Research

3. EFFECT OF CENTRIFUGAL PRETREATMENT, pH AND WATER ACTIVITY ON
THE PRODUCTION OF PROTEASE BY Pediococcus acidilactici
Mushafau A. OKE1
and Mutiat B. ODEBISI1
1. Department of Microbiology, University of Ilorin, Ilorin, Nigeria.
ABSTRACT
Pediococcus acidilactici and other lactic acid bacteria (LAB) have wide applications in the food
industry due to the beneficial characteristics they impact on fermented foods. The proteolytic
system of LAB contributes to their use as starter cultures. The proteolytic system of the pediococci
has not been extensively researched like that of other LAB. This work was therefore aimed at
studying the effect of centrifugation, pH and water activity on the growth and production of
protease enzyme by Pediococcus acidlactici. The organism was grown in MRS broth and
subjected to a pretreatment of 2000 rpm and 3000rpm centrifugation speed, pH 4.5 and 6.5 and
water activity levels of 0.99, 0.97, 0.95 and 0.93. The effect of these treatments on growth and
protease production were determined separately and in combination. Centrifugation speed of 3000
rpm, pH 6.5 and water activity of 0.99 favoured growth and protease production by the organism.
However, lower protease production was observed when the three treatments were combined. The
results support the use of P. acidilactici as starter culture in meat fermentations.
Keywords: Lactic acid bacteria, Pediococcus acidilactici, protease, food fermentation.
INTRODUCTION
Pediococcus acidilactici belongs to a group of organisms known as lactic acid bacteria (LAB).
They are gram-positive, non-endospore forming, usually non-motile and metabolise glucose by
lactic acid fermentation (Maczulak, 2011). Other members of the group include Streptococcus,
Enterococcus, Lactococcus, Lactobacillus and Leuconostoc (Willey et al., 2009). LAB have
received much attention owing to their role in diary and food industry like baking, brewing, cheese
manufacturing and meat tenderization (Hans, 1993). They are particularly important in food
fermentation and their proteolytic system is considered to be one of the major processes involved
in texture and flavor development (Fadda et al., 2001).
The inability of LAB to synthesize many of the amino acids required for protein synthesis
necessitates the active functioning of a proteolytic system in those environments where protein
constitutes the main nitrogen source (Pritchard and Coolbear, 1993). The proteolytic system is
composed of proteinases which initially cleave the protein to peptides, peptidases which cleave
the peptides thus formed into smaller peptides and amino acid transport systems which are
involved in the cellular uptake of small peptides and amino acids (Law and Haandrikman, 1997).
Several environmental factors have been found to affect the growth and metabolism of P.
acidilactici but most of the studies have been mainly on bacteriocin production by the organism
(Calderon-Santoyo, 2001; Guerra and Pastrana, 2002; Vasquez et al., 2003). Few reports exist
about the proteases of the genus Pediococcus compared to other LAB genera (Llorente-Bousquets
et al., 2008). Due to the importance of P. acidilactici in the fermentation of vegetables, meat and
other fermented foods, this work was designed to determine the effects of centrifugation, water
activity and pH on the growth and protease production by the organism.
EXPERIMENTAL

4. Source of organism: Pediococcus acidilactici used in this work was obtained from the culture
collection stock of the Department of Microbiology, University of Ibadan. Pure cultures of the
organism were stored on MRS agar slants at 4o
C.
Effect of centrifugal pretreatment on growth and protease production
A loopful of the organism was inoculated into each of 2 McCartney bottles containing 10ml of
sterile MRS broth. These were incubated at 350
C for 24 hours after which they one of the bottles
was centrifuged at 2000 rpm and the other at 3000 rpm for 5 minutes. The bottles were tapped
slightly and shaken gently to disperse the settled cells. From each bottle, an inoculum load of 2.4
x 105
cfu/ml was taken and inoculated into 10ml of sterile MRS broth. These were then incubated
at 350
C for 48 hours after which the cruse enzyme was obtained by centrifugation at 2000 rpm for
15 minutes. The supernatant was obtained for protease assay and the cells were plated out for
growth determination.
Effect of pH on growth and protease production
Using either 0.1M NaOH or 0.1M HCl where necessary, 2 sets of McCartney bottles each
containing 10ml of MRS broth were set at pH 4.5 and 6.5. The bottles were sterilized and allowed
to cool. Each of the bottles was inoculated with 2.4 x 105
cfu/ml of the organism. Each set was
incubated for 24 hours and 48 hours. At the end of each interval, aliquots of the culture were
plated out. The cultures wee centrifuged at 2000 rpm for 5 minutes and the supernatants were
obtained for protease assay.
Effect of water activity (aw) on growth and protease production
Four sets of McCartney bottles containing 10ml of MRS broth each were adjusted to aw of 0.99,
0.97, 0.95 and 0.93 using NaCl (humectant) concentrations of 1.2%, 4.3%, 7.5% and 10.6%
respectively following the method of Troller and Stinson (1981). The bottles were sterilized,
cooled and then inoculated with the organism (2.4 x 105
cfu/ml). They were incubated for 24 hours
at 350
C and then plated out. Supernatants were obtained as described earlier and used for protease
assay.
Enzyme production under optimal conditions
P. acidilactici was grown in MRS broth for enzyme production under the treatments that gave the
highest protease production i.e. 3000 rpm centrifugation speed, pH 6.5 and aw of 0.99. The
organism was grown in sterile broth for 24 hours. The culture was centrifuged for 15 minutes at
3000 rpm. Inoculums from this culture was then used to inoculate 250ml of sterile MRS broth
which had been adjusted to a pH of 6.5 and aw of 0.99. This was incubated at 350
C for 24 hours
and at the end, it was centrifuged at 10000 rpm for 20 minutes. The supernatant (crude enzyme)
was obtained for and protease assay.
Protease assay
Protease assay was done using a modification of the method of Kunitz (1947). One percent casein
solution was prepared in 0.1M citrate phosphate buffer (pH 5.5) and was heat-denatured at 1000
C
for 15 minutes in a water bath and allowed to cool. The reaction mixture consisted of 1 ml
substrate (1% casein) thoroughly mixed with 0.5ml of the enzyme extract. Incubation was for 1
hour at 370
C after which the reaction was terminated by adding 3ml of cold (20
C) 10%
trichloroacetic acid (TCA). The reaction tubes were allowed to stand for 1 hour at 20
C to allow
the undigested protein to precipitate. Control tubes contained 0.5ml of enzyme extract incubated
for 1 hour at 370
C before adding 3ml of cold TCA and 1ml of 1% casein. The reaction mixtures
were centrifuged at 10000 rpm for 5 minutes at 40
C. Optical density readings of the carefully
decanted supernatant fluids were measured with a spectrophotometer at 660nm wavelength

5. against a blank containing the control. One unit of protease activity was defined as the amount of
enzyme that released 1 µg tyrosine per ml per minute from 1 mg casein under the specified assay
conditions.
RESULTS AND DISCUSSION
Effect of centrifugal pretreatment on growth and protease production
Table 1 shows the results of the effect of centrifugation on P. acidilactici.
Table1: Effect of centrifugation on growth and protease production by P. acidilactici.
Centrifugation speed (rpm) Growth (cfu/ml) Protease production
(units/ml)
2000 4.2 x 107
87.63
3000 4.8 x 107
103.98
The highest protease activity was observed at 3000 rpm probably because the higher
centrifugation speed dislodged cell surface-associated proteinases hence causing their release into
the surrounding medium. Some LAB are known to have proteinases located at the outer surface
of the cell (Pritchard and Coolbear, 1993). Centrifugation is known to have several effects on
bacterial cells such as affecting the hydrophobicity and electrophoretic mobility (Pembrey, 1991),
destabilization of the envelope (Gilbert, 1991) and alteration of cellular surface macromolecules
(Tsunada et al., 2003) which can be stripped off or compressed during centrifugation. An
important application of this finding is in the use of centrifugation as a pretreatment step for P.
acidilactici starter culture preparation for meat fermentations.
Effect of pH on growth and protease production
From the results in table 2, it can be observed that the growth of the organism at pH 4.5 and 6.5
at 24 hours was the same (1.4 x 108
cfu/ml). However, after 48 hours, higher growth was recorded
at pH 6.5. according to Harvey (1965), LAB grow best when the medium is near neutral pH and
growth rate declines as the extracellular medium becomes more acidic.
Table 2: Effect of pH on growth and protease production by P. acidilactici
Growth (cfu/ml) Protease production (units/ml)
pH 24 hours 48 hours 24 hours
4.5 1.4 x 108
3.2 x 105
71.57
6.5 1.4 x 108
4.1 x 105
82.37
The highest protease activity was recorded at pH 6.5 (82.37 units/ml) while the lowest was at pH
4.5 (71.57 units/ml). Other researchers have also found pH 6.5 to favour the growth and
metabolism of P. acidilactici. This pH has been found to favour cell mass, acid and pediocin
production after 16 hours of growth in TGE broth (Biswas et al., 1991) and it was also found to
favour higher biomass production in MRS broth containing 5.0% sugar cane strap molasses as
carbon source by P. acidilactici (Sant’ Anna and Torres, 1998).
Effect of water activity (aw) on growth and protease production
Growth of the organism showed a gradual decline as aw level was reduced (table 3). The highest
growth (6.5 x 108
cfu/ml) was at aw 0.99 while the lowest was at aw of 0.93.

6. Water activity (aw) Growth (cfu/ml) Protease production
(units/ml)
0.99 6.5 x 108
71.57
0.97 5.4 x 108
0
0.95 2.7 x 108
0
0.93 2.4 x 108
0
A similar trend was observed by Troller and Stinson (1981) in a test to determine the effect of
reduced water activity levels on growth and metabolite production by some LAB using NaCl,
glycerol and sucrose as humectants. They recorded the highest growths at aw of 0.998 and the
lowest growth at 0.95.
Protease production was only detected at aw of 0.99 and the growth slowed down while enzyme
production was halted. According to Braun et al. (1999), a decrease in temperature, pH or water
activity is usually associated with a loss of enzyme activity. Furthermore, Willey et al. (2009)
stated that microorganisms differ greatly in their ability to adapt to low aw and different organisms
present varying responses (in terms of growth and metabolite production) to reduced aw. The
organism has thus shown a promising feature in meat fermentation due to its favourable protease
production at aw 0.99 which is the aw of fresh meat (Ray, 2005).
Enzyme production under optimal conditions
The protease production observed under combination of the optimal conditions was 63.39 unit/ml.
this value is lower than that recorded when the treatments were treated separately thus suggesting
that a combination of the treatments would not favour protease production.
In conclusion, this study has shown that a centrifugal pretreatment of 3000 rpm supports protease
production by the organism. This could be applicable in preparation of P. acidilactici as starter
culture in the fermentation of meat and other products. Also the organism would do well in
initiating the hydrolysis of meat proteins in meat fermentation so as to enhance the qualities of
the product. This is as a result of its high protease production at physicochemical conditions
similar to that of fresh meat.
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