protease biosynthesis from lactobacillus species: fermentation parameters and kinetics

PROTEASE BIOSYNTHESIS FROM LACTOBACILLUS SPECIES:FERMENTATION PARAMETERS AND KINETICS

IKRAM-UL-HAQ and HAMID MUKHTAR1

Institute of Industrial BiotechnologyGovernment College University

Lahore, Pakistan

Accepted for Publication October 4, 2006

ABSTRACT

In the present study, 15 strains of Lactobacillus species, isolated from25 samples of curd, were screened for their ability to produce extracellularprotease. The proteolytic activities of these strains based on casein hydrolysisshowed a variation of 1.26–5.80 U/mL with Lactobacillus IH8 showing amaximum of all. Different cultural conditions for enhanced production ofprotease by Lactobacillus IH8 were optimized. It was found that optimalconditions for the production of enzyme were an incubation temperature of35C and a medium pH of 5.5. The maximum proteolytic activity of Lactoba-cillus IH8 (7.28 U/mL) was achieved after 48 h of cultivation. The kineticparameters such as product yield coefficient (Y p/x), growth yield coefficient(Y x/s), specific product yield coefficient (qp) and specific growth yield coef-ficient (qx) also revealed that the values of experimental results were highlysignificant when analyzed kinetically.

PRACTICAL APPLICATIONS

The studies have been proved helpful in the isolation and identification ofa Lactobacillus strain which can be used as potent starter culture for cheesemanufacturing. The strain can also be used for protease production which canthen be used for coagulating milk casein in the cheese manufacturing as thestrain has a high caseinolytic activity.

1 Corresponding author. TEL: 092-3334245581; FAX: 092-42-9211634; EMAIL: [email protected]

Journal of Food Processing and Preservation 31 (2007) 102–115. All Rights Reserved.© 2007, The Author(s)Journal compilation © 2007, Blackwell Publishing

102

INTRODUCTION

Proteases are the enzymes that hydrolyze the peptide linkages of proteinsto simpler proteins, peptides and amino acids. The activity of proteolyticenzymes produces not only one product but also a number of end products,which is due to the fact that proteases are a mixture of enzymes (Lee et al.2002). These include proteinases, peptidases and amidases hydrolyzing intactproteins, peptides or peptones and amino acids, respectively. Proteases aregenerally divided into three broad categories depending on whether theseare active in acidic, neutral or alkaline pH range (Haque et al. 1990). Theseinclude acidic proteases that show maximum activity between pH 2.0 and 5.0,neutral proteases with a maximal activity at pH 7.0 or around 7.0 and inhibitedby metal-chelating agents (Tsuru et al. 1967) and alkaline proteases havingmaximum activity at pH 9.0–11.0, unaffected by metal-chelating agents andcleave a wide range of peptide bonds (Keay 1971).

All three types of proteases are produced by a wide range of microor-ganisms in their extracellular environment. Extracellular proteases are impor-tant for the hydrolysis of protein substrates in the cell-free environment andenable the cell to absorb and utilize the hydrolytic products of the substrates(Kalisz 1988). At the same time, these extracellular enzymes have also beenexploited commercially to carry out protein degradation in a number of indus-trial processes (Kumar and Tagaki 1999) and hence have a wide application invarious industries.

Lactic acid bacteria (LAB) play an important role in dairy and meatfermentation processes, and have a great influence on the quality and preser-vation of the end products. The main roles of LAB are the production of lacticacid, thus causing the lowering of pH and the proteolysis of the substrate torelease peptides and free amino acids, which subsequently affect the flavor andtexture of the end products (Piuri et al. 2003). A large number of LAB specieshave been employed for the production of yogurt and different types ofcheeses; therefore, the knowledge of the overall proteolytic system of thesebacteria has been the main subject of investigation in the past two decades. Theproteolytic system of LAB consists of cell envelope-associated proteinases(PrtP), specific peptide and amino acid transport systems and several cytoplas-mic peptidases (Piuri et al. 2003).

A fairly large number of Lactobacillus species are known to producevarious types of proteolytic enzymes (Wick et al. 2004). However, the mostcommonly used species for this purpose include Lactobacillus bulgaricus(Courtin et al. 2002), Lactobacillus rhamnosus (Pastar et al. 2003), Lactoba-cillus casei (Benech et al. 2003; Piuri et al. 2003), Lactobacillus paracasei(Bintsis et al. 2003), Lactobacillus helveticus, Lactobacillus delbrueckii(Oberg et al. 2002; Germond et al. 2003), Lactobacillus brevis, Lactobacillus

103KINETICS OF PROTEASE PRODUCTION FROM LACTOBACILLUS

cellobiosus, Lactobacillus fermentum and Lactobacillus plantarum (Mugulaet al. 2003). Many of these species are used as starters in cheese manufactur-ing; however, some are the nonstarter usual floras of cheeses. The proteolyticenzymes of Lactobacillus species are highly specific and can be looselygrouped into a PI/PIII type of classification (Kunji et al. 1996). However,studies also suggest that some Lactobacillus proteases have relatively broadspecificity resulting in a large number of possible cleavage sites on the sub-strate (Juillard et al. 1995).

The present investigation was undertaken to evaluate 15 strains of Lac-tobacillus isolated from curd based on their protease activity, and to furthercharacterize the strain with the highest protease activity by manipulatingenvironmental conditions and kinetic analysis of the results so that a potentstrain can be identified and enzyme production can be optimized, which wouldbe helpful for its use as a starter culture in cheese manufacturing.

MATERIALS AND METHODS

Organism, Growth and Maintenance

A potent strain of Lactobacillus was isolated from curd using nutrientagar casein plates. The strain was isolated on the basis of clear zone of caseinhydrolysis. The culture was grown and maintained on nutrient agar-lactosemedium. The bacterial slants were incubated at 37C for 48 h and then storedat 4C. The bacterial strains were transferred to new slants on a weekly basisand immediately prior to fermentation experiments.

Culture Characterization

All the selected isolates were morphologically and biochemically char-acterized. Cell morphology, Gram staining, biochemical tests, carbohydratefermentation profile and the ability to produce lactic acid were studied toconfirm the genus designation.

Fermentation Experiments

The inoculum of Lactobacillus was prepared in 250-mL Erlenmeyerflasks containing 50 mL of the nutrient broth–lactose medium (pH 7.0). Theflasks were sterilized in an autoclave at 121C (15 Ib/in2 pressure) for 15 min.After cooling, the medium was aseptically inoculated with a loopful of bac-teria from a 48-hour-old slope. The flasks after inoculation were incubated for24 h on a rotary incubator shaker (Gallenkamp, U.K.) at 37C and 200 rpm.

104 I. UL-HAQ and H. MUKHTAR

The fermentation batches for the production of protease from Lactoba-cillus IH8 were carried out in 250-mL shake flasks. Fifty milliliters of thefermentation medium containing 10 mg/mL of Tryptone, 4.0 mg/mL of yeastextract, 8.0 mg/mL of meat extract, MgSO4.7H2O (0.2 mg/mL), MnSO4.4H2O(0.05 mg/mL), 1 mL of Tween 80 (pH 6.5) and 2% lactose separately auto-claved was added to each flask and sterilized in an autoclave. After cooling, themedium was inoculated with 1 mL of the inoculum containing 3–4 ¥ 108 cfu.The flasks were then placed in a rotary type incubator shaker (Gallenkamp)for growth and fermentation. After incubation, the fermented broth wascentrifuged at 2,350 ¥ g for 10 min (Sigma Laboratory Centrifuge, Model3K30, Germany) and the supernatant was assayed for protease activity.

Analytical Methods

Assay of Protease. The method of McDonald and Chen (1965) was usedfor the assay of protease. Casein (1% solution in 0.1-M phosphate buffer of pH6.0) was incubated with 1 mL of the enzyme sample at 30C for 1 h. Thereaction was arrested by the addition of 5 mL of 5% trichloroacetic acidsolution. The mixture was centrifuged at 5,000 rpm for 10 min and 1 mL ofsupernatant was mixed with 5 mL of alkaline reagent. To this mixture, 1 mL of1-N NaOH was added to make the contents of the tube alkaline. After 10 min,1.0 mL of Folin and Ciocalteau reagent (diluted with distilled water with theratio 1:1) was added to the test tubes and mixed. The blue color produced wasmeasured with UV spectrophotometer (CE 7,200, CECIL, Cambridge,England) at 700 nm after 30 min.

One unit of protease activity is defined as the amount of enzyme requiredto produce an increase of 0.1 in optical density at 700 nm under the definedconditions.

Analysis of Residual Sugar. The amount of residual sugar in the fer-mented broth was measured by DNS method (Miller 1959).

Measurement of Dry Cell Mass. After fermentation, the fermentedbroth was centrifuged in preweighed glass tubes at 2,350 ¥ g for 10 min. Thesupernatant was removed and the pellet was dried at 105C overnight in a hotair oven. After drying, it was again weighed to determine the dry cell mass(DCM) of the bacteria.

Kinetic and Statistical Analysis. The experimental data were statisti-cally analyzed following Snedecor and Cochran (1980). Duncan’s multiplerange test was applied under one-way analysis of variance. Significance hasbeen presented in the form of probability (P � 0.05) values.


Kinetic parameters for batch fermentation experiments were determinedaccording to the methods as described by Pirt (1975) and Lawford and Roseau(1993). The following parameters of kinetics were studied:

(1) Maximum specific growth rate (m)max per hour – the value of (m)max wascalculated from plot of lnx versus time of fermentation.

(2) Product yield coefficient (Y p/x) U/mL/mg – the value of Y p/x wasdetermined by the equation:

Y p x dp dx=

(3) Growth yield coefficient (Y x/s) U/mL/g – the value of Y x/s was deter-mined by the equation:

Y x s dx ds=

(4) Specific product yield coefficient (qp) U/ml/h – the value of qp wasdetermined by the equation:

Y p x. maxμ( )

(5) Specific growth yield coefficient (qx) U/mL/g – the value of qx wasdetermined by the equation:

Y x s. μ( )max

RESULTS AND DISCUSSION

Screening of Isolates for the Biosynthesis of Protease

Fifteen different strains of Lactobacillus, which were capable of produc-ing protease, isolated from different curd samples were screened for higherproduction of the enzyme (Table 1). Among all the strains screened, Lactoba-cillus IH8 gave the maximum production of protease (5.80 U/mL) and wasselected as the most potent producer for subsequent investigations.

Culture Characterization

The proteolytic isolates were morphologically and biochemically charac-terized. As shown in Table 2, the strains were nonmotile, anaerobic, Grampositive and rod-shaped bacteria, showing negative catalase and benzidine tests.The strains were capable of fermenting glucose, fructose, lactose and mannose


but not galactose, and produced lactic acid as the main end product. It was clearfrom Bergey’s Manual of Determinative Bacteriology (Buchanan and Gibbons1974) that the isolates belonged to the genus Lactobacillus.

Effect of Medium pH on the Protease Production by Lactobacillus

In most studies on microbial cultures, the pH of the culture medium isone of the most important factors that influence the microbial growth and

TABLE 1.SCREENING OF LACTOBACILLUS SPECIES FOR THE

PRODUCTION OF PROTEASE IN SHAKE FLASKS

S.No. Strain Protease activity (U/mL)

1 Lactobacillus IH1 2.34 � 0.015j

2 Lactobacillus IH2 4.40 � 0.030c

3 Lactobacillus IH3 1.82 � 0.005l

4 Lactobacillus IH4 3.22 � 0.02f

5 Lactobacillus IH5 2.88 � 0.01h

6 Lactobacillus IH6 1.42 � 0.030m

7 Lactobacillus IH7 5.20 � 0.030b

8 Lactobacillus IH8 5.80 � 0.02a

9 Lactobacillus IH9 4.00 � 0.041d

10 Lactobacillus IH10 2.24 � 0.020k

11 Lactobacillus IH11 1.26 � 0.020n

12 Lactobacillus IH12 3.68 � 0.015e

13 Lactobacillus IH13 2.94 � 0.011g

14 Lactobacillus IH14 2.72 � 0.005i

15 Lactobacillus IH15 3.20 � 0.041f

The values given are mean of three parallel replicates with an SD ofeach. Incubation period = 48 h; incubation temperature = 35 � 1C;medium pH = 6.5; least significant difference = 0.03349; signifi-cance level = 0.05. The letters in subscripts show that these valuesdiffer significantly. S.No. is serial number.

TABLE 2.MORPHOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF THE ISOLATES

Morphology Straight rodsMotility Non-motileGram’s staining PositiveGrowth in anaerobic conditions YesProduction of lactic acid as main end product YesCatalase NegativeBenzidine reaction NegativeCarbohydrate fermentation profile Capable of fermenting glucose, fructose, lactose and

mannose but not galactose


metabolism. So, the effect of pH of medium on the production of protease byLactobacillus IH8 was investigated. Figure 1 shows the results according towhich the optimum pH of the fermentation medium for maximum growth ofLactobacillus IH8 (8.60 mg/mL) and subsequent production of protease(7.20 U/mL) was 5.5. It was inferred that the organism was an acidophile andgrew well in an acidic medium. The organism showed less growth and enzymebiosynthesis above or below the medium pH of 5.5.

The results of the present parameter were analyzed kinetically and it wasfound that the values of product yield coefficient (Y p/x) coincided with theexperimental values (Fig. 2); however, the maximum value of growth yieldcoefficient (Y x/s) was observed at pH 6.5 instead of 5.5, the optimal growthpH. Therefore, it can be inferred that the organism showed a maximum growthat pH 6.5 in terms of cell mass production per unit substrate consumption. Thevalues of the specific product yield coefficient (qp) and specific growth yieldcoefficient (qx) also showed similar results (Fig. 3).

It was also found in the present experiments that the organism was verysensitive to pH change especially toward alkaline values because a slightincrease in optimum pH resulted in much-reduced biomass and protease pro-duction. Rhee and Pack (1980) and Frey et al. (1986) have also reported thesimilar results but they found the optimum pH for production of protease byLactobacilli as 6.0, a little higher than the one in the present studies. Similarly,

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FIG. 1. EFFECT OF MEDIUM pH ON THE PRODUCTION OF PROTEASE BYLACTOBACILLUS IH8

Each value is an average of three parallel replicates. Y error bars indicate the standard error of themean. Incubation period = 48 h; incubation temperature = 35 � 1C.


Pastar et al. (2003) have found pH 6.5 as the best medium pH for proteaseproduction from L. rhamnosus.

Effect of Incubation Temperature on the Protease Production byLactobacillus

The effect of incubation temperature on the production of protease byLactobacillus IH8 was studied by varying the incubation temperature of fer-mentation flasks from 25 to 50C. It was found that the maximum growth

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FIG. 2. PRODUCT AND GROWTH YIELD COEFFICIENTS (Y p/x AND Y x/s) FORPROTEASE PRODUCTION BY LACTOBACILLUS IH8 AT DIFFERENT MEDIUM pHs

0.045

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FIG. 3. SPECIFIC PRODUCT AND GROWTH YIELD COEFFICIENTS (qp AND qx) FORPROTEASE PRODUCTION BY LACTOBACILLUS IH8 AT DIFFERENT MEDIUM pHs


(8.40 mg/mL) and production of protease (7.10 U/mL) were achieved at anincubation temperature of 35C (Fig. 4). The strain was found to be able togrow even at 50C (5.02 mg/mL DCM) but the optimum temperature forenzyme production was 35C.

The kinetic analysis of the results revealed that the maximum value ofproduct yield coefficient (Y p/x) was found at the incubation temperature of35C, which was in accordance with the experimental results (Fig. 5), but thevalues of growth yield coefficient (Y x/s) at the incubation temperatures of 35and 40C were almost the same, which showed that the growth of LactobacillusIH8 in terms of substrate consumption was the same at both these temperatures.The values of specific product yield coefficient (qp) and specific growth yieldcoefficient (qx) also showed the similar values as were obtained during theexperiments (Fig. 6).

Environmental temperature is a very important parameter that is inescap-ably linked to the biomass production of an organism because cell temperatureis always equal to the temperature of the culture medium. It affects the rates ofcell reactions, the nature of the metabolism, the biomass composition and thenutritional requirements of the organism. Therefore, incubation temperaturemust always be considered an important parameter while carrying out thefermentation experiments.

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FIG. 4. EFFECT OF INCUBATION TEMPERATURE ON THE PRODUCTION OF PROTEASEBY LACTOBACILLUS IH8

Each value is an average of three parallel replicates. Y error bars indicate the standard error of themean. Incubation period = 48 h; medium pH = 5.5.


Effect of Incubation Period on the Protease Production by Lactobacillus

The incubation period of a fermentation experiment has a direct rela-tionship with the growth of microorganism and production of enzymes. Theexperimental flasks were incubated for variable time intervals ranging from

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FIG. 5. PRODUCT AND GROWTH YIELD COEFFICIENTS (Y p/x AND Y x/s) FORPROTEASE PRODUCTION BY LACTOBACILLUS IH8 AT DIFFERENT INCUBATION

TEMPERATURES

25 30 35 40 45 50

Incubation temperature (C)

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FIG. 6. SPECIFIC PRODUCT AND GROWTH YIELD COEFFICIENTS (qp AND qx) FORPROTEASE PRODUCTION BY LACTOBACILLUS IH8 AT DIFFERENT INCUBATION

TEMPERATURES


12 to 72 h. It was found that the maximum production of protease (7.14 U/mL) and bacterial biomass (8.66 mg/mL) were achieved after 48 h of incu-bation (Fig. 7), which corresponded to the logarithmic growth phase of thebacterium. It is clear from the results that the incubation period of more orless than 48 h did not show the promising results as far as production of theenzyme is concerned. Moreover, there was a steady decrease in the amountof protease produced by Lactobacillus IH8 after 48 h of incubation. Theprevious reports have also shown that Lactobacilli start producing proteasesin the early exponential growth phase but their maximum production usuallyoccurs in the logarithmic growth phase (Back et al. 1983; Hebert et al.1996).

The kinetic analysis of this parameter showed that the maximumamount of enzyme per unit biomass (Y p/x) was produced after 48 h ofincubation (Fig. 8). The data also reveal that the value of growth yield coef-ficient (Y x/s) was at maximum in the beginning of the fermentation, afterwhich it declined gradually and reached the minimum after 72 h. The kineticstudy of this parameter depicted a rapid growth of organism at the start offermentation, and then the growth profile decreased gradually, which wasfollowed by the more secretion of enzyme. The values of the specificproduct yield coefficient (qp) and specific growth yield coefficient (qx) alsoshowed similar values (Fig. 9).

12 24 36 48 60 720

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FIG. 7. EFFECT OF INCUBATION PERIOD ON THE PRODUCTION OF PROTEASE BYLACTOBACILLUS IH8

Each value is an average of three parallel replicates. Y error bars indicate the standard error of themean. Medium pH = 5.5; incubation temperature = 35 � 1C.


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protease biosynthesis from lactobacillus species: fermentation parameters and kinetics

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