Bacterial control with Mentha spicata organic compounds in a

Bioethanol producing unit

Keywords:
Peppermint essential oil, yeast antibiotics, microbial control.
057-064 | JRM | 2012 | Vol 1 | No 1
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Journal of Research in
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Authors:
Marcia Nalesso Costa
Harder
1
.


Institution:
1.Professor Dr. - Technology
College of Piracicaba -
FATEC-Piracicaba - Rua
Dicono Jair de Oliveira, s/n
Santa Rosa CEP 13414-141 -
Piracicaba - SP-Telefone 55
(19) 3413-1702. Post-
Doctoral IPEN/CENEN/
USP.



Corresponding author:
Marcia NC. Harder.



E-mail:
marcia.harder@fatec.sp.gov.br.








Web Address:
http://ficuspublishers.com/
documents/MI0009.pdf.


Dates:
Received: 14 Mar 2012 Accepted: 01 Jun 2012 Published: 09 Jul 2012
Article Citation:
Marcia NC. Harder.
Bacterial Control with Mentha spicata organic compounds in a Bioethanol producing
unit.
Journal of Research in Microbes (2012) 1(1): 057-064
An International Online Open Access
Publication group
Original Research
Journal of Research in Microbes
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ABSTRACT:

Currently, the world has started focused on environmental concerns and the
nations have adopted government policies to mitigate and find solutions to such
problems. One of the main factors that contributes to this environmental degradation
is the use of fossil energy sources and high emission of Carbon dioxide in the
atmosphere. Brazil is appointed as one of the leading producers of the world
sustainable energy sources from sugar cane. The product obtained from this process
yields a highly combustible compound; the bioethanol. Although, there are renewable
energy sources which make an example of sustainability, their production requires
some care to increase their productivity. Microbial contamination is a peculiar
problem undesirable for the production, as the invading microorganisms compete for
nutrients and substrate with the promoters of ethanol. The aim of this research is to
use organic compounds for optimizing the production of bioethanol which assessed
the use of peppermint essential oil as antibiotic agent in the fermentation process
since this is the most critical step of the process to obtain bioethanol. With the
presented results we can be noted that the peppermint is a potential replacement for
the synthetic antibiotics but there still needs more conclusive studies.


INTRODUCTION
One of the key factors to obtain a good
production of bioethanol is the fermentation and in this
process there are several factors that increase or decrease
the rate of production and one of the factors is the
bacterial presence that compete with the yeast
(Saccharomyces cerevisiae) during this process thus
prove a considerable decrease in production.
The decreased production of ethanol
fermentation by contaminating bacteria in a production
unit shows the largest impact on productivity on an
industrial scale.
Due to the difficulty of asepsis in large volumes
working with a living population of bacteria in the
fermentation environment is inevitable.
Several drawbacks are associated with bacterial
contamination. Not only the consumption of sugar for the
formation of microbial biomass which is negligible but
also diversion of sugar for the formation of lactic acid
and acetic acid. In the case of heterofermentative
bacteria, it also should consider the formation of
mannitol and so far no one has an overview of the
formation of this polyol in industrial fermentations.
Fermentations conducted in the laboratory showed
significant fraction of the sugar being diverted to the
formation of this bacterial product (Basso, 2012).
According to the same author depending on the
level of contamination, bacteria may also cause
flocculation of the yeast resulting in operational
problems especially in the centrifuge and decreasing the
concentration of yeast in cream. Flocculation also
reduces the contact surface between the yeast and the
environment increasing the fermentation time.
Lactic acid increases the acidity produced
damaging the yeast. Also the increase in the proportion
of walk-in foot tub wine (due to lower concentration of
yeast in the centrifuge) results in an increase in the
power of the same buffer resulting in a higher
consumption of sulfuric acid in the treatment of yeast
(Basso, 2012).
There is a great biodiversity in the predominant
lactic acid bacteria in bacterial fermentation but few are
those have been characterized according to their
metabolic profile and its effect on fermentation. More
frequently encountered are the Lactobacillus
(L. fermentum, L. plantarum, L. buchneri, L. fructosus,
L. fructivorans, L. vaccinostercus) and Bacillus
(B. subtilis, B. coagulans) but the ratio is large. This
biodiversity is certainly greater than that already
established since techniques have shown the presence of
molecular species of uncultured in conventional means
(Basso, 2012).
Few studies address the relationship of
coexistence between yeasts and bacteria as occurred in
the fermentation, and most reports tend to explain the
deleterious effect of bacteria on the yeast. However, the
yeast also show antagonistic effects against bacteria, the
same effect was attributed, amongst others, by the action
of synergism with succinic acid in ethanol; both were
formed by the yeast. Just curious citing as an example,
but that leads us to a reflection in the industrial
production of lactic acid by bacterial fermentation was
observed that the presence of yeast (for their invertase
activity) stimulates bacterial growth and production of
this acid (Basso, 2012).
The fermentation process incorporating a number
of characteristics that differentiate them from chemicals
which may explain the difficulties encountered in
formulating mathematical models that represent them
adequately contrary to what occurs in conventional
chemical processes. These characteristics may be
mentioned the following: low concentrations and low
reaction rate as a result of the use of a diluted medium;
complexity of the reaction mixture and the system
capacity (microbial cells) to synthesize their own
catalyst; insufficient knowledge of various phenomena
limiting the production speed sensors and lack of on-line
automation; problems of sterility; safety and toxicity may
Harder, 2012
058 Journal of Research in Microbes (2012) 1(1): 057-064
be the fermentation itself.
The knowledge accumulated in recent decades by
biochemists on the metabolic pathways of
microorganisms together with the experience of
engineers in the mathematical description of chemical
reactors were catalyzed by the increasing availability and
processing capacity of microcomputers nowadays. The
result of this interdisciplinary synergy was a quantum
leap in an attempt to quantify some processes catalyzed
by microorganisms (biocatalysts) culminating in the
emergence of biotechnology and more recently the
bioengineering. The interdisciplinary nature of food
engineering presents a major challenge, however recent
advances in their individual areas of expertise make
interdisciplinarity to present itself an exciting task.
Biotechnology also includes the study of
microbiological systems, their products and their
applications. Between traditional biotechnology and
recent innovations there is a long history of research and
development, with a large number of ventures and
collaborations between microbiologists, chemists and
engineers. Being a "clean" technology and safe when
compared to traditional chemical processes and provide
extremely useful and valuable products for various
industries (pharmaceuticals; food; fuel; etc)
Biotechnology is becoming increasingly present and
important activities of contemporary society.
Despite the wide variety of antimicrobial agents
that act on various pathogenic microorganisms
antimicrobial studies searching for an ideal ie one that
has broader spectrum of action; lower toxicity; lower
cost and reduced evidence of bacterial resistance
considering that already exists bacterial resistance some
antimicrobial products (Nascimento et al., 2000;
Pazhani, 2004). The antimicrobial activity desired can be
found in species of medicinal plants. The present flora is
highly diverse in species that mostly has not been
scientifically studied for their antimicrobial activity
(Simes et al., 2001; Auricchio & Bacchi, 2003;
Carretto, 2007).
There were scientifically used popular plant in
order to obtain a variety of pharmacological effects
including their use as antimicrobials (Pozetti et al., 1972;
Kumar & Berwal, 1998, Castro et al., 2000;
Koo et al., 2003; Burt, 2004). The use of medicinal
plants has made many to grown it in the backyard as a
secular practice, based on popular knowledge and oral
transmission in most situations (Nascimento et al., 2000,
Castro et al., 2000).
Carretto (2007) tested various medicinal essences
and found that the 20% aqueous extracts of ginger and
mint showed zones of inhibition greater than that
observed by the action of vancomycin on
Staphylococcus aureus. The remaining aqueous extracts
studied showed inhibition zones below those given by
antibiotics corresponding to each bacterial species.
The halos found in this study are higher than the values
found in literature on the genus Shigella sp. when
subjected to the action of extracts from guava whose halo
was 15mm (Carvalho et al., 2002).
According to this author, the alcoholic extracts of
all plants tested showed inhibitory activity against
Shigella flexineri. For most of the extracts, it showed a
halo formation. These can be researched with further
concentration or test plants during the chemical
fractionation of the components as prospects for better
results and application of these extracts as antibiotics.
Bacteria and fungi are able to acquire and
transmit genetic resistance to drugs that are used as
therapeutic agents. The problem of microbial resistance
is growing and the future prospect of the use of
antimicrobials are uncertain. Therefore it becomes
necessary to adopt measures to address this problem
since the control of antimicrobial use the development of
research to better understand the genetic mechanisms of
microbial resistance and the development of research on
new synthetic and natural drugs (Loguercio et al., 2005).
The plants synthesize chemical compounds from
Harder, 2012
Journal of Research in Microbes (2012) 1(1): 057-064 059


the nutrients; water and light receiving. Many of these
compounds can cause reactions in organisms as they are
the active ingredients. Some of these substances may or
may not be toxic depending on the dosage that are used.
Thus a medicinal plant is one that contains at least one
active ingredient conferring therapeutic activity
(Martins et al., 1998).
Many extracts and essential oils isolated from
plants have shown antimicrobial activity. This
antimicrobial activity in most cases represents an
extension of the role they play in plants defending
them from phytopathogenic bacteria and fungi
(Siani et al., 2000).
Plants are important sources of biologically
active substances ie substances that have some activity
on the metabolism of a living organism different from
that in which it was produced which may have
pharmacological activity. The structural diversity and
chemical properties of these substances occurring in
nature is striking and can often be used directly or
indirectly to the synthesis and development of a large
number of drugs.
The mint (Mentha piperita L.) and peppermint
(Mentha spicata) aromatic plants of the Labiatae family
produce essential oils rich in menthone (14-32%) and
menthol (30-50%). Menthol has wide application in
food; pharmaceutical; toiletries and tobacco.
Commercialized a year over five thousand tons of
essential oil of mint for a total of fifty million dollars a
year (Garlet, 2007).
The species is named in the Amazon region as
pepper, peppermint and in the Forest Atlantic as just
peppermint; a name given in Brazil. Among its
synonyms are little peppermint; mint-the-kitchen; mint
and peppermint true.
It is an aromatic herb; annual or perennial for
about 30cm tall; semi-erect; with branches of dark green
to purple. The leaves are elliptical-acuminate; toothed
and pubescent. It was migrated originally from Europe in
the period of colonization of the country being highly
cultivated as a medicinal plant in beds; gardens and
backyards all over Brazil. In the Brazilian Northeast, it is
the only plants that grown in humid mountain with
mountain climate bloom once a year. Its cultivation is
made from pieces of branches underground, and must be
replanted every six months to ensure the good quality of
the plant.
Essential oils are associated with various
functions necessary to the survival of the plant in its
ecosystem playing a key role in defense against
microorganisms and predators and also in attracting
insects and other fecund agents protection against
water loss and temperature rise (Siani et al., 2000;
Cechinel, 2003).
Chemically most of them essential oils consist of
terpenic substances and possibly phenylpropanoids plus
smaller molecules such as alcohols; esters; ketones;
aldehydes and short chain. The profile provides generally
terpenic substances consisting of molecules of ten and
fifteen carbons (monoterpenes and sesquiterpenes)
(Siani et al., 2000).
In his research Carretto (2007) concluded that:
a) the hydroalcoholic extract of Mentha piperita L.
showed fungistatic activity for some strains of Candida
albicans; C. tropicalis and C. glabrata when compared
to the control group; b) the hydroalcoholic extract of
Mentha piperita L. showed antifungal activity on all
strains of Candida species in the control group; c) the
essential oil of Mentha piperita L. exhibited higher
inhibitory activity for strains of C. albicans; C. tropicalis
and followed by C. parapsilosis; d) the infusion of
Mentha piperita L. had no effect on the adherence of
Candida to the acrylic resin in the strains observed.

MATERIAL AND METHODS
Cane sugar was used for obtaining ethanol by
fermentation broth. All experiments are done on the
Department of plant production of the College of
Harder, 2012
060 Journal of Research in Microbes (2012) 1(1): 057-064
Agriculture "Luiz de Queiroz"; University of Sao
Paulo - ESALQ/USP.
This crop was harvested and immediately taken
to the premises of FATEC-Piracicaba where it was
milled to extract the juice poured into the fermentation
vats.
The yeast (Saccharomyces cerevisiae) used in the
experiment was a commercial bakery lyophilized type in
the proper ratio for fermentation commonly used in
FATEC-Piracicaba.
The active ingredients of peppermint were
obtained from essential oil plant extracted by the method
of entrainment of steam rotary vacuum evaporator
according to a methodology adapted from Almeida
(2006).
The fermentation of each treatment was
standardized and performed according to the standard
protocol of the institution.
We used two treatments: 0.2 and 0.5% of
essential oil extracted from peppermint and the control
(no oil) (CTC, 2005). Each treatment had three replicates
mounted on the statistical design of random blocks.
The microbiological analyzes are performed at
each periods in accordance with standards established by
the institution and according to the protocol manual
Sugarcane Technology Center (CTC) to check the
effectiveness of essences.
Methodology was used for light microscopy by
staining the viable cell of yeasts with methylene blue in a
Neubauer chamber adapted from the methodology
(Antonini, 2004).
The assessment of cell viability was given by the
formula described by Antonini (2004), where:
Viability (%) = (Number of living cells / total cells) X 100

RESULTS AND DISCUSSION
The results are shown in Figures 1, 2 and 3
below:
According to Figure 1 it can be noted that the
indicated element blank it is contaminating bacteria, the
element indicated in red yeast is a viable blue and the
indicated element is some viable yeast.
It can be seen that there are a number of viable
cells and a greater number of viable cells moreover
presents different structures of microbial cells of yeasts
characterized contamination of the environment.
On Figure 2 despite having some non-viable
yeast cells, it has a very large number of viable cells;
visible in greater quantities than the control. Also it is
noted that the yeast cells were multiplicated (green)
indicating that the substance used in the treatment had
little significant effect on the development of
microorganism.
No bacteria were found indicating a possible
elimination of bacterial contaminants from the material
analyzed.
In Figure 3 it is noted that, it does not have non-
viable cells that there is also the presence of cell
multiplication and absence of contaminating cells but the
number of viable cells is presented in number and
reduced as compared to the control and the treatment of
0.2% indicating that the this dose can influence the
number of viable cells of yeast during fermentation.
According to data presented in Table 1 it can be
observed that the presence of the essential oil of
spearmint does not adversely affect cell viability of yeast
cells.

Harder, 2012
Journal of Research in Microbes (2012) 1(1): 057-064 061
Figure 1 - Evaluation of the control fermentation


The results do not agree with the results obtained
by Drumond et al., (2004) who studied with several
natural products including a peppermint based product
for bacterial control that found no action on the
microorganisms analyzed.
Peixoto (2010) in studies with peppermint
essential oil to control Candida sp. found that in addition
to efficiency in controlling this organism and non-
toxicity pappermint against other cells present in the
environment which is to reaffirm the information
obtained in this work.
Moreover; Ernandes and Garcia-Cruz (2007) and
Trajano et al., (2009) evaluated the active principles of
spices that includes peppermint for food conservation
and found efficient use of this plant confirming once
again what has been achieved in this work.
This research is according to Dhole et al., (2011)
and Mydeen et al., (2011) that say that is necessary to
determine the active phytochemicals ingredients
contributes for antimicrobial activity from weed and
herbal species and is necessary to confirm the possible
use as a source of antimicrobial agent. Also to determine
their minimum inhibitory concentration (MIC) values
required for demonstrating the antimicrobial activity.

CONCLUSION
In analyzing the results obtained we observed
that the use of peppermint essential oil as an inhibitor of
bacterial growth during the ethanolic fermentation can be
a good choice to replace chemical additives and
antibiotics during the fermentation process. However we
suggest further studies with respect to confirmation and
possible need for concomitant use with other active
ingredients.


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Harder, 2012
062 Journal of Research in Microbes (2012) 1(1): 057-064
Table 1 - Mean values of yeast cell viability
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Treatment Control 0.2% 0.5%
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Harder, 2012
064 Journal of Research in Microbes (2012) 1(1): 057-064
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