International Dairy Journal 14 (2004) 495–504

ARTICLE IN PRESS

*Correspondin

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and Molecular B

St. Louis, Misso

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doi:10.1016/j.ida

Characteristics of proteinases and lipases produced by sevenBacillus sp. isolated from milk powder production lines

L. Chena,1, T. Coolbearb,*, R.M. Daniela

a Thermophile Research Unit, University of Waikato, Hamilton, New Zealandb Fonterra Research Centre, Private Bag 11029, Palmerston North, New Zealand

Received 17 October 2002; accepted 27 October 2003

Abstract

Seven Bacillus strains isolated from milk powder production lines were grown in two selective media. All the strains produced

extracellular and intracellular proteinase and lipase activity, but at different levels. Proteolytic activity was generally highest at

neutral to alkaline pH. Inhibitor studies showed that two types of proteinase were produced — a serine proteinase and a metallo-

proteinase. Not all of the strains produced both types of activity and those that did produced different levels. All strains produced a

1,3-specific lipolytic activity that showed a preference for short-chain fatty acids. The lipolytic activity had greater heat stability than

the proteinase activities, but all would clearly survive pasteurisation at 72�C for 2 min in milk. Further, the enzymes are likely to

survive any heat treatments applied during commercial milk powder manufacture. The enzymes remain active in milk powder during

storage, and are therefore likely to be active in milk products made from recombined milk powder.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Bacillus species; Proteinase; Lipase; Characterisation; Milk; Milk product manufacture

1. Introduction

Bovine milk is a biologically active product. Apartfrom the indigenous enzymes such as plasmin (EC3.4.21.7) and liproprotein lipase (EC 3.1.1.34), milk (rawor processed) also contains enzymes originating fromcontaminating bacteria. Bacillus species are widelydistributed in the environment, and can be easilyintroduced into milk and milk products during handlingand processing. Many Bacillus species, such as B. cereus,B. circulans, B. coagulans, B. licheniformis, B. stear-

othermophilus and B. subtilis, are commonly isolatedfrom pasteurised milk, ultra-high heat treated (UHT)milk and milk powders (Stadhouders, Hup, & Hassing,1982; Chopra & Mathur, 1984; Champagne, Laing,Roy, Mafu, & Griffiths, 1994; Hammer, Lembke,Suhren, & Heeschen, 1995; Matta & Punj, 1996, 1999;Garc!ıa-Armesto & Sutherland, 1997; Eneroth, Chris-

g author. Tel.: +64-6-350-4614; fax: +64-6-350-

s: tim.coolbear@fonterra.com (T. Coolbear).

ess: Edward A. Doisy Department of Biochemistry

iology, School of Medicine, Saint Louis University,

uri, USA.

front matter r 2004 Elsevier Ltd. All rights reserved.

iryj.2003.10.006

tiansson, Brendehaug, & Molin, 1998). These Bacillus

species include both mesophilic and thermophilic strains(the latter growing at X45�C) and some of themesophilic strains are thermoduric.

It is generally accepted that microbial growth doesnot take place in milk powder because of its low watercontent. Langeveld, van Montfort-Quasig, Weerkamp,Waalewijn, and Wever (1995) suggested that althoughenzyme-producing bacteria might be killed duringprocessing, their enzymes would still be present andactive in a final milk product. Shamsuzzaman, Modler,and McKellar (1986) reported that freeze-dryingand spray-drying had little effect on the activity ofeither native milk lipase (LPL) or lipase produced byP. fluorescens B521 in raw milk. Their study showed thatheating raw milk at 70�C for 2 min (equivalent to high-temperature short-time pasteurisation) inactivated LPL,but did not affect the activity of Pseudomonas lipase.Hence, the powder showed no loss of lipolyticactivity after storage for 2 months at 20�C (Shamsuzza-man, Modler, & McKellar, 1986). Celestino, Iyer,and Roginski (1997a,b) confirmed the prediction ofStadhouders, Hup, and Hassing (1982) that proteinasesand lipases found in milk survived all the heat

ARTICLE IN PRESSL. Chen et al. / International Dairy Journal 14 (2004) 495–504496

treatments applied during the manufacture of milkpowder (e.g., pasteurisation and spray-drying) andsubsequent UHT treatment. These enzymes remainedactive in reconstituted UHT milk even after a further 6months storage at 25�C (Celestino et al., 1997b).

Generally speaking, the thermostability of microbialproteins (enzymes) shows a positive correlation with theoptimum growth temperature of the source microorgan-ism (Owusu, Makhzoum, & Knapp, 1991). Manyproteinases and lipases produced by psychrotrophs(mainly Pseudomonas) of dairy origin have beenreported to survive pasteurisation and UHT and theseenzymes cause functionality and flavour defects duringstorage (Cogan, 1977; Fox & Stepaniak, 1983; Choi &Jeon, 1993; Makhzoum, Owusu-Apenten, & Knapp,1996). However, the major viable bacterial contami-nants of whole milk powder production lines are nowknown to be seven Bacillus strains (Ronimus, Parker, &Morgan, 1997), and few researchers have reported thecharacteristics of enzymes produced by any Bacillus

species of dairy origin (Chopra & Mathur, 1984, 1985).This paper reports the characteristics of proteinases andlipases produced by these seven Bacillus strains andinvestigates the possible effects of these enzymes in milkpowder during storage.

2. Materials and methods

2.1. Chemicals

All general chemicals were analytical grade andsupplied by BDH Laboratory Suppliers (Poole, BH151 TD, England) and all assay buffers were from Sigma(St. Louis, MO, USA), unless stated otherwise.

2.2. Enzyme production

Three strains of Bacillus stearotheromophilus, Am, Bm

and Cm, three strains of B. licheniformis, Dm, F/G andFta and one strain of B. subtilis, B. sub, were from theThermophile Research Unit Culture Collection (Uni-versity of Waikato, Hamilton, New Zealand). All sevenstrains were originally isolated from milk powderproduction lines and identified by random amplifiedpolymorphic DNA analysis (Ronimus et al., 1997). Thestrains were grown from freeze-dried ampoules onsterilised Tryptic Soy Broth (TSB; DIFCO Labora-tories, Detroit, MI, USA) agar plates, pH 7.3. The plateswere made with 30 g L�1 TSB and 5 g L�1 agarsupplemented with 1 g L�1 soluble potato starch.

For the production of proteinase, strains weregrown in sterilised C-medium containing 5 g L�1 tryp-tone (DIFCO), 1.5 g L�1 yeast extract (BBL, BectonDickinson & Co., Cockeysville, MD, USA), 5 g L�1

NaCl, 1 g L�1 soluble starch and 2.5 g L�1 K2HPO4, pH

7.2. For the production of lipase, strains were grown inC-medium supplemented with 10 g L�1 monoacylglycer-ol. The monoacylglycerol was first homogenised into 1 Lof C-medium containing 20 g gum arabic using an Ultra-turrax homogeniser (Janke & Kunkel Co. GmbH, IKALabortechnik, Staufen, Germany) at 8000 rev min�1 for3 min, and then sterilised.

To culture the bacteria for enzyme production, aloopful of each bacterium from 20 h TSB agar plates wasfirst inoculated into a 10 mL tryptic soy broth, and thentransferred to 100 mL C-medium containing monoacyl-glycerol, as required. B. licheniformis and B. subtilis weregrown at 37�C, and B. stearothermophilus was grown at55�C, with shaking at 110 rpm (air to medium ratio of5 : 1) for B24 h. Cultures were centrifuged after growthat 4500 g for 20 min at 4�C, and the supernatants wereultra-filtered to a 12-fold final concentration using aYM3 membrane (Millipore Corporation, Bedford, MA,USA) to give the extracellular enzyme fraction. The cellpellets were washed twice with 10 mL saline solution(8 g L�1 NaCl) by vortexing followed by centrifugation(4500 g, 20 min, 4�C). Intracellular enzymes werereleased into 5 mL saline solution by disrupting thecells on ice using a Vibra cell (Sonics & Materials Inc.,Danbury, CO, USA) at 50% duty cycle for 2 min. Thelysed cells were centrifuged at 4500 g for 20 min at 4�C,and the supernatant retained as the intracellular enzymefraction. Each enzyme activity was named after theisolate, e.g., proteinase Am and lipase Am were bothfrom B. stearothermophilus strain Am.

2.3. Enzyme assays

2.3.1. Proteinase activity

Two methods were used for determining proteinaseactivity. The first method (Method 1) was based on thatof Twining (1984) using 10 g L�1 fluorescein thiocarba-moyl-b-casein (FTC-b-casein) in 50 mm Tris-HCl buffer,pH 7.5. For each assay, 20 mL FTC-b-casein wasdispensed into a 1.5 mL Eppendorf tube with 20 mL0.1m Hepes (N-2-hydroxyethylpiperazine-N0-2-ethane-sulfonic acid) buffer, pH 7.2, containing 0.2 mg mL�1

penicillin-G (Sigma; PEN-NA). To this, 20 mL samplewas added and incubated at 40�C for the required time.The reaction was stopped by addition of 150 mL ice cold150 g L�1 TCA, the stopped reaction mixture thenchilled on ice for 10 min and centrifuged at 10,000 g

for 5 min. With care, 150 mL of supernatant wereremoved and pipetted into 3 mL 0.5m Tris-HCl buffer,pH 8.5. After thorough mixing, the fluorescence wasread at 490 nm excitation (525 nm emission; slit width5 nm) on a Luminescence Spectrometer LS50B (Perkin-Elmer Ltd., Beaconsfield, Buckinghamshire, England).The assay had an extinction coefficient of17,089 mm

�1 cm�1 and one unit of proteinase activitywas defined as the amount of enzyme that released

ARTICLE IN PRESSL. Chen et al. / International Dairy Journal 14 (2004) 495–504 497

1 mmol fluorescein isothiocyanate (FITC) per min underthe assay conditions.

The second method (Method 2) was based on thosereported by Sarath, de La Motte, and Wagner (1989), andToogood (1998). N-Succinyl-alanyl-alanyl-prolyl-pheny-lalanyl-p-nitroanilide (Suc-AAPF-pNA, Sigma S7388)was prepared at 1 mm by dissolving 1.87 mg Suc-AAPF-pNA in 0.3 mL ethanol, then adding 0.1m Hepes buffer,pH 7.2, to give a final volume of 3 mL. Fifty microlitres ofSuc-AAPF-pNA were dispensed in a 100mL micro-cuvette and warmed at 40�C for 2 min in a Lamba Bspectrophotometer fitted with a thermoelectric cell holder(Perkin-Elmer Ltd.), then 50mL of sample were added,mixed well and the absorbance at 400 nm measuredcontinuously. The assay had an extinction coefficient of11.2 mm

�1 cm�1 for p-nitroaniline (pNA) and one unit ofproteinase was defined as the amount of enzyme thatreleased 1mmol pNA per min under the assay conditions.

2.3.2. Lipase activity

A method adapted from Janssen, Monk, and Morgan(1994) was used to determine lipase activity. A substrateemulsion was first prepared by dissolving 1 mg of thep-nitrophenyl ester of the required fatty acid (C4:0–C18:0)in 1 mL ethanol. This was then homogenised into 8 mLof 0.1m Mops (3-[N-Morpholino] propanesulfonic acid)buffer, pH 7.2, containing 5 mm CaCl2 and 1 g L�1

Triton X-100, using a Ultra-turrax T25 blender at8000 rev min�1 continuously for 1 min. To each 0.9 mLsubstrate emulsion, 0.1 mL sample were added andincubated at 40�C or 60�C for 0.5–3 h. When a definiteyellow colour had developed (or at 3 h, whichever wasthe sooner), the reaction was stopped by adding 0.5 mLstop-reagent (detailed below), chilled on ice for 10 min,centrifuged at 10,000 g for 5 min, and the absorbance ofthe supernatant measured at 400 nm. The assay had anextinction coefficient of 12.6 mm

�1 cm�1 for p-nitrophe-nol (pNP) and one unit of lipase activity was defined asthe amount of enzyme that released 1 mmol pNP per minunder the assay conditions.

Stop reagent was prepared by slowly adding 20 mLCuCl2 � 2H2O (28 g L�1) to 40 mL Na3PO4 � 12H2O(68.5 g L�1) with continuous stirring. The mixture wascentrifuged at 3000 g for 5 min at room temperature.The supernatant was discarded and the pellet washedtwice with 100 mL Na2B4O7 � 10H2O (19.1 g L�1), andfinally suspended in 100 mL of the same solutioncontaining 6 g NaCl. Triton X-100 (100 mL L�1) wasincluded in the final solution for assay samples contain-ing fat. The stop-reagent was stored for at least 2 daysand mixed well before use.

2.4. Enzyme characterisation

To obtain an indication of the type of proteinaseobtained from each strain, proteinase activity was

determined in the presence and absence of 12.5 mm

phenylmethyl sulphonyl fluoride (PMSF; an irreversibleinhibitor of serine proteinases), 4 mm 1,10-phenanthro-line (a reversible inhibitor of metallo-proteinases) and amixture of the two. Enzyme solutions were first treatedwith the inhibitors at room temperature for 30 min, andthen assayed for activity using FTC-b-casein. Thermo-lysin (from B. thermoproteolyticus, Sigma; P1512) wasused as the positive control. Lipase activities weredetermined after the enzyme solutions were treated with1 mm each of PMSF, Hg2+ or p-chloromercuribenzoicacid (PCMB) at room temperature for 60 min. Lipaseactivity was determined using pNP-caproate (C6:0) as thesubstrate.

Hydrolysis of milk lipids was determined using a thinlayer chromatography (TLC) method (Chen, 2000).Partially hydrolysed milk lipid (0.4 g, containing162 g L�1 monoacylglycerols, 276 g L�1 1,2- and 1,3-diacylglycerols and 545 g L�1 triacylglycerols; IndustrialResearch Ltd., Wellington, New Zealand) was firstdissolved in 1 mL ethanol, and then dispersed in 10 mL0.1 m Mops buffer, pH 7.5, containing 0.2 g gum arabic,10 mg Triton X-100 and 2 mg Penicillin G, using a Vibracell (Sonics & Materials Inc.) at 50% cycle for 2 mincontinuously. To 1 mL of milk lipid emulsion, 3 mL ofsample were added, and then incubated at 60�C for upto 43 h. The reaction was stopped by addition of 0.1 mL5m H2SO4 to 1 mL reactant, and the organic layer wasextracted using 1 mL hexene/tert-methyl butyl ether (1:1v/v). After mixing for 2 min, the samples werecentrifuged at 10,000 g for 5 min. The organic layerobtained was applied to an aluminum-backed 0.2 mmMerck Silica Gel 60 F-254 (AllTech 5539, Deerfield, IL,USA).

Prior to loading, the TLC plate was marked at 1 cmintervals horizontally for each loading spot and condi-tioned by developing in a solvent mixture comprising300 mL chloroform and 150 mL iso-propyl alcohol(IPA) until the solvent front reached 2 cm below thetop edge of the plate. The plate was then air-dried.Samples of the organic extractants (5 mL each) wereloaded onto the plate using a micro-pipette (DrummondScientific Co., Broomall, PA, USA). A TLC standard(18-1-A TLC reference standard, Nu-Chek-Prep Inc.MN, USA), supplemented with 4.2 mg oleic acid (C18:1,Sigma O1008) and 11 mg 1,2-dipalmitin (Sigma D2135),was dissolved in 10 mL hexene/tert-methyl butyl ether(1:1, v/v). The standard contained (final concentrations)1.27 mm monoolein, 1.25 mm 1,3-diolein, 1.25 mm trio-lein, 1.24 mm methyl oleate, 1.48 mm oleic acid and1.32 mm 1,2-dipalmitin. The loaded TLC-plate was runin a solvent mixture comprising 450 mL toluene and50 mL acetone until the solvent front reached 3 cmbelow the top edge of the plate. The plate was air-driedand stained with 100 mL of 80 mL L�1 H3PO4 contain-ing 10 g Cu2SO4 for 2 s, air-dried for 20 min, then heated

ARTICLE IN PRESS

Table 1

Growth and production of proteinase and lipase activitiesa by seven

Bacillus strains isolated from milk powder production streams

Isolateb Medium A650nm Final Proteinasec Lipased

pH (mU mL�1) (mU mL�1)

Am TSBe+starch 0.47 5.22 0.01 0.20

C-mediumf 0.75 5.02 ndg 0.05

Bm TSB+starch 0.47 5.15 0.02 0.11

C-medium 0.49 5.88 0.06 0.003

Cm TSB+starch 1.66 5.69 0.07 0.07

C-medium 0.78 5.29 0.01 0.10

Dm TSB+starch 0.26 6.90 nd 0.07

C-medium 0.67 5.71 0.001 0.25

F/G TSB+starch 1.13 6.45 0.08 0.05

C-medium 1.76 7.46 0.24 0.01

Fta TSB+starch 1.19 7.01 0.48 0.08

C-medium 1.59 7.64 0.68 0.03

L. Chen et al. / International Dairy Journal 14 (2004) 495–504498

on a hot plate until yellow spots appeared, indicating thepresence of acylglycerols and free fatty acids (FFAs).

The effect of pH on proteinase activity was deter-mined in 0.1m potassium phosphate buffer (KH2PO4/K2HPO4) over the pH range 4.5–11.5. In the case oflipase activity, 0.1m sodium citrate/citric acid, bis-Trisand bis-Tris-propane (BTP) buffers over the pH range4–8 were used to determine the pH optimum. The heat-stabilities of the crude enzyme preparations (containingB3 mg mL�1 protein) were determined by heating theenzymes in sealed glass ampoules at various tempera-tures from 50�C to 90�C for 10 min, chilling on iceimmediately, and assaying for residual activity using themethods described above.

2.5. Determination of protein concentration

Protein concentration was determined following themethod of Bradford (1976) using bovine serum albumin(0–1 mg mL�1) as the standard.

B. sub TSB+C-medium 2.09 7.26 0.19 0.15

2.03 7.77 1.06 0.24

a All assays were done in triplicate on at least two separate occasions,

the data presented being the average activities observed, with generally

less than 8% variation between results.b Bacteria Am, Bm and Cm were grown at 55�C for B24 h; bacteria

Dm, F/G, Fta and B. subtilis strain B. sub were grown at 37�C for

B24 h.c Proteinase activity was assayed using fluorescein thiocarbamoyl-b-

casein in 0.1 m Hepes buffer, pH 7.0, at 40�C for 0.5–3 h; one unit of

activity is defined as the release of 1mmol fluorescein isothiocyanate

per min under the assay conditions.d Lipase activity was assayed using p-nitrophenyl palmitate in 0.1 m

Mops buffer, pH 7.2, containing 5 mm CaCl2 at 40�C for 40 min; one

unit of activity is defined as the release of 1mmol p-nitrophenol (pNP)

per min under the assay conditions.e Tryptic soy broth (DIFCO) at 30 g L�1, containing 1 g L�1 potato

soluble starch, pH 7.3.f C-medium (1 L) contains 5 g tryptone (DIFCO), 1.5 g yeast extract

(BBL), 5 g NaCl, 1 g soluble starch and 2.5 g K2HPO4, pH 7.2.g nd: not detected.

3. Results and discussion

3.1. Enzyme production

Bacillus species generally synthesise a variety ofextracellular enzymes (e.g., proteinases and lipases),the maximum synthesis of which normally occurs in thelate exponential and early stationary phases of growth,before sporulation (Priest, 1977). Different types ofproteinases, i.e. extracellular serine proteinases andmetallo proteinases, are commonly produced in thesame medium (Egorov, Vybornykh, Kreier, & Loriya,1982; Chopra & Mathur, 1985). The synthesis ofextracellular serine proteinase has been associated withBacillus sporulation (Egorov et al., 1982; Strongin &Stepanov, 1982).

The results showed that the seven Bacillus strainsisolated from the milk process streams both grewdifferently and produced different levels of enzymes inthe TSB-starch and C-media (Table 1). In general, theB. stearothermophilus strains (Am, Bm and Cm) tended togrow with a reduction in the pH of both media. StrainAm grew better in C-medium, while strain Cm grewbetter in TSB-starch. All B. licheniformis strains (Dm,F/G and Fta) showed better growth in C-medium andgenerally the final pH of the culture was higher than thatfound for the B. stearothermophilus strains. B. subtilis

showed the best growth of all the strains, with a finalabsorbance greater than 2 and little change in pH duringgrowth. The cultures of B. licheniformis F/G and Fta,and the B. subtilis strain, were all capable of high levelsof proteinase production. B. stearothermophilus Am,B. licheniformis Dm, and the B. subtilis strain producedthe highest levels of lipase.

However, both extracellular and intracellular protei-nases and lipases were produced by six of the sevenstrains when grown in C-medium (data not shown). Theexception was B. stearothermophilus Am, which, underthe conditions used, did not appear to produceproteinase. Most of the proteinase activity detectedwas extracellular, except in the case of B. stearothermo-

philus Bm for which the intracellular activity was higherthan the extracellular activity.

In the case of lipase activity, most strains producedconsiderably more extracellular activity. Only B. liche-

niformis Fta produced a significantly higher level ofintracellular lipase than extracellular lipase, whileB. stearothermophilus Bm produced approximately equalamounts.

ARTICLE IN PRESS

Table 2

Reaction to inhibitors of proteinase activity from the seven Bacillus

strains

Enzyme Activitya in the presence of

No inhibitors

(mU mL�1)

12.5 mm PMSFb

(% remainingc)

4 mm

1,10-Phenanthroline

(% remaining)

Thermolysin 3.6 22 0

Am 1.0 26 21

Bm 0.2 42 21

Cm 0.8 43 6

Dm 0.4 33 77

F/G 0.3 0 66

Fta 0.3 2 88

B. sub 1.1 40 25

a Samples were first treated with the inhibitors at room temperature

for 30 min, then assayed for proteinase activity using fluorescein

thiocarbamoyl-b-casein in 0.1 m Hepes buffer, pH 7.0, at 40�C for

30 min; one unit of activity is defined as the release of 1mmol

fluorescein isothiocyanate per min under the assay conditions. All

assays were done in triplicate on at least two separate occasions, the

data presented being the average activities observed, with no more

than 5% variation between results.b PMSF: phenylmethylsulphonyl fluoride.c Activity obtained in the absence of inhibitors was taken as 100%.

L. Chen et al. / International Dairy Journal 14 (2004) 495–504 499

The seven Bacillus species were also inoculated intosterile reconstituted skim and whole milk powders at125 g L�1 and incubated at 60�C (data not shown). Milkinoculated with B. stearothermophilus Am, Bm or Cm orB. licheniformis Dm curdled after 10 h, and the pH of themilk dropped below pH 6. Milk inoculated with B.

licheniformis F/G or Fta or B. subtilis B. sub. showed novisible changes in appearance and the pH of the milkincreased slightly. These results are generally consistentwith the different growth characteristics found in TSB-starch and C-medium.

One point to consider in the context of milkprocessing is that these results indicate that not allthermophilic strains may pose the same risk to processor product quality. If a plant is contaminated with alow-enzyme producing thermophile, then the quality ofthe product may not be compromised until bacterialnumbers are much greater than in a plant contaminatedby a high-enzyme producing strain. Consequently, theformer plant could, theoretically, be run for muchlonger periods than the latter, especially if the low-enzyme producer occupied the same ecological niche inthe plant and out-competed any high-enzyme producingstrains.

3.2. Characteristics of proteinases

The differences in response of the crude proteinasepreparations from the seven Bacillus isolates to inhibi-tors are shown in Table 2. In all cases, a combination ofthe two inhibitors, at the concentrations shown, reducedactivity to less than 2%. The activity from strains F/Gand Fta was especially sensitive to PMSF, but resistantto 1–10 phenanthroline, suggesting a predominance ofserine proteinase. Strain Cm was sensitive to 1–10phenanthroline, suggesting a predominance of metalloproteinase. These inhibitor data indicate that both serineand metallo-proteinase were present, but the propor-tions of the enzymes differed among the strains. Thischaracteristic is similar to that in the previously reportedBacillus isolate, B. stearothermophilus RM-67, from rawmilk that produced both a serine and a metalloproteinase with a similar pH optimum (Chopra &Mathur, 1985). The results are also consistent withprevious findings on the extracellular production of bothserine proteinases and metallo proteinases by differentstrains of Bacillus species in the same medium (Egorovet al., 1982; Chopra & Mathur, 1985).

In general, the proteinases from the seven bacilli had apH optimum in the alkaline range, except the proteinasefrom Bacillus B. sub, which had a neutral pH optimum(data not shown). Proteinases Bm, Cm, F/G and Fta gavesimilar activity at pH 11.5 as they did at pH 8.0, and insome cases even higher activity. In the case ofproteinases F/G and Fta, this response is consistentwith an alkaline serine proteinase as indicated by the

inhibitor studies, and in the case of proteinase Bm isconsistent with an alkaline serine proteinase being oneof two enzymes produced by B. stearothermophilus Bm.The activity of the proteinase from B. stearothermophi-

lus Cm at alkaline pH values was surprising, since theinhibitor data suggested that this strain producedpredominantly a metallo proteinase which for Bacillus

species are generally neutral enzymes. Otherwise, thetrends seen are consistent with those reported for manyproteinases produced by Bacillus species (Coolbear,Eames, Casey, Daniel, & Morgan, 1991; Peek et al.,1993, Kobayashi et al., 1995; Ferrero, Castro, Abate,Baigor!ı, & Sineriz, 1996).

The thermal stability of the proteinase activity fromthe seven Bacillus strains at 70�C indicated that at least50% of the initial activity would survive pasteurisationof 72�C for 2 min in all cases. The apparent activation ofthe proteinases seen in some cases after heating for10 min at the lower temperatures used (remainingactivity greater than that found before heating) is notan uncommon finding with thermophilic proteinases.The stabilities shown in Table 3 were obtained usingbuffer systems—it is highly likely that milk proteinswould confer much greater stability to the enzymes.Pseudomonas proteinases from dairy origins have beenreported to be more heat stable in milk than in buffersbecause of protection by milk proteins, mainly caseins(Barach, Adams, & Speck, 1978; Griffiths, Phillips, &Muir, 1981; Stepaniak, Zakrzewki, & S^rhaug, 1991).Such substrate stabilisation is a general phenomenon. Itis likely that all the proteinase activity would survive not

ARTICLE IN PRESS

Table 3

Thermal stability of proteinase activities from the seven Bacillus strains

Heat

treatment

(�C)

Proteinase activitya remaining (%)

Am Bm Cm Dm F/G Fta B. sub

50 114b 99 85 57 98 110 170

60 119 84 29 70 98 63 53

70c 75 59 35 64 70 6 47

80 19 24 24 58 4 6 4

90 3 24 22 64 3 6 5

a All proteinases (containing B3 mg mL�1 protein) were first heated

at 50–90�C for 10 min, chilled on ice immediately, and then (except

that from strain F/G) assayed for proteinase activity using N-Succinyl-

Ala-Ala-Pro-Phe p-nitroanilide in 0.1 m Hepes buffer, pH 7.0, at 40�C

for 1–3 h. F/G proteinase was assayed using fluorescein thiocarba-

moyl-b-casein (FTC-b-casein) in 0.1 m Hepes buffer, pH 7.0, at 40�C

for 5 h. All assays were done in triplicate on two separate occasions,

the data presented being the average activities observed, with no more

than 5% variation between results.b Activity values for unheated proteinases were taken as 100%.

Actual activities for unheated proteinases were (mU mL�1): Am, 1.0;

Bm, 0.3; Cm, 0.5; Dm, 0.2; F/G, 0.3; Fta, 3.0; B. sub, 2.7. One unit of

activity is defined as the release of 1mmol p-nitroaniline or fluorescein

isothiocyanate per min under the assay conditions.c Half-lives for the proteinases (time required for a 50% reduction of

the initial activity) at 70�C were (in min): Am, 24; Bm, 13; Cm, 6.6; Dm,

15.5; F/G, 19.4; Fta, 2.5; B. sub, 9.1.

Table 4

Hydrolysis of p-nitrophenyl esters of fatty acids by lipase activities

from the seven Bacillus strains

Substratea

(pNP-)

Lipase activitiesb (%)

Am Bm Cm Dm F/G Fta B. sub

Butyrate 100 100 100 100 57 100 100

(C4:0)

Caproate 68 61 54 75 36 50 21

(C6:0)

Caprylate 89 18 15 20 76 1 1

(C8:0)

Caprate 76 6 5 7 100 ndc nd

(C10:0)

Laurate 15 8 8 8 74 3 nd

(C12:0)

Myristate 3 3 4 3 37 3 nd

(C14:0)

Palmitate 1 2 3 2 23 1 nd

(C16:0)

Stearate nd 1 2 nd 9 1 nd

(C18:0)

a Assays were carried out in 0.1 m Mops buffer, pH 7.2, containing 5

mm CaCl2 and 1 g L�1 Triton X-100, at 60�C for 10–150 min using p-

nitrophenyl esters of butyrate, caproate, caprylate, caprate, laurate,

myristate, palmitate and stearate; one unit of activity is defined as the

release of 1 mmol p-nitrophenol per min under the assay conditions. All

assays were done in triplicate on at least two separate occasions, the

data presented being the average activities observed, with no more

than 5% variation between results.b Highest activity was taken as 100%. Actual highest activities were

(mU mL�1): Am, 1.74; Bm, 0.36; Cm, 0.22; Dm, 0.26; F/G, 1.52; Fta,

0.81; B. sub, 0.24.c nd: not detected.

L. Chen et al. / International Dairy Journal 14 (2004) 495–504500

only pasteurisation but also heat treatments used in milkpowder production, and therefore any proteinaseactivity generated will be present in milk powders. Thisis particularly true of the proteinase activities from B.

stearothermophilus strains Bm and Cm and B. licheni-

formis Dm, which were the most stable and retained >20% of their initial activities even after 10 min at 90�C.Overall, these findings are consistent with the detectionof protease in milk powders (Chen, 2000).

The heat stability data are also consistent with theinhibition results suggesting that two different protei-nases were produced by some of the Bacillus strains. Inthe case of the proteinases from B. licheniformis Cm thisis particularly clear, with one of the proteinasesseemingly being far more heat stable than the other.The fact that the thermal decay curves (data not shown)were biphasic for many of the proteinase activities mayalso reflect the presence of more than one enzyme.

3.3. Characteristics of lipolytic activities

The activities of the lipase preparations from theseven Bacillus isolates on substrates with different chainlengths are shown in Table 4. With one exception, thelipolytic activities were highest against short chain pNPesters. The lipolytic activities from B. licheniformis strainFta and the Bacillus subtilis strain B. sub had a narrowlydefined specificity for short chain pNP esters of C4:0 andC6:0. The B. stearothermophilus strain F/G lipase activity

was the least specific and hydrolysed all pNP estersregardless of the chain length, but with maximal activityagainst the medium chain C10:0. All the other activitieswere intermediate in specificity, but with highest activityon short chain pNP esters (C4:0). However, of these four,only the B. stearothermophilus Am lipolytic activity hadhigh activity up to a chain length of C10:0. It is possibleto account for the specificities shown by the differentlipase activities by the presence of various mixtures ofthe B. sub type of lipase with the strain F/G type oflipase.

The short chain specificity of the lipase activity fromBacillus strain Am was confirmed by studies usingsynthetic triacyglycerols (data not shown). Since thisspecificity probably extrapolates to an overall preferencefor short chain acylglycerols of milk fat by the Bacillus

lipases, it can be expected to have a significant potentialimpact on flavour because the short-chain fatty acidscontribute most to flavour (Al-Shabibi, Langner,Tobias, & Tuckey, 1964; Vulfson, 1994).

With triacylglycerols as substrates, all seven Bacillus

lipases appeared to be 1,3-specific, acting on the outer 1and 3 positions of the triacylglycerol moiety to give 1,2-diacylglycerols or 2,3-diacylglycerols, 2-monoacylglycer-ols and free fatty acids (FFAs). For example, lipase F/G

ARTICLE IN PRESS

1 2 3 4 5 6 7 8 9 10 1 2 3 4

Triolein

Methyl oleate

1,3-Diolein

1,2-Diolein

Monoolein

Linoleic acid

(A) (B)

Fig. 1. Thin layer chromatography of milk fat following digestion by Bacillus lipases. Panel A shows milk fat incubated with each of the seven

Bacillus lipases (approximately 0.07–0.10 U mL�1, where one unit of activity is defined as the amount of enzyme required to release 1mmol p-

nitrophenol under the conditions used) at 60�C for 72 h. Lane 1, Nu-Chek 18-1-A TLC reference standard; lane 2, milkfat at zero time; lane 3, milk

fat after incubation without enzyme; lanes 4–10, milkfat incubated with (4) Am lipase, (5) Bm lipase, (6) Cm lipase, (7) Dm lipase, (8) F/G lipase, (9) Fta

lipase, (10) B. sub lipase. Panel B shows milk fat incubated with F/G lipase at 60�C for 10 min (lane 3) and 20 min (lane 4) compared with Nu-Chek

18-1-A TLC reference standard (lane 1) and milk fat at zero time (lane 2).

Table 5

Effect of inhibitors on lipase activity from the seven Bacillus strains

Enzyme Activitya in the presence of

no inhibitors 1.0 mm PMSFb 1.0 mm PCMBd

(mU mL�1) (% remainingc) (% remaining)

Am 0.19 58 100

Bm 0.11 88 106

Cm 0.08 82 102

Dm 0.11 78 90

F/G 0.09 85 90

Fta 0.10 37 72

B. sub 0.10 38 87

a Samples were first treated with each inhibitor at room temperature

for 60 min, then assayed for lipase activity using p-nitrophenyl

caproate in 0.1 m Mops buffer, pH 7.2, containing 1 g L�1 Triton X-

100 at 60�C for 10–15 min; one unit of activity is defined as the release

of 1mmol p-nitrophenol per min under the assay conditions. All assays

were done in triplicate on at least two separate occasions, the data

presented being the average activities observed, with no more than 5%

variation between results.b PMSF: phenylmethylsulphonyl fluoride.c Activity obtained in the absence of inhibitors was taken as 100%.d PCMB: p-chloromercuribenzoic acid.

L. Chen et al. / International Dairy Journal 14 (2004) 495–504 501

hydrolysed all monoacylglycerols and 1,2- and 1,3-diacylglycerols after 10 min incubation at 60�C, andcompletely hydrolysed all the triacylglycerols after afurther 10 min incubation (Fig. 1). These substratespecificities on milk lipids are consistent with previousdata (Macrae, 1983), which showed that extracellularBacillus lipase activity hydrolysed different chain lengthfatty acids with a preference for short-chain fatty acids.In some cases, diacylglycerols and monoacylglycerolswere hydrolysed faster than triacylglycerols.

When added to reconstituted whole milk powders andthen freeze-dried back to powdered form and stored at37�C, the Bacillus lipases were found to be able tohydrolyse the milkfat (lipids) in the powders duringstorage (Chen, Daniel, & Coolbear, unpublished). Thesame pattern of release of FFAs from the milkfat wasseen in the powders as seen in buffer solutions.Hydrolysis of triacylglycerols, the main component ofmilk lipids at 98.3% (Walstra, Geurts, Noomen,Jellema, & van Boekel, 1999), resulted in the release ofpredominantly short chain FFAs, which affected theflavour of the milk powders (Chen, 2000).

Because pNP-caproate is not stable at pH X 8 and60�C, the pH optimum of the Bacillus lipases could notbe determined accurately. However, the data (notshown) were consistent with literature informationsuggesting that Bacillus lipases generally have pHoptima of 7–9 (Wood, Fernandez-Lafuente, & Cowan,1995; Schmidt-Dannert, R !ua, Atomi, & Schmid, 1996;Schmidt-Dannert, R !ua, & Schmid, 1997; Sidhu, Shar-ma, Soni, & Gupta, 1998; Kim, Park, Lee, & Oh, 1998;Dharmsthiti & Luchai, 1999). Therefore, if theseBacillus lipases are present in milk, they would bereasonably active in milk at its pH of B6.7.

Inhibitor studies showed that all seven Bacillus lipaseswere sensitive to PMSF (Table 5), with higher concen-trations of PMSF leading to higher levels of inhibition

(data not shown). This sensitivity is similar to that of anesterase cloned from B. stearothermophilus (Amaki,Tulin, Ueda, Ohmiya, & Yamane, 1992). Studies onthe three-dimensional structures of bacterial lipolyticenzymes have shown that a nucleophilic serine residue isusually located within a highly conserved pentapeptideGly X-Ser-X-Gly active site (Arpigny & Jaeger, 1999;Jaeger, Dijkstra, & Reetz, 1999). This catalytic triad issimilar in its function (rather than its structure) to thetriad of serine proteinases (Kordel, Menge, Morelle,Erdmann, & Schmid, 1991; Arpigny & Jaeger, 1999;Jeager et al., 1999), which are sensitive to PMSF. ManyPseudomonas lipases have been reported to be sensitive

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to Hg2+ and PCMB (Fox, Power, & Cogan, 1989).However, most of the seven Bacillus lipases wererelatively insensitive to PCMB: even those from strainsFta and B. sub lost only 28% and 13% of initial activity,respectively, after treatment for 60 min at room tem-perature with 1 mm PMCB. Similar results (not shown)were obtained using 1 mm Hg2+ as an inhibitor.

All the lipase preparations from the Bacillus isolatesshowed relatively high thermal stability (Table 6). Forexample, the lipase from B. stearothermophilis Am had acomparatively high half-life of 11.5 h at 70�C in bufferat pH 7. However, although this was the longest half-lifegiven by any of the enzymes at 70�C and therefore themost stable of the activities at this temperature, thelipase activities from all the other strains survived highertemperatures better. The extreme cases were the lipasesfrom B. stearothermophilis Bm and B. licheniformis Dm,which retained 97% of their initial activity after 10 minat 90�C. Such high heat stability is not uncommon forBacillus lipases, and the range of stability is consistentwith previous findings. For instance, the purifiedcarboxylesterase produced by B. stearothermophilus

Tok19A1 (Wood et al., 1995) and a crude lipaseproduced by a thermophilic Bacillus sp. H1 (Handels-man & Shoham, 1994) had half-lives of 5 and 3 h,respectively, at 90�C. In comparison, the alkaline lipaseproduced by a thermophilic soil Bacillus sp. RS-12 wasmuch less stable with a half-life of 18 min at 70�C andpH 8.0 (Sidhu et al., 1998). As was the case for theproteases, the pattern of activity loss with temperaturesuggests the presence of more than one enzyme. This

Table 6

Thermal stability of the lipase activities from the seven Bacillus strains

Heat

treatment

Lipase activitya remaining (%)

(�C) Am Bm Cm Dm F/G Fta B. sub

50 99b 103 99 96 94 98 83

60 100 104 100 95 95 75 58

70c 99 97 94 97 91 64 42

80 18 98 90 97 82 64 40

90 15 97 89 97 87 62 34

a All lipase preparations (containing B3 mg mL�1 protein) were first

heated at 50�C to 90�C for 10 min, chilled on ice immediately, and

then assayed for lipase activity using p-nitrophenyl caproate in 0.1 m

Mops buffer, pH 7.2, containing 1 g L�1 Triton X-100 at 60�C for 10–

15 min. All assays were done in triplicate on two separate occasions,

the data presented being the average activities observed, with no more

than 8% variation between results.b Activities of unheated lipases were taken as 100%. Actual activities

for unheated lipases were (mU mL�1): Am, 0.19; Bm, 0.11; Cm, 0.08;

Dm, 0.11; F/G, 0.09; Fta, 0.10; B. sub, 0.10. One unit of activity is

defined as the release of 1 mmol p-nitrophenol under the assay

conditions.c Half-lives for the lipases (time required for a 50% reduction of the

initial activity) at 70�C were (min): Am, 687; Bm, 227; Cm, 112; Dm,

227; F/G, 73; Fta, 15; B. sub, 8.

was especially so in the case of B. sub activity, wherethere was a substantial loss at 60�C, but relatively littlefurther loss as the temperature was increased from 60�Cto 90�C.

All seven Bacillus lipases appeared to be more heatstable than the proteinases from the same strains. Thiswas most readily reflected by the half-life values at 70�Cand the levels of residual activity after treatment at 90�Cfor 10 min shown in Tables 3 and 6 for proteinases andlipase, respectively. This is not unexpected. Lipases areoften produced concomitantly with proteinases by thesame microorganism, and they are frequently more heatstable than the proteinases (Andersson, Hedlund, &Jonsson, 1979; Fox & Stepaniak, 1983; Stepaniak &Fox, 1983; Kumura, Mikawa, & Saito, 1993a,b). InBacillus species, the first glycine residue in the highlyconserved pentapeptide Gly-X-Ser-X-Gly active site isoften replaced with an alanine (Ala) residue and isassociated with the high heat stability of Bacillus lipases.Replacement of this alanine residue in the lipase from B.

subtilis 168 with glycine using site-directed mutagenesisleads to a marked reduction in the thermostability of theenzyme (Eggert, Pencreac’h, Douchet, Verger, & Jaeger,2000).

Overall, even if the stability in milk is no higher thanin buffer systems, all seven Bacillus lipases would beexpected to completely survive pasteurisation/heattreatment at 72�C for 2 min in milk. Heat stabilities ofother Bacillus lipases are known to increase withincreasing enzyme concentration and with addition ofmilk proteins (Kumura, et al., 1993b; Wood et al.,1995). In addition, some lipases produced by Bacillus

showed activities in a cell-bound form even at 100�C(Jaeger et al., 1994). Stead (1986) reported that oncelipase from psychrotrophic bacteria is present in milk, itis not possible to inactivate it completely. Shamsuzza-man et al. (1986) reported that spray-drying of skimmilk after heating at 70�C for 2 min had no significanteffect on the bacterial lipase activity, which remained inthe powder after the spray-drying process. Therefore,once the Bacillus lipases are present in milk, they willclearly survive pasteurisation, evaporation and spray-drying processes and remain in milk powders, poten-tially contributing to spoilage during storage.

4. Conclusions

The seven Bacillus strains isolated from milk powderproduction lines produced both extracellular andintracellular proteinases and lipases. Both serine protei-nases and metallo-proteinases were expressed and a1,3-specific lipase with preferential specificities formono- and diacylglycerols was common to all strains.Both the proteinases and lipases were sufficiently heat-stable to survive any of the heat treatments applied

ARTICLE IN PRESSL. Chen et al. / International Dairy Journal 14 (2004) 495–504 503

during a milk product manufacturing process andtherefore would remain active in the final products,including milk powders.

The results indicate that counting bacterial (e.g.,thermoduric or thermophiles) numbers in milk powders,for example, will not necessarily provide any correlationwith, or predictive indicator of, product quality orspoilage potential. This is because the bacteria found inthe powder are not necessarily an indication of thosewhich may have contributed any enzyme burden to thepowder. It is clear from this work that different strainsof the thermophilic bacilli contaminating the milkpowder production line have the potential to producedifferent types and levels of proteinase and lipase. A keyfactor in predicting the keeping properties of milkpowder, and its quality, is likely to be the determinationof actual enzyme levels in the final product.

Acknowledgements

The award to LC of a Graduate Researcher inIndustry Fellowship, and financial support for this workfrom the Foundation for Research, Science andTechnology, and from the New Zealand Dairy Board(now part of Fonterra Co-operative Group Ltd.), isgratefully acknowledged.

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