.14 0.90 0.06—4.73 1.82 0.34—1.87 1.10 1.46—4.58 3.02 0.24—0.77 0.50 0.01—1.04 0.52 2.90—4.89 3.89 1.10—1.16 1.13 0.68—2.05 1.36 1.71 3.67 (0.01 2.01 3.68

Gly Pro pNA

! Mean specific activities (nmol p-nitroaniline formed mg~1 protein min~1 and range of activities for the number of strains indicated. " Mean specific activities (nmol alanine or # proline and leucine released mg~1 protein min~1) and range of activities for the number of strains indicated with the following exceptions: ¸b. paracasei, 4; ¸b. plantarum, 4; ¸b. curvatus, 3; the dipeptides Pro-Leu and Leu-Pro were substrates for prolinase and prolidase (prolyl dipeptidase and proline dipeptidase) respectively. $ ND, No detectable activity; the number of strains in which activity was undetectable is indicated in parentheses.

Strains tested

NSLAB

Table 4. Peptidase Activities in ¸actobacillus spp. Isolated from Cheddar Cheese

Hydrolytic enzymes of NSLAB 769

770

A. G. Williams, J. M. Banks Table 5. Esterase Activities! in NSLAB Isolated from Cheddar Cheese

NSLAB

Strains tested

Acetate

Butyrate

Caproate

Myristate

Palmitate

¸b. paracasei subsp. paracasei ¸b. plantarum

9 9

¸b. curvatus

4 2

¸b. helveticus

2

¸b. buchneri

2

¸b. parabuchneri

2

¸b. farciminis

2 2

¸b. kefir

2

¸b. bifermentans ¸b. brevis ¸b. collinoides ¼. halotolerans ¼. viridescens

1 1 1 1 1

0.74—8.59 2.74 ND(5)—4.26 0.62 0.98—9.86 3.45 0.22—0.42 0.32 1.69—16.2 8.94 0.46—1.47 0.96 0.46—1.22 0.84 0.44—7.70 4.07 1.97—2.34 2.15 0.73—5.28 3.00 3.47 1.15 ND 12.12 0.86

(0.01—0.53 0.12 ND(8)—0.24 0.027 ND(3)—0.06 0.014 0.22—0.44 0.33 0.19—0.44 0.31 0.11—0.55 0.33 0.19—0.27 0.23 ND

¸b. fermentum

0.63—5.90 2.12 ND(1)—3.77 1.29 0.09—4.40 1.84 1.44—1.50 1.47 2.59—11.1 6.84 0.96—3.45 2.20 0.90—1.00 0.95 2.25—4.74 3.49 3.24—3.99 3.61 5.08—8.04 6.56 4.96 0.78 0.99 2.31 2.31

0.01—0.37 0.13 ND(7)—0.79 0.098 ND

¸b. casei

0.41—4.47 1.62 ND(1)"—16.7 7.50 ND(2)—4.68 1.80 0.29—0.87 0.58 1.64—7.96 4.80 0.25—7.95 4.10 6.15—6.70 6.42 0.46—23.44 11.95 4.05—4.45 4.25 7.42—10.20 8.81 6.76 0.16 0.66 3.75 3.75

ND—0.06 0.032 ND—0.04 0.020 ND ND—0.40 0.20 ND—0.06 0.030 ND

0.10—0.13 0.11 ND—0.18 0.090 0.64 ND 0.40 0.76 0.76

ND—0.12 0.060 0.09 ND 0.087 0.11 0.19

! Mean specific activities (nmol p-nitrophenol released mg~1 protein min~1) and activity range for the number of isolates indicated. " ND, no detectable activity; the number of strains in which activity was not detected is indicated in parentheses.

Table 6. Glycoside Hydrolase Activities! in NSLAB Isolated from Cheddar Cheese NSLAB

Strains tested

a-D-Galactosidase

b-D-Galactosidase

N-Acetyl-a-DGalactosaminidase

N-Acetyl-b-DGalactosaminidase

¸b. paracasei subsp. paracasei ¸b. plantarum

9

¸b. curvatus

4

¸b. casei

2

ND(2)—0.42 0.051 ND(8)—0.05 (0.01 ND(3)—0.064 0.016 ND

¸b. helveticus

2

¸b. buchneri

2

¸b. parabuchneri

2

¸b. farciminis

2

¸b. fermentum

2

¸b. kefir

2

¸b. bifermentans ¸b. brevis ¸b. collinoides ¼. halotolerans ¼. viridescens

1 1 1 1 1

ND(1)—1.43 0.90 0.52—32.00 12.58 ND(1)—29.48 11.24 0.17—7.00 3.58 2.71—17.55 10.13 6.62—16.19 11.40 ND—24.84 12.42 ND—1.07 0.53 3.39—13.72 8.55 59.3—147.4 103.3 19.7 15.40 1.01 1.15 0.030

ND

9

ND(1)"—1.56 0.58 ND(8)—5.16 0.57 ND(3)—0.10 0.025 ND 2.22—2.92 2.57 ND—18.87 9.43 ND—1.01 0.50 ND—0.71 0.35 9.26—14.04 11.6 ND—113.8 56.9 27.5 ND ND ND ND

ND(7)—0.20 0.039 ND(3)—0.03 (0.01 ND—0.04 0.021 ND—0.31 0.15 ND—0.03 0.015 0.02—0.12 0.068 0.02—0.18 0.10 ND ND—0.05 0.025 0.070 ND ND 0.010 0.020

ND ND ND ND—0.18 0.09 ND—0.02 0.011 ND—0.02 0.010 0.070 ND 0.047 0.58 ND

! Mean specific activities (nmol p-nitrophenol released mg~1 protein min~1) and activity range for the number of strains indicated. " ND, no detectable activity; the number of strains in which activity was not detectable is given in parentheses.

Hydrolytic enzymes of NSLAB

monitored in this study were mesophilic ¸actobacillus species; the population of 105—107 cfu g~1 cheese is typical of numbers found in Cheddar cheese of the same age produced in other countries (Peterson and Marshall, 1990; Martley and Crow, 1993; McSweeney et al., 1993, 1994). Although the species profiles of the cheeses were variable, ¸b. paracasei subsp. paracasei and ¸b. plantarum were detected in 87 and 67% of the cheeses, respectively. ¸b. paracasei subsp. paracasei was the dominant NSLAB in 7 (47%) of the cheeses. The predominance of these two species in the NSLAB populations of Cheddar cheese is in agreement with other surveys (Peterson and Marshall, 1990; Peterson et al., 1990; Jordan and Cogan, 1993). Pediococcuus and ¸euconostoc spp. that are also considered to be NSLAB (Franklin and Sharpe, 1963) were not recovered on LBS from the cheeses surveyed. Pediococcus spp. can be isolated on LBS medium from Cheddar cheese using the protocols applied in this study (Williams unpublished results) but ¸euconostoc spp. do not grow well on LBS agar and, therefore, would not be recovered easily when present in the cheese unless more selective media are used (Martley and Crow, 1993; Mathot et al., 1994). Thirteen different species of ¸actobacillus and two species that have been reassigned as ¼eissella were identified in the cheese isolates using commercially available bacterial identification systems. The complexity of the NSLAB population was variable, ranging from 2 to 7 different identified species of lactic acid bacteria in a cheese. The majority of the species identified, with the exception of ¸b. helveticus and ¸b. farciminis, are classified as either obligately or facultatively heterofermentative (Hammes and Vogel, 1995), although it is not uncommon for facultative species to be described as homofermentative as in the presence of glucose, the facultatively heterofermentative lactobacilli ferment hexoses exclusively to lactate. However, as Cheddar cheese matures, the appearance of heterofermentative types in the NSLAB population has been reported (Naylor and Sharpe, 1958) and the presumptive identification of so many heterofermentative species (¸b. brevis, ¸b. buchneri, ¸b. collinoides, ¸b. fermentum, ¸b. kefir, ¸b. parabuchneri, ¼. halotolerans, ¼. viridescens) may be due to the maturity status of the cheeses examined. In this study, the NSLAB populations were monitored at only a single point during maturation. During these latter stages of maturation, the dominant species present in the cheese are likely to be strains that are not susceptible to lysis and are better adapted to the nutritional and physical constraints on growth in the cheese. There is a need to examine more fully the interactions and changes that occur in the NSLAB populations during maturation as these shifts in the microbial population may impact on flavour development and the ultimate quality of the product. This study highlighted that the NSLAB populations differed not only in cheeses produced at different creameries but also in different batches of cheese produced at the same plant. These population shifts may account for some of the betweenbatch variation in cheese quality; an improved understanding as to control of the establishment, nature and development of the NSLAB population could eliminate one potentially important variable from the cheesemaking process. The precise role of NSLAB in cheese flavour development is equivocal, but it is believed that they may be

771

involved in proteolysis and amino acid turnover. Strain selection for use as adjunct cultures has been random, although isolates from good-quality cheeses have generally been used. The sensory characteristics of the 15 cheeses used in the present study confirmed their acceptability (Muir and Banks, unpublished results); nevertheless, the NSLAB population profiles differed. A study of the range and activity of the proteolytic enzymes produced by over 40 NSLAB isolates was, therefore, undertaken both to assess their enzymic potential for involvement in proteolysis during ripening and as a selection criterion for strains to be used as adjuncts in subsequent cheese-making trials. Proteolytic activity was detected in all of the NSLAB isolates studied, although both species and strain differences were evident in the range and level of activity present. The isolates were grown on MRS broth and it is possible that the activity profiles may be affected by the composition of the growth medium, as has been shown with ¸c. lactis (Laan et al., 1993). Enzyme activities may also be growth stage-dependent (Sasaki et al., 1995), and assay conditions were selected for comparative purposes rather than being optimised for individual enzymes. Higher or other activities may, therefore, be detectable by manipulation of the growth or assay conditions, although this study extends the range of NSLAB species that are known to be proteolytic. Diagnostic substrates for metallo, serine and cysteine protease activity were degraded by lysates of many of the isolates. The presence of activity against such substrates is indicative of the involvement of a specific mechanistic type of protease; the evidence, however, is not definitive and requires confirmation with inhibitors and by the characterization of purified enzymes. To date, only a limited number of proteinases have been purified from a restricted range of ¸actobacillus species (Kunji et al., 1996; Law and Haandrikman, 1997), although the existence of both serine and cysteine-type activities has been confirmed. In spite of this evident proteolytic capacity, the addition of adjunct lactobacilli with the starter culture does not necessarily increase the extent of proteolysis in the cheese (Lane and Fox, 1996). These authors concluded that the starter enzymes were principally responsible for the production of small peptides, although the enzymes of the lactobacilli may have different specificities and thereby contribute to the range of peptides formed from casein. The formation of amino acids from the peptide products of the proteolysis of casein is an important stage in cheese maturation since the released amino acids are important flavour constituents that develop during ripening. It was evident that wide-ranging peptidolytic activities were present in the NSLAB isolates although there were marked intra- and inter-species variations in the levels of activity detected. Such within- and betweenspecies differences in activity have been reported in earlier surveys of the peptidase systems of cheese NSLAB (e.g. Peterson et al., 1990; Sasaki et al., 1995). The range of activities present in the different NSLAB species was similar and high-activity isolates were not restricted to a single species. The selection of strains for potential use as adjuncts must therefore be based on derived information rather than random selection. Peterson et al. (1990) have also advocated peptidase profiling of lactobacilli as an important aid to strain selection for cheese manufacture.

772

A. G. Williams, J. M. Banks

Many peptidases of the starter ¸actococcus spp. have been purified and characterised but it is only relatively recently that such studies have been attempted with NSLAB lactobacilli (Bockelmann, 1995; Kunji et al., 1996). The enzyme profiles of the starter lactococci and NSLAB have many similarities and even if the NSLAB do not have an important role in the initial breakdown of casein (Lane and Fox, 1996), they would appear to possess the enzymic potential, like the lactococci, to be involved in the release of amino acids during the ripening process. The diagnostic substrates degraded by the lysates suggest the presence of an array of peptidolytic enzymes. However, the breakdown of a specific substrate is only indicative of the presence of an enzyme in a crude preparation, as substrate degradation may be due to non-specifc action of a broad-specificity enzyme or the sequential actions of more than one enzyme within the preparation. Confirmatory studies will be necessary to unequivocally confirm the occurrence of specific enzymes. Nevertheless, the range of diagnostic substrates degraded indicates that the NSLAB lactobacilli, like the starter cultures, produce tripeptidase, dipeptidase, dipeptidyl peptidase and aminopeptidases. Furthermore, there is evidence from specific substrate utilisation for the presence of prolinase- and prolidase-like activities, and in addition to general aminopeptidase action, the NSLAB would appear to possess both prolyl and proline aminopeptidase (aminopeptidase P; Mars and Monnet, 1993), glutamyl aminopeptidase (PepA) and the omega peptidase pyroglutamyl peptidase (syn PCP, pyrrolidonecarboxylyl peptidase). The presence of only a limited number of these peptidase activities in lactobacilli has so far been confirmed (Bockelmann, 1995; Kunji et al., 1996; Law and Haandrikman, 1997). Other hydrolytic activities were detected in the NSLAB isolates which through their role in substrate generation for the bacteria may have consequences in the cheese maturation process. The i-casein molecule is glycosylated and the release of these sugars may provide a potential energy source to the microorganisms. The presence of the sugars may also impede proteolytic enzyme action and their removal would also facilitate more effective proteolysis. The principal sugars associated with the i-casein molecule are galactose, N-acetylgalactosamine and N-acetyl neuraminic acid (van Halbeek et al., 1980); appropriate glycoside hydrolase activities were detectable in the NSLAB. b-Galactosidase activity was widely detected using the non-specific substrate p-nitrophenyl-b-D-galactoside. The carbohydrate side chain of casein contains a b1-3 linked galactose moiety, whilst lactose is degraded by a galactosidase with a b1-4 specificity. Both enzymes would degrade the p-nitrophenyl galactoside and thus further studies are required to confirm the configuration of the b-galactosidase present. However, N-acetyl-b-D-galactosaminidase was detected in some strains of nine of the NSLAB species although neuraminidase activity was detected in only three of the species. It would, therefore, appear that the NSLAB ¸b. paracasei subsp. paracasei strains may have a role in protein deglycosylation. The carbohydrate substrate obtained may afford some nutritional advantage to this dominant species in the cheese ecosystem, although the benefits may be limited as the glycosylated residues are associated after renneting with the macropeptide fraction which is not extensively retained in the curd.

The release of fatty acids from lipids retained in the cheese curd contributes to flavour development in Cheddar cheese. A tributyrin esterase has been purified from ¸actococcus lactis subsp. lactis and ¸c. lactis subsp. cremoris (Holland and Coolbear, 1996; Chich et al., 1995) and esterolytic enzymes of ¸b. plantarum and ¸b. fermentum have been characterized (Andersen et al., 1995; Gobbetti et al., 1997). Esterase activity has also been detected in other surveys of enzyme profiles in various lactobacilli (El Soda et al., 1986a, b; Khalid et al., 1990; Gobbetti et al., 1996). Esterase activity was also detected in studies using the APIZYM profiling technique to differentiate and characterize lactic acid bacteria (Lee et al., 1986; Arora et al., 1990). The NSLAB isolates from Cheddar cheese likewise had esterase activities that were highest against esterified short-chain fatty acids. The potential, therefore, exists that the NSLAB contribute to the release of fatty acids during the ripening of Cheddar cheese. This aspect of their activities warrants further investigation with adjunct cultures in cheese-making trials. The effects of the inclusion of adjunct cultures of lactobacilli in Cheddar cheese have been variable (Peterson and Marshall, 1990), but this variability may only reflect the random manner in which strain selection occurred. There is an obvious need to use well-characterized strains as the results presented here confirm that inter-species and strain differences in enzyme profiles and levels of activity exist. Strain selection for specific characteristics, such as sulphur amino acid or proline turnover, may be of value in influencing flavour development and the level of bitterness in the mature cheese. The NSLAB have wide-ranging hydrolytic activities and are well-adapted to the cheese environment. Further insights into their metabolic capabilities should enable the effective use of NSLAB as adjunct cultures, with traditional starters, in the manipulation of flavour development.

ACKNOWLEDGEMENT The financial support for these investigations was provided by the EC-AAIR Programme (Contract 93-1531) and The Scottish Office, Agriculture, Environment and Fisheries Department. The technical support of Mrs A. Limond and Mr S. Hamilton is acknowledged.

REFERENCES Andersen, H. J., "stdal, H. and Blau, H. (1995) Partial purification and characteristics of a lipase from ¸actobacillus plantarum MF32. Food Chemistry 53, 369—373. Appel, W. (1974) Peptidase. In Methods of Enzymatic Analysis, 2nd edn., Academic Press, London, pp. 949—978. Arora, G., Lee, B. H. and Lamoureux, M. (1990) Characterisation of enzyme profiles of ¸actobacillus casei species by a rapid APIZYM system. Journal of Dairy Science 73, 264—273. Bhowmik, T. and Marth, E. H. (1990) Role of Micrococcus and Pediococcus species in cheese ripening: a review. Journal of Dairy Science 73, 859—866. Bockelmann, W. (1995) The proteolytic system of starter and non-starter bacteria: components and their importance for cheese ripening. International Dairy Journal 5, 977—994. Bradford, M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein—dye binding. Analytical Biochemistry 72, 248—254.

Hydrolytic enzymes of NSLAB Broome, M. C., Krause, D. A. and Hickey, M. W. (1990) The isolation and characterization of lactobacilli from Cheddar cheese. Australian Journal of Dairy ¹echnology 45, 60—66. Chich, J-F., Marchesseau, K. and Gripon, J-C. (1997) Intracellular esterase from ¸actococcus lactis subsp. lactis NCDO 763: purification and characterisation. International Dairy Journal 7, 169—174. Dacre, J. C. (1953) Cheddar cheese flavour and its relation to tyramine production by lactic acid bacteria. Journal of Dairy Research 20, 217—223. De Man, J. C., Rogosa, M. and Sharpe, M. E. (1960) A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology 23, 130—135. Doi, E., Shibata, D. and Matoba, T. (1981) Modified colorimetric ninhydrin methods for peptidase assay. Analytical Biochemistry 118, 173—184. Drake, M. A., Boylston, T. D., Spence, K. D. and Swanson, B. G. (1996) Chemical and sensory effects of a ¸actobacillus adjunct in Cheddar cheese. Food Research International 29, 381—387. Dunn, B. M. (1990) Determination of protease mechanism. In Proteolytic Enzymes, A Practical Approach, eds R. J. Beynon and J. S. Bond. IRL Press, Oxford, pp. 57—81. El Soda, M. A. (1993) The role of lactic acid bacteria in accelerated cheese ripening. FEMS Microbiology Reviews 12, 239—252. El Soda, M. A., Fathallah, S., Ezzat, N., Desmazeaud, M. J. and Abou Donia, S. (1986a) The esterolytic and lipolytic activities of lactobacilli. Detection of the esterase systems of ¸actobacillus casei, ¸actobacillus plantarum, ¸actobacillus brevis and ¸actobacillus fermentum. Sciences des Aliments 6, 545—557. El Soda, M., El Wahab, A., Ezzat, N., Desmazeaud, M. J. and Ismail, A. (1986b) The esterolytic and lipolytic activities of the lactobacilli. II. Detection of the esterase system of ¸actobacillus helveticus, ¸actobacillus bulgaricus, ¸actobacillus lactis and ¸actobacillus acidophilus. ¸e ¸ait 66, 431—443. Feltham, R. K. A., Power, A. K., Pell, P. A. and Sneath, P. H. A. (1978) A simple method for storage of bacteria at !76°C. Journal of Applied Bacteriology 44, 313—316. Franklin, J. G. and Sharpe, M. E. (1963) The incidence of bacteria in cheese milk and their association with flavour. Journal of Dairy Research 30, 87—99. Gobbetti, M., Fox, P. F. and Stepaniak, L. (1996) Esterolytic and lipolytic activities of mesophilic and thermophilic Lactobacilli. Italian Journal of Food Science 2, 127—135. Gobbetti, M., Smacchi, E. and Corseti, A. (1997) Purification and characterisation of a cell surface-associated esterase from ¸actobacillus fermentum DT41. International Dairy Journal 7, 13—21. Hammes, W. P. and Vogel, R. F. (1995) The genus ¸actobacillus. In ¹he ¸actic Acid Bacteria, Vol. 2. ¹he Genera of ¸actic Acid Bacteria, eds B. J. B. Woods and W. H. Holzapfel. Blackie Academic & Professional, Glasgow, pp. 19—54. Holland, R. and Coolbear, T. (1996) Purification of tributyrin esterase from ¸actococcus lactis subsp. cremoris E8. Journal of Dairy Research 63, 131—140. Johnson, J. A. C., Etzel, M. R., Chen, C. M. and Johnson, M. E. (1995) Accelerated ripening of reduced-fat Cheddar cheese using four attenuated ¸actobacillus helveticus CNRZ-32 adjuncts. Journal of Dairy Science 78, 769—776. Jordan, K. N. and Cogan, T. M. (1993) Identification and growth of non-starter lactic acid bacteria in Irish Cheddar cheese. Irish Journal of Agricultural and Food Research 32, 47—55. Khalid, N. M. and Marth, E. H. (1990) Lactobacilli—their enzymes and role in ripening and spoilage of cheese: a review. Journal of Dairy Science 73, 2669—2684. Khalid, N. M., El Soda, M. and Marth, E. H. (1990) Esterases of ¸actobacillus helveticus and ¸actobacillus delbrueckii sp. bulgaricus. Journal of Dairy Science 73, 2711—2719.

773

Kleter, G. (1977) The ripening of Gouda cheese made under strictly aseptic conditions. 2. The comparison of the activity of different starters and the influence of certain ¸actobacillus strains. Netherlands Milk and Dairy Journal 31, 177—187. Kunji, E. R. S., Mierau, I., Hagting, A., Poolman, B. and Konings, W. N. (1996) The proteolytic systems of lactic acid bacteria. In ¸actic Acid Bacteria: Genetics, Metabolism and Applications, eds G. Verema, J. H. J. Huis in’t Veld and J. Jugenholtz. Kluwer Academic Publishers, Dordrecht, pp. 91—125. Laan, H., Bolhuis, H., Poolman, B., Abee, T. and Konings, W. N. (1993) Regulation of proteinase synthesis in ¸actococcus lactis. Acta Biotechnologica 43, 327—345. Lane, C. M. and Fox, P. F. (1996) Contribution of starter and adjunct lactobacilli to proteolysis in Cheddar cheese during ripening. International Dairy Journal 6, 715—728. Law, J. and Haandrikman, A. (1997) Proteolytic enzymes of lactic acid bacteria. International Dairy Journal 7, 1—11. Lee, B. H., Hache´, S. and Simard, R. E. (1986) A rapid method for differentiation of lactic acid bacteria by enzyme systems. Journal of Industrial Microbiology 1, 209—217. Lloyd, G. T., Horwood, J. F. and Barlow, I. (1980) The effect of yogurt culture YB on the flavour and maturation of Cheddar cheese. Australian Journal of Dairy ¹echnology 35, 137—139. Lynch, C. M., McSweeney, P. L. H., Fox, P. F., Cogan, T. M. and Drinan, F. D. (1996) Manufacture of Cheddar cheese with and without adjunct lactobacilli under controlled microbiological conditions. International Dairy Journal 6, 851—867. Mars, I. and Monnet, V. (1995) Aminopeptidase P from ¸actococcus lactis with original specificity. Biochimica et Biophysica Acta 1243, 209—215. Martley, F. G. and Crow, V. L. (1993) Interactions between non-starter microorganisms during cheese manufacture and ripening. International Dairy Journal 6, 461—483. Martley, F. G., Jayashankar, S. R. and Lawrence, R. C. (1970) An improved agar medium for the detection of proteolytic organisms in total bacterial counts. Journal of Applied Bacteriology 33, 363—370. Mathot, A. G., Kihal, M., Prevost, H. and Divies, C. (1994) Selective enumeration of ¸euconostoc on vancomycin agar media. International Dairy Journal 4, 459—469. McDonald, J. K. and Barrett, A. J. (1986) Mammalian Proteases: A Glossary and Bibliography, Vol. 2, Exopeptidases. Academic Press, London, pp. 111—144. McSweeney, P. L. H., Fox, P. F., Lucey, J. A., Jordan, K. N. and Cogan, T. M. (1993) Contribution of the indigenous microflora to the maturation of Cheddar cheese. International Dairy Journal 3, 613—634. McSweeney, P. L. H., Walsh, E. M., Fox, P. F., Cogan, T. M., Drinan, F. D. and Castelo-Gonzalez, M. (1994) A procedure for the manufacture of Cheddar cheese under controlled bacteriological conditions and the effect of adjunct lactobacilli on cheese quality. Irish Journal of Agricultural and Food Research 33, 183—192. Muir, D. D., Banks, J. M. and Hunter, E. A. (1996) Sensory properties of Cheddar cheese: effect of starter type and adjunct. International Dairy Journal 6, 407—423. Naylor, J. and Sharpe, M. E. (1958) Lactobacilli in Cheddar cheese. 1. The use of selective media for isolation and serological typing for identification. Journal of Dairy Research 25, 92—103. Peterson, S. D. and Marshall, R. T. (1990) Nonstarter lactobacilli in Cheddar cheese: a review. Journal of Dairy Science 73, 1395—1410. Peterson, S. D., Marshall, R. T. and Heymann, H. (1990) Peptidase profiling of lactobacilli associated with Cheddar cheese and its application to identification and selection of strains for cheese ripening studies. Journal of Dairy Science 73, 1454—1464.

774

A. G. Williams, J. M. Banks

Puchades, R., Lemieux, L. and Simard, R. E. (1989) Evolution of free amino acids during the ripening of Cheddar cheese containing added lactobacilli strains. Journal of Food Science 54, 885—888. Rogosa, M., Mitchell, J. A. and Wiseman, R. F. (1951) A selective medium for the isolation and enumeration of oral and fecal lactobacilli. Journal of Bacteriology 62, 132—133. Sarath, G., De La Motte, R. S. and Wagner, F. W. (1990) Protease assay methods. In Proteolytic Enzymes. A Practical Approach, eds R. J. Beynon and J. S. Bond, IRL Press, Oxford, pp. 25—55. Sasaki, S., Bosman, B. W. and Tan, P. S. T. (1995) Comparison of proteolytic activities in various lactobacilli. Journal of Dairy Research 62, 604—610. Sherwood, I. R. (1939) The bacterial flora of New Zealand Cheddar cheese. Journal of Dairy Research 8, 224—237. Tittsler, R. P., Sanders, G. P., Walter, H. E., Geib, D. S., Sager, O. S. and Lochry, H. R. (1947) The effects of lactobacilli on the quality of Cheddar cheese made from pasteurised milk. Journal of Bacteriology 54, 276.

Tittsler, R. P., Sanders, G. P., Lochry, H. R. and Sager, O. S. (1948) The influence of various lactobacilli and certain streptococci on the chemical changes, flavour, development and quality of Cheddar cheese. Journal of Dairy Science 31, 716. Tre´panier, G., Simard, R. E. and Lee, B. H. (1991a) Effect of added lactobacilli on composition and texture of Cheddar cheese during accelerated maturation. Journal of Food Science 56, 696—700. Tre´panier, G., Simard, R. E. and Lee, B. H. (1991b) Lactic acid bacteria relation to accelerated maturation of Cheddar cheese. Journal of Food Science 56, 1238—1241. Tre´panier, G., El Abboudi, M., Lee, B. H. and Simard, R. E. (1992) Accelerated maturation of Cheddar cheese: microbiology of cheeses supplemented with ¸actobacillus casei subsp. casei L2A. Journal of Food Science 57, 345—349. van Halbeek, H., Dorland, L., Vliegenthart, J. F. G., Fiat, A-M. and Jolles, P. (1980) A 360-MHz 1H-NMR study of three oligosaccharides isolated from cow i-casein. Biochimica et Biophysica Acta 123, 295—300.