Download APPLICATION OF LACTIC ACID BACTERIA TO CONTROL

Sept. 2014. Vol. 2, No.5
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
APPLICATION OF LACTIC ACID BACTERIA TO CONTROL
MICROBIAL CONTAMINANTS DURING FERMENTATION OF
COCOA BEANS
1
JOKO SULISTYO, 2HO EFFIE CAHYANINGSIH, 3BETTY SRI LAKSMI JENNY
1
Faculty of Food Science and Nutrition, University Malaysia Sabah,
Faculty of Agriculture, Artha Wacana Christian University, Kupang, Indonesia
3
Faculty of Food Technology, Bogor Agriculture University, Bogor, Indonesia
Email: [email protected] and [email protected]
2
ABSTRACT
Cocoa is one of the leading commodity plantation sector. Not only has the potential to increase the country's
foreign exchange income but also a major source of income for cocoa farmers in many centers of cocoa
producer, but the quality of cocoa beans produced have not been uniform and not in accordance with
international standard, resulting in low international price of cocoa market. One of the opportunities to
improve the quality of cocoa is through development on fermentation and preservation technology of cocoa
beans, using cultures of lactic acid bacteria (LAB) isolated from palm sap-based fermented products. Some
LAB strains those had been isolated from palm sap fermented liquid, identified as Leuconostoc mesenteroides,
Leuconostoc pseudomesenteroides, Lactobacillus plantarum and L. fermentum. Results of assay on their
antimicrobial activities showed that only L. fermentum and L. plantarum were effective on inhibiting against the
growth of some microbial contaminants in cocoa beans. Strain of L. plantarum was able to produce as much as
2.05% lactic acid and hydrogen peroxide as much as 24.87 g/ml, but did not produce bacteriocins. Strain of L.
plantarum was also able to reduce the presence of microbial pathogens S. Typhimurium and Aspergillus flavus
by 2 log units at concentrations of 107-109 CFU / ml, so that it can meet the quality standards of cocoa that has
been established.
Key words: lactic acid bacteria, palmyra-sap, cocoa fermentation, antimicrobial, antimycotic.
1.
INTRODUCTION
Lactic acid bacteria (LAB) has long been known
and used by humans in food processing through
fermentation process, thus contributing greatly to
improvement of flavor, texture and shelf life of the
food product. Preservation effects caused by LAB
are generally caused by the production of
metabolites and a decrease in pH in the
fermentation environment, thus preventing the
growth of other microbes that are not desired
during the fermentation (Rahayu et al, 1999).
LAB is known to inhibit growth of spoilage and
pathogenic bacteria by producing organic acids,
bacteriocins, CO2, diacetyl and H2O2, while also
has antimycotic activity (Jenie et al, 2002;
Salminen et al. 2004). Lactococcus lactis and L.
casei can inhibit growth and aflatoxin production
by Aspergillus parasiticus, which was allegedly
caused by the production of lactic acid (El Gendy
and Marth, 1981; El Gendy and Marth 1981). The
results of study by Karunaratne et al (1990)
showed that isolates of L. acidophillus, L.
bulgaricus and L. plantarum can inhibit the growth
of A. flavus and aflatoxin production.
According to Paavola et al (1999), nowadays, the
use of LAB culture increasingly in demand, since
the antimicrobial component produced by LAB
could serve as a natural preservative that is safe for
health. Thus, application of this metabolites
derived from LAB cultures will continue to evolve
in the future, since it has a fairly wide spectrum on
utilization. Study on some LAB cultures that have
been isolated from several fermented food
demonstrated antimicrobial and antimycotic
activity against microbial pathogens and spoilage.
These isolates were generally dominated by L.
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Sept. 2014. Vol. 2, No.5
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
plantarum,
Pediococcus
acidilactici,
Streptococcus thermophillus (Lie, 1995).
and
spontaneously fermented palmyra sap, and thus it
is expected to reduce the number of
microorganisms that are pathogenic contaminants
in cocoa beans.
A research conducted by Anonymous (2005)
microbial cultures which had been isolated from
palmyra sap showed antimicrobial activity against
several microbial pathogens. The results showed
that these isolated LAB and acetic acid bacteria
cultures had ability to inhibit the growth of
Escherichia coli, S. Typhimurium and Bacillus sp.
Up to present, this indigenous palmyra sap LAB
cultures have not been extensively studied for use
as a source of potential isolates as biological
preservative (Rahayu et al, 1999). In fact, the
cultures of LAB have the ability to produce
antimicrobial metabolites as important natural
metabolites which is useful as part of an effort to
combat contamination caused by microbial
pathogens, especially on cocoa commodities.
2.
MATERIALS AND METHODS
The materials used are palmyra sap, fresh cocoa
fruit, S. Typhimurium and A. flavus. GYP medium,
NA, NB, 0.85% NaCl, 3% H2O2 solution, PDA,
LTB medium consisting of 1% glucose, 1% yeast
extract, 1% beef extract, 1% tryptone, 0.5% NaCl
and 0.2% Na2HPO4, peptone water, lactose broth,
XLD, TSI, SCB media and Cary-Blair media.
2.1. Isolation of LAB From Palmyra Sap
The spontaneously fermented sap (10 ml) was
diluted to 102-105 dilution in 90 ml of 0.85% saline
and the pH was measured. A total of 1 ml of each
dilution was inoculated on GYPA media
containing liquid CaCO3 and incubated at 37C for
2 days. Colonies with clear zones were tested by
Gram staining and catalase test. For the purpose of
identifying species, culture was inoculated on
GYPB media and incubated for 24 hours at a 37C.
The culture was subsequently identified based on
morphological
characteristics
observation,
biochemical and physiological bacteria.
Decrease in number of cocoa exports coming from
some cocoa exporting countries were due to
foreign consumers consider the quality of these
cocoa beans are low. The low quality of such kinds
of cocoa beans among other things due to the high
contamination caused by microbial pathogens,
especially Salmonella, causes salmonellosis
affecting economic losses in many countries
(Kapperud, 1989; Torres-Vitela, 1995).
Most traditional farmers dry the cocoa beans by
means of sun drying, so if not dried immediately,
then within one day will grow mold that can
damage the appearance and flavor of cocoa beans
(Amin, 2005). The existence of a family mold
Aspergillus (A. glaucus, A. niger, A. flavus, A.
tamarii), Penicillium and Mucor, which could
potentially lead to mycotoxins in cocoa beans that
could potentially hydrolyze fats into short-chain
fatty acids (Christensen, 1987; Harrison, 2000).
2.2. Preparation Stock Culture of LAB
The isolated LAB cultures were transferred onto 50
ml GYPB and incubated for 2 days at 37C. A
positive LAB growth is characterized by the
presence of turbidity in the medium after
incubation. Stock cultures were inoculated on
GYPA semisolid agar medium containing 0.1%
(w/v) CaCO3, and incubated for 2 days at 37C.
The stock culture was prepared by adding a sterile
glycerol (1: 1, w/v). One ml of the stock cultures
were inoculated into 9 ml of GYPB medium and
incubated for 2 days at 37C. Cultures were diluted
with 0.85% NaCl until the desired concentration
(Jenie et al, 2002).
Another negative impact of the presence of mold in
cocoa beans, in addition to mycotoxins is the
emergence of off-flavor due to the activity of
lipolytic enzymes (Collins et al, 1981; Lund,
2000). In addition, spoilage microbes that grow
during fermentation of cocoa beans is Aerobacter
spp, Pseudomonas, Enterobacter and E. coli that
cause off-flavor. However, bacterial pathogens
commonly found in cocoa beans, dairy products
and cause disease is Salmonella (Lund, 2000).
2.3. Selection for LAB Cultures
Antibacterial activity test was conducted using
diffusion wells (Schved et al, 1993), whereas
antimycotic activity assay by using method of
contact (Gourama and Bullerman, 1995). LAB
cultures were inoculated in 10 ml GYPB medium
and incubated at 37C for one day. Culture of S.
This study aims to obtain alternative solutions in an
effort to increase the security of cocoa beans, by
utilizing LAB cultures were isolated from
17
Sept. 2014. Vol. 2, No.5
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
Typhimurium was inoculated into NB medium and
incubated at 37C for one day. A total of 0.2 ml
was transferred into 100 ml of NA medium (0.2%),
mixed and transferred onto a petri dish and allowed
to solidify. Hereafter solidified, diffusion wells
were made within a diameter of 6 mm.
2.5. Submersion of Cocoa Beans with LAB
Fresh cocoa beans were placed in a container, then
stir until evenly distributed. At random, cocoa
beans as much as 6 kg were replaced onto a box
and fermented for 5 days. The selected LAB
cultures (approximately 9 ml) in GYPB media
were inoculated into the soaking beans as starter
cultures to ferment cocoa beans while incubation at
37C for 24 h. The number of colonies of LAB
which was applied was equal to 106 CFU/ml.
A total of 50 mL of LAB in GYP broth culture was
added to the wells and incubated for 2 days at
37C. Diameter of clear zone around the wells
were measured as LAB inhibitory effect against the
test bacteria.
2.6. Application of LAB for Preservation of
Cocoa Beans
To test the activity of antimycotics, molds of the
stock cultures were grown on PDA slant for 10
days at room temperature. Separaton of mold
spores that grown on surface of PDA slant was
done by adding of 10 ml of diluting solution. The
number of mold spores counted by Petroff-Hauser
method to obtain 106 spores/ml mold solution.
A total of 500 grams of fermented cocoa beans
were soaked in water (1: 1, w/v) for 1h, then
drained and soaked again with a solution of LAB
and pathogenic microbial strains with six
treatments: (1) soaked in water, (2) soaked with
LAB (107 and 109 CFU/ml), (3) inoculated with S.
Typhimurium (103 CFU/ml), (4) inoculated with A.
flavus (103 CFU/ml), (5) inoculated with S.
Typhimurium (103 CFU / ml) and addition with
LAB (107 CFU/ml) and (6) inoculated with A.
flavus (103 CFU/ml) and addition with LAB (107
CFU/ml). Cocoa beans then drained and dried in an
oven at 50C for 3 days.
One ml of the stock culture of LAB was inoculated
into 9 ml GYPB medium and incubated for 2 days
at 37C, then diluted with 0.85% NaCl to obtain
the desired concentration (108 CFU/ml). A total of
0.5 ml of LAB culture was inoculated into 50 ml
LTB medium and incubated for 24 hours at 37C,
then added with 0.5 ml of mold spores solution
(106 spores/ml) and incubated at room temperature
for 14 days. Mycelial dry weight was measured by
filtering mycelia was collected using a vacuum
filter and washed with distilled water and then
dried at 95C until a constant weight was obtained.
2.7. Assay for S. Typhimurium and A. flavus
Test for existence of S. Typhimurium and A. flavus
was done after drying cocoa beans. Samples were
first crushed by weighing 50 g of sample into a
sterile plastic bag and added with 450 ml of diluent
(0.1% peptone water) and blended for 2 min. The
crushed samples was then replaced into a sterile
vial and diluted to 108. To determine the presence
of S. Typhimurium which infected cocoa beans, as
many as 1 ml of sample was furthermore pipetted
into some petri dish and then poured respectively
with 12 ml to 15 ml of PDA which had been
acidified with tartaric acid. The media containing
inoculum was thoroughly mixed by by shaking and
rotating the petri dish so that the media solidified
then compiled them upside down and incubated at
25C for 5 days. Colonies were counted on a petri
dish containing number of colonies 30-300 for
each dilution (Fardiaz, 1989).
2.4. Test of Bacteriocin
The strain of S. Typhimurium was grown on NA
medium and incubated at 37C for 24 h, and
approximately 1 ose of culture was transferred into
NB and incubated at 37C for 18 h. Approximately
0.2% S. Typhimurium culture which had been
grown in NB medium was poured onto a melted
NA media on a sterile petri dish and allowed to set,
then 6 mm in diameter of diffusion wells were
made on the solidified media. LAB cultures in
GYPB media (24 h) were neutralized with 0.01 N
of NaOH up to pH 7.0. Cells were harvested by
centrifugation and then filtered with paper milipore
2 m. A total 50μl of supernatant was dropped
into the wells and incubated at 37C for 24 h.
Observations were made by measuring the
diameter of the clear zone formed around the wells
as a zone of inhibition (Wolf and Gibbons, 1996).
18
Sept. 2014. Vol. 2, No.5
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
3.
RESULTS AND DISCUSSION
3.1. Identification of LAB of Palmyra Sap
From various ranges of fermentation, we have
obtained 33 isolates consisting of yeast, Bacillus,
and 10 isolates of bacteria that were Gram-positive
and catalase negative, which allegedly were
cultures of LAB. Based on observations of cell
form, ability to grow at different temperatures and
salt concentration, the ability to produce CO2, the
ability to produce ammonia, the ability to produce
dextran and the ability to ferment various kinds of
carbohydrates, the 10 isolates of these bacteria
were belonging to genus Lactobacillus and
Leuconostoc. The presence of these cultures of the
genus Lactobacillus and Leuconostoc on the
palmyra sap, due to adaptability of these LAB
cultures in such conditions with high sugar
concentration (Sumanti, 1994).
Figure 2. Performance of Clear Zone of Isolates of LAB
In Inhibiting Growth of S. Typhimurium.
The NL-249 isolates was identified as
Lactobacillus plantarum showed the highest
inhibitory activity against S. Typhimurium. This
was allegedly due to presence of lactic acid which
was a main product of L. plantarum NL-249 as
homofermentatif LAB (Jay 1996). This was
supported by a statement of Nousianen et al (2003)
that organic acids and H2O2 which were produced
by LAB showed inhibitory activity against
Salmonella. According to Jacobsen (1999), LAB
has been declared to have antimicrobial activity, if
it has a minimum inhibitory area of 1 mm, and was
stated to has a positive inhibitory activity (+) when
the area of inhibition between 2mm to 5 mm, and
has a high inhibitory activity (++) when the area of
inhibition was more than 5 mm.
Tests on the ability of LAB strains in the
production of bacteriocins was performed by well
diffusion test, where test showed that there was no
antibacterial activity resulted by bacteriocins
against S. Typhimurium. Jack et al (1995) states
that bacteriocins are compounds produced by most
of the genera of LAB is a protein or protein
complex (agegrat protein, lipokarbohidrat proteins,
glycoproteins, etc.) which are easily degraded by
proteolytic enzymes, and has spesific ability to
inhibit microbial growth of strains which is closely
related to the producing bacterium.
Figures 1 and 2 showed that only isolate NL-128
and NL-249 out of 10 isolates of LAB were
isolated from fermented palmyra sap belongs to the
genus Lactobacillus showed inhibitory activity
against S. Typhimurium, with a diameter of
inhibition were 3.3 mm and 9.3 mm, respectively.
While other LAB isolates belongs to the genus
Leuconostoc (NL-2410) were not able to inhibit the
growth of S. Typhimurium.
Figure 1. Ability of Strains of LAB Isolated from
Fermented Palmyra Sap in Inhibiting Growth of S.
Typhimurium.
S. Typhimurium is a Gram-negative bacterium, and
therefore it was possible that bacteriocins produced
by L. plantarum NL-249 as Gram-positive bacteria
become ineffective. According to Lindgren and
Dobrogosz (1990), plantaricin A was bacteriocin
produced by L. plantarum strains which had a
narrow spectrum of activity, so it wass only
19
Sept. 2014. Vol. 2, No.5
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
effective against closely related microbes. Another
possibility was due to that L. plantarum NL-249
did not produce bacteriocins, so that the inhibitory
activity against S. Typhimurium, may be caused by
the presence of organic acids and hydrogen
peroxide (Nousianen et al, 2003).
and acidification of the cytoplasm (Ostling and
Lindgren 1993; Alakomi et al, 2000)
According Lazarova (1994) undissociated acid
levels of lactic acid would be higher at the low pH
range of pH 3-4, where percentage of undissociated
acid was about 86.6% and therefore the
antimicrobial activity was also high (Fardiaz and
Jenie, 1989). According to Chung and Goepfert
(1980), Salmonella would be stunted by lactic acid
at pH <4.4. Jay (1996) and Lin et al (2000) also
stated that S. Typhimurium is a bacteria that was
quite resistant to acids, but below pH 4.0 the
growth of this bacteria would be disrupted and
gradually become extinct.
3.2. Antimicrobial Activity Due to Lactic Acid
Figure 3 showed that the strain L. plantarum NL249 increasingly produced lactic acid during 4 days
of incubation, but then exhibited a decline after day
4 of incubation. Assay for the ability of NL-249 in
inhibiting S. Typhimurium was tested using 24 h
cultured of NL-249 where the production capacity
of lactic acid was about 0.54%, and furthermore
incubated with S. Typhimurium for 2 days, where
production of lactic acid was approximately 1.42%.
According to Salminen et al (2004) L. plantarum is
included in the homofermentatif group of LAB, in
which pyruvic acid is generated from a glycolytic
pathway (Embden-Meyerhorf-Parnas), play a role
as receiver of hydrogen, where the reduction of
pyruvic acid by NADH2, will produce lactic acid
by the following reaction:
Glycolysis
2 pyruvic acid + 2 (NADH + H+)
2 (lactic acid + 2NAD+)
Strain of L. plantarum isolated from pikel which
produced lactic acid on day-3 at 0.181%, showed
higher inhibitory activity against S. typhimurium
compared to other isolates of LAB from different
sources, despite they produced higher lactic acid
(Lie, 1995). As well as with strain of L. Plantarum
isolated from fish sauce which produced lactic acid
at 0.961% on day-3 of incubation was also able to
inhibit S. Typhimurium effectively. It seemed that
the inhibitory activity of LAB against S.
Typhimurium was not only due to the organic acids
production, but there was also a possibility
influence due to a combination effect with H2O2
(Nousianen et al, 2003).
3.3. Antimicrobial-Hydrogen Peroxide
According to Lindgren and Dobrogosz (1990),
H2O2 produced by LAB strains was related to the
presence of O2 through the activity of flavoproteins
oxidation or peroxidation of NADH. In anaerobic
conditions, LAB will ferment glucose to lactic
acid, while in a condition of adequate oxygen,
glucose will be used for respiration. During
incubation, glucose oxidation will produce CO2
which will make an anaerobic condition by
replacing existing of O2. Decreasing production of
H2O2 also caused due to nature of H2O2 which is
very unstable, as a consequence of presence of
some components such as catalase, heavy metal
ions, organic matter and lactoperoxidase, which
will decompose H2O2 rapidly into water and O2
(Branen and Davidson, 1983).
Figure 3. Total Production of Lactic Acid By L.
Plantarum NL-249 During Incubation.
Lactic acid is the main metabolite resulted in
fermentation of homofermentatif LAB which
converts glucose into lactic acid more than 90%,
whereas heterofermentatif LAB produces about
50% lactic acid. Inhibitory effect of this organic
acids mainly depending on the amount of
undissociated acid that can passively diffuse into
the microbial cell where it will break down into
anions and protons, this leads to the disruption of
important metabolic functions such as inhibition of
substrate transport, energy production, synthesis of
macromolecules, impaired motor skills of protons,
Bactericidal effect of H2O2 associated with strong
oxidation effect and the destruction of bacterial
cells on the basis of molecular structure of cell
protein. Various bacterial cultures can produce
H2O2 although the production is low but its
20
Sept. 2014. Vol. 2, No.5
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
presence can help to inhibit the growth of
unwanted bacteria.
Figure 4 showed the production of H2O2 by L.
plantarum NL-249 that was likely to increase on
the second day of incubation, however then went
into decline due to decreasing amount of oxygen.
Lactobacillus produces H2O2 which could inhibit
the growth of some Gram-negative bacteria
(Pseudomonas and Salmonella) and Gram-positive
bacteria, such as Staphylococcus (Gililand and
Speck, 1975). Lactobacillus produces H2O2 in high
quantity through pyruvate, L-lactate oxidase, Dlactate dehydrogenase and NADH oxidase
(Salminen et al, 2004). The concentration of H2O2
which is bactericidal against Gram-negative
bacteria is amount 25-30μg/ml, while the isolate of
L. plantarum NL-249 produced highest H2O2 on
the second day of incubation at 24.87μg/ml, and
therefore the possibility of antibacterial compounds
produced by isolate of L. plantarum NL-249 which
was effective against S. Typhimurium was the
combination effect of organic acids and H2O2, even
though such this concentration is actually
insufficient to eradicate S. Typhimurium.
Figure 5. Effect of LAB on Mycelial Growth of A.
Flavus.
Figure 5 showed that isolate L. plantarum NL-249
produced the highest antimycotic activity. This
antimycotic compounds produced by the isolate
were most likely organic acids and H2O2. Few
studies have shown that some strains of LAB
produced antimycotics compounds such as lactic
acid, acetic acid, caproic acid, formic acid, phenyl
lactic acid and 4-hydroxy phenyl lactic acid, cyclic
dipeptide such as cyclo (Gly-L-Leu), cyclo (L-PheL- Pro), and cyclo (L-Phe-trans-4-OH-L-Pro),
benzoic acid, methyl hydantoin, mevalono lactone,
short-chain fatty acids, low molecular weight of
proteins, bacteriocins, hydrogen peroxide (Corsetti
1998; Lavermicocca et al, 2000; Storm, 2002).
Sjogren et al (2003) reported that L. plantarum
produced a kind of fatty acids such as 3-(R)decanoic
hydroxy
acid,
3-hydroxy-5-cisdodecenoic acid, 3-(R)-dodecanoic hydroxy acid
and 3-(R)-hydroxy tetradecanoic which had
antimycotic property, however mechanism of
inhibition has been unclear. Allegedly these fatty
acids have activity such as detergents that will
affect structure of cell membrane of fungi, which
increase membrane permeability and release of
electrolytes and intracellular proteins that lead to
disintegration of cytoplasm of fungal cells. Isolates
of L. plantarum NL-249 exhibited the highest
inhibitory activity both against S. Typhimurium
and A. flavus, and therefore this LAB isolate was
then selected for further experiment that was used
for fermentation and preservation of cocoa beans.
Figure 4. Production of H2O2 By L. Plantarun NL-249
During 6-Days of Incubation.
3.4. Test for Activity of Antimycotics
One of parameter that can be used to determine
antimycotic properties of LAB is by measuring of
dry weight of mycelia, since a mold growth is
directly proportional to the production of mycelia.
The media used for the purpose is Lablemco
tryptone broth that can be used for the growth of
LAB and molds simultaneously (Gourama and
Bullerman 1995), where the lowest mycelial dry
weight was obtained showed the highest
antimycotic activity.
3.5. Effect of LAB Growth of S. Typhimurium
The number of cells of LAB culture that could be
absorbed by the cocoa beans during submersion for
1 hour was decreasing 1 log unit from initial
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Sept. 2014. Vol. 2, No.5
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International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
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amount of 2.7 x 107 CFU/ml to 1.4 x 106 CFU/ml.
In general, the higher concentration of LAB
suspension was applied and the length of time of
soaking was performed, causing the number of
LAB cells were absorbed more and more. The
number of cells of S. Typhimurium was also
decreased by 1 log unit, from the initial amount of
2.9 x 104 to 1.4 x 103. Decreasing in the number of
LAB cells that could be absorbed by these cocoa
beans was due to protective effect of shell of cocoa
beans. Submersion cacao beans included isolate L.
plantarum NL-249 after fermentation using isolate
L. plantarum NL-249 was tehrefore able to reduce
S. Typhimurium by 3 log units.
CFU/ml could reduce A. flavus by 1 log unit.
Concentration of isolates L. plantarum NL-249
could be increased up to 109 CFU/ml for 2h
submersion to reduce the number of A. flavus as
amount of 2 log units, however impact on the
decrease in pH to 4.8 was not desired since
requirements for the quality cocoa should has a
lowest pH at pH5 (Amin, 2005).
Submersion of cocoa beans resulted the pH of dry
beans could still be maintained above pH 5, and it
still meets the quality requirements of cocoa beans,
however it was able to decrease amount of A.
flavus by 2 log units. Figure 7 showed Performance
of dry cocoa beans after submersion with isolate L.
plantarum NL-249 in existence of A. flavus. The
isolate L. plantarum NL-249 exhibited its ability to
reduce A. flavus in cocoa beans due to production
of metabolites which inhibited germination of
spores and growth, such as phenyllactic acid, phydroxy phenyl lactate, cyclic dipeptide such as
cyclo (Gly-L-Leu), cyclo (L-Phe-L-Pro), and cyclo
(L-Phe-trans-4-OH-L-Pro), benzoic acid, methyl
hydantoin, mevalonolactone and short chain of
fatty acids (Corsetti, 1998; Lavermicocca et al,
2000; Storm, 2002).
The ability of L. plantarum NL-249 to reduce S.
Typhimurium in cocoa beans may be caused by
accumulation of organic acid and production of
H2O2 by NL-249 during submersion. Organic acids
produced by NL-249 will passively diffuse into the
microbial cells in undissociated form, and then
there will be a separation of anions and protons
which will penetrate cell membrane and will affect
the integrity of the cytoplasmic membrane,
resulting in cell acidification and denaturation of
proteins, and therefore the cytoplasmic membrane
will be damaged. This will cause a disruption in the
metabolic system, such as the inhibition of
transport of substrate, energy production and
synthesis of macromolecules (Lazarova, 1994).
On dry beans which were not treated with isolate L.
plantarum NL-249 indicating the presence of S.
Typhimurium, eventhough they have received a
treatment of drying resulted in moisture content
decreased up to about 7.2% (± 0.7 aw). This is
because of resistance of S. Typhimurium to
warming and drying, as consequences of a
presence of a protective effect derived from fat of
cocoa beans (Lund, 2000).
In the treatment without application of LAB
culture, the amount of A. flavus was still high (79 x
102 CFU/ml) due to high ability for adaptation of
A. flavus in cocoa beans, caused by fat content of
beans, even if the moisture of beans were dried up
to about 7.0-7.3% (± 0.70 aw), A. flavus was still
able to survive. The optimum temperatures for A.
flavus to grow is approximately 24-28C, however
it can grow at range of 10-55C to produce
aflatoxin within a range of 7.5 to 40C. In starchy
grains such as corn and wheat, the moisture content
for the growth of A. flavus is 18.5%, whereas in
oily seeds such as beans is about 8-9%. A. flavus
can not grow well if the growth medium has a pH
below 4.0 (Christensen and Kaufman, 1974).
The existence of A. flavus in the cocoa beans has
an impact not only on performance of the cocoa
beans, but also in decreasing of lipid content from
51.2% to 40.6%. The decrease in lipid content of
cocoa beans is caused by the ability of this A.
flavus in producing lipase (Fardiaz, 1992). In
general, a mold including A. flavus can utilize
some nutrient components of food as for its growth
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Figure 6. Performance of Cocoa Beans After
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S.Typhimurium.
3.6. Effect of LAB on Growth of A. flavus
Submersion of cocoa beans in a suspension
containing isolates L. plantarum NL-249 at 107
22
Sept. 2014. Vol. 2, No.5
ISSN 2311 -2476
International Journal of Research In Agriculture and Food Sciences
© 2013 - 2014 IJRAFS & K.A.J. All rights reserved
http://www.ijsk.org/ijrafs.html
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