Journal of Life Sciences Volume 8 Number
J LS
Journal of Life Sciences
Volume 8, Number 12, December 2014 (Serial Number 80)
Contents
Microbiology
925
Antibacterial Activity of Lyngbya and Chroococcus Species Isolated from Koya (Hizoop River)
Sewgil Saaduldeen Anwer and Parween Mohsin Abdulkareem 931
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
Maciej Grabowski, Agnieszka Strzelczak and Waldemar D ąbrowski
940 Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors
Ignatova-Ivanova Tsveteslava and Radoslav Ivanov
Botany and Zoology
946
Morphogenesis of Oil Palm (Elaeis guineensis Jacq.) Fruit in Seed Development
Hermine Bille Ngalle, Joseph Martin Bell, Georges Franck Ngando-Ebongue, Hernild Eman-Evina, Godswill Ntsefong Ntsomboh and Armand Nsimi-Mva
955
Characterization in Greenhouse Conditions of Two Salt Tolerant Citrumelo (Citrus paradisi Macf. x Poncirus trifoliata (L.) Raf.) Cultivars
Anas Fadli, Ouiam Chetto, Abdelhak Talha, Rachid Benkirane, Raphaël Morillon and Hamid Benyahia
Interdisciplinary Researches
967
Economic Viability of Production of Tree Paricá (Schizolobium amazonicum Huber ex. Ducke) of Reforestation Project in the Municipality Paragominas-PA, Brazil
Manoel Tavares de Paula, Altem Nascimento Pontes, Hélio Raymundo Ferreira Filho, Lucy Anne Cardoso Lobão Gutierrez, Ismael Matos da Silva, Maria da Conceição Silva Damasceno and Aline Lima de Sena
972
Electric Energy Saving
Gordana Pintar and Marijana Magdi ć
Journal of Life Sciences 8 (2014) 925-930
doi: 10.17265/1934-7391/2014.12.001 DAVID PUBLISHING
Antibacterial Activity of Lyngbya and Chroococcus Species Isolated from Koya (Hizoop River)
Sewgil Saaduldeen Anwer and Parween Mohsin Abdulkareem Department of Biology, Faculty of Health and Science, University of Koya, Erbil 44001, Iraq
Received: December 15, 2014 / Accepted: December 28, 2014 / Published: December 30, 2014.
Abstract: In the study cyanobacterial strains were isolated from different sites of Hizoop rivers, Koya-Iraq and identified according to their morphological characters by using microscope, two genera which were in filamentous form identified as Chroococcus sp. and Lyngbya sp.. After identification of genera their optimum growth condition studied by using the effect of temperature and pH to their dry weight. In the result, the optimum temperature and pH for both filamentous cyanobacteria were 25 °C and pH 7.5. Both cyanobacterial strains were extracted with ethanol, methanol and diethyl ether at various concentrations (0.2 g/mL, 0.1 g/mL, 0.005 g/mL) which exhibited the antibacterial activities against Staphylococcus aureus, E. coli and B. subtilus. Inhibition activities of the two cyanobacterial extracts were more effective at high concentration against the tested pathogens at the low concentration, especially those of Lyngbya sp. The higher inhibition zone showed with extract by ethanol.
Key words: Cyanobacteria, pH, temperature, antibacterial activity.
1. Introduction In general, isolation of bioactive compounds from cyanobacteria is done to discover new compounds. A
Cyanobacteria are the most prokaryotic algae and number of cyanobacteria produce toxins that may have they are found in virtually every type of environment potential pharmaceutical application [4]. The authors including terrestrial, fresh water, marine habitats. had found that various strains of cyanobacteria are Since cyanobacteria are prokaryotes, they lack known to produce intracellular and extracellular membrane bound organelles however the external metabolites with diverse biological activities such as structure can change from unicellular or colonial to antibacterial, antialgal, antifungal and antiviral activity branched or unbranched and filamentous [1]. Like [5-8]. As an efficient strategy of investigation, organic rhodophytes, the cyanophytes possess no flagellated solvents have been used to extract the possible lipid or ciliated cell at any stage of their life cycle. They are soluble active principles from microalgae [8, 9]. The heavily pigmented with chlorophyll a, beta carotene, antimicrobial substances involved may target various and several xanthophylls [2, 3]. kinds of microorganisms, prokaryotes as well as The bacterial infections are still the major problem in eukaryotes. The properties of secondary metabolites in the world today, because these bacteria which causes nature are not completely understood [6, 10]. disease will eventually develop ways to resist the drugs
as well. To help preventing and treating these illnesses,
2. Materials and Methods
many researchers have studied the antimicrobial effects
2.1 Isolation and Identification
of various plants extracts, as well as antimicrobial activity of algae.
Samples were collected from sites along the Hizoop rivers Fig. 1. Water samples diluted and plated onto Corresponding author: Sewgil Saaduldeen Anwer,
plates of BG11 medium solidified with %1.5 associate professor, research field: industrial microbiology. E-mail: sevgil75@yahoo.com.
agar-agar. According to Rippka and Castenholz et al.,
Antibacterial Activity of Lyngbya and Chroococcus Species Isolated from Koya (Hizoop River)
the cultures were incubated under continues light at
10 min and kept in room temperature for 10 h. After pH 7.2 and 23 °C. Two weeks later, following the
that, the solvent was removed by incubation at 60 °C growth of colonies on the agar media, the colonies
and redissolved in water (ratio 0.2 g/mL, 0.1 g/mL were removed with pasture micropipettes and were
and 0.05 g/mL) and kept at 4 °C until use for further gently blown into liquid medium then incubated at
assay [18].
23 °C, at pH 7.2 [11, 12].
2.3.3 Antibacterial Bioassay
After 15 days single cells and filamentous Staphylococcus aureas, E. coli and Bacillus subtilus, removed with pasture micropipette and examine under
were used as test microorganisms. Antibacterial light microscope and identified as described by [10,
activity was determined by the disc method as
13, 14]. described by ghasemi et al., Filter paper disc were saturated with 20 μL of test bacteria and dried under
2.2 Effect of Environmental Factors laminar air flow and placed on nutrient agar, the plates
Strains were cultivated at different temperature were incubated at 37 °C for 24-28 h. Ampicilin,
20 °C, 23 °C, 25 °C & 30 °C and different pH 6, 6.5, 7, gentamicin and amphoterin were used as positive
7.5, 8 & 8.5 [15, 16]. control. The diameter of inhibition zones were determined and used as an indication of antibacterial
2.3 Analytical Method
activity [19].
2.3.1 Cell Dry-Weight and Fresh-Weight Estimation
3. Results
According to Oswald, samples grown at 100 mL BG11 medium After 2 weeks 10 mL of samples were
Growing cells were observed and photographed taken and cells harvested after centrifugation for 10
with light microscope, and the mode of division of min. To determine the cell dry weight, cells were
strain examined in slide culture showed the following harvested after centrifugation for 10 min, the collected
characters:
sample were dried in oven at 80 °C and weighed Filament erect or less curved, rarely solitary, ends quickly after drying [17].
not constricted and not attenuated, cross walls marked
2.3.2 Preparation of Cyanobacterial Extracts with one or two large granules on either side; and is Cyanobacterial cells were dried at 70 °C, and then
identified as Lyngbya sp. Spherical or ovate colony of the cells were grinded in sterile tubes. As described by
2-4 spherical cells. Evenly arranged cell sheath usually Thummajitsakul, cells were mixed with ethanol,
is well defined with colorless lamellate Chroococcus diethyl ether and methanol for 1 mL with shaking for
sp. (Fig. 2).
Fig. 1 Hizoop River.
Antibacterial Activity of Lyngbya and Chroococcus Species Isolated from Koya (Hizoop River)
(a)—Lyngbya sp. (b)—Chroococcus sp.
Fig. 2 Isolated unicellular cyanobacteria.
3.1 Effect of pH to Growth Rate those of Lyngbya sp. The higher inhibition zone showed with extract by Ethanol.
The higher cell dry weight was at pH 7.5 for two cyanobacteria and the lowest growth rate shown at pH
4. Discusion
8.5 for both cyanobacteria isolates (Fig. 3). Cyanobacteria grow well within the temperature
3.2 Effect of Temperature to Growth Rate range of 25-30 °C, these temperatures displays a short exponential phase, along linear phase and stationary
In Fig. 4, the temperature role in the growth of phase from about 14 days on. In this study the cyanobacterial strain, at 20 °C and 35 °C, the higher optimum temperature for the growth of filamentous growth shown at 25 °C for both cyanobacterial strains,
and unicellular cyanobacteria were around 20-35 °C, while in 20 °C, the cyanobacteria strains showed low the highest growth found at 25 °C, and the lowest
growth. growth found at 35 °C which show denaturized of
3.3 Antibacterial Activity of Chroococcus sp. and pigment after 10 days of cultivation. This result shows Lyngbya sp.
that increase of temperature from 20 °C to 30 °C caused increase of chlorophyll and cell dry weight but
The effect of ethanol, diethyl ether and methanol higher this value caused decreasing of chlorophyll-a extractions of cyanobacteria Lyngbya sp. and and cell density with cell dry weight. This result is
Chroococcus sp. on the inhibition of tested pathogens agreed with finding of Donmez et al. [21-23]. was shown in Tables 1-3. The results showed that
The effect of pH on growth rate and chlorophyll ethanol, diethy ether and methanol extracts of
content was shown in Fig. Cyanobacteria grow at all Lyngbya sp. and Chroococcus sp. at various pH values just the best growth determined at pH 7.5
concentrations (0.2 g/mL, 0.1 g/mL and 0.005 g/mL) for both cyanobacterial strain. This pH is near the pH exhibited the antibacterial activities against of isolated place. Staphylococcus aureus, E. coli and B. subtilus.
Several different organic solvents have been used to Inhibition activities of the two cyanobacterial extracts
screen cyanobacteria for antibacterial activity. The were more effective at high concentration against the
species Lyngbya and Chroococcus demonstrated the tested pathogens at the low concentration, especially
effective extracts for antibacterial activity against
Antibacterial Activity of Lyngbya and Chroococcus Species Isolated from Koya (Hizoop River)
Chroococcus sp. Chroococcus sp. 2
Lyngbya sp. Lyngbya sp. 1.5
PH7.5 g/L
PH8 g/L
PH8.5 g/L
Fig. 3 Effect of pH to dry weight of cyanobacterial strains.
2 Lyngbya sp. Chroococcus sp. Chroococcus sp. Lyngbya sp.
Fig. 4 Effect of temperature to dry weight of cyanobacterial strains.
Table 1 Antibacterial activity of Lyngbya sp. and Chroococcus sp. extracted with ethanol (diameter of inhibition zone in mm).
Chroococcus sp. (g/mL) Bacterial species 0.05 0.1 0.2 0.05 0.1 0.2
Lyngbya sp. (g/mL)
Escherichia coli 4 7 10 5 7 9 Staphylococcus aureus,
4 9 11 6 9 9 B. subtilus
Antibacterial Activity of Lyngbya and Chroococcus Species Isolated from Koya (Hizoop River)
Table 2 Antibacterial activity of Lyngbya sp. and Chroococcus sp. extracted with diethyl ether (diameter of inhibition zone in mm).
Chroococcus sp. (g/mL) Bacterial species 0.05 0.1 0.2 0.05 0.1 0.2
Lyngbya sp. (g/mL)
Escherichia coli 7 9 8367 Staphylococcus aureus
9 9 9567 B. subtilus
Table 3 Antibacterial activity of Lyngbya sp. and Chroococcus sp. extracted with methanol (diameter of inhibition zone in mm).
Chroococcus sp. (g/mL) Bacterial species 0.05 0.1 0.2 0.05 0.1 0.2
lyngbya sp. (g/mL)
Escherichia coli 579 5 7 8 Staphylococcus aureus,
598 4 6 7 B. subtilus
Escherichia coli, Staphylococcus aureus and Bacillus
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5. Conclusions and Recommendation
Compounds from Microalgae.” Enz. Microbiol. Tech. 8: 386-394.
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Phycol. 29: 125-130.
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Staphylococcus aureus and Bacillus subtilus.
Bot. Marina. 37: 357-360.
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“Antibiotic Activity of New Cyanobacterial Isolates from Australia and Asia against Green Algae and
agricultures, and for further investigations. Cyanobacteria.” J. Appl. Phycol. 10: 471-479.
The authors suggested that further work should be [10] Dakshini, K. M. M. 1994. “Algal Allelopathy.” Botanical performed on the isolation and characterization of the
Rev. 60: 182-196.
active components responsible for the antibacterial [11] Rippka, R. 1988. “Isolation and Purification of Cyanobacteria.” Method of Enzymology 167: 3-27.
activities need to be evaluated. [12] Castenholz, R., Boom, W., and Gerry, G. 2001. “The
Reference Archae and the Deepl Branching and Phototrophic
Bacteria.” Bergeys Manual of Systematic Bacteriology 2: [1] Vashishta, B. R., Sinha, A. K., and Sinh, V. P. 2002.
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[13] Prescott, G. W. 1963. Algae of the Western Great Lakes African Journal of Microbiology Research 6 (10): Area Michigan: Wm. C. Brown.
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[14] Desikachary, T. V. 1968. Cyanophyta, Chrococcales. [20] Ghasemi, Y., Yazdi, T. M., Shokravi, S., Soltani, N., and New York and London academic press.
Zarrini, G. 2003. “Antifungal and Antibacterial Activity [15] Holt, J. G., Rieg, N. R., Smeath, P. H. A., Staley, J. T.,
of Paddy Fields Cyanobacteria from the North of Iran.” J. and Williams, S. T. 1994. Bergeys Manual of
Islamic Republic of Iran 14: 203-209. Determinative Bacteriology, 9th ed.
[21] Donmez, G., Obali, A., Ozturk, A., Elmaci, A., and [16] Ratkowsky, D. A., Olley, J., Mcmeekin, T. A., and Ball,
Cakmakci, L. 1999. “Isolation and Abundance of B. 1982. “Relationship between Temperature and Growth
Unicellular Cyanobacteria from Mosquito Development Rate of Bacterial Culture.” Journal of Bacteriology 149
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[22] Dhargalkar, U. K. 2004. “Effect of Different Temperature [17] Becker, E. W. 1994. Micro Algae Biotechnology and
Regimes on the Chlorophyll-A Concentration in Four Microbiology, Measurement of Algal Growth. Cambridge
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Light Intensity, Salinity and Temperature on Growth in Browizka, M. A., and Browizka, L. J. Cambridge University.
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Turkey.” J. of Biological Sciences 8: 1356-1359. 2012. “Antibacterial Activity of Crude Extracts of
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Journal of Life Sciences 8 (2014) 931-939
doi: 10.17265/1934-7391/2014.12.002 DAVID PUBLISHING
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
1 2 Maciej Grabowski 1 , Agnieszka Strzelczak and Waldemar D ąbrowski
1. Department of Microbiology and Applied Biotechnology, West Pomeranian University of Technology in Szczecin, Szczecin 70-310, Poland 2. Department of Food Process Engineering, West Pomeranian University of Technology in Szczecin, Szczecin 70-310, Poland
Received: November 30, 2014 / Accepted: December 10, 2014 / Published: December 30, 2014.
Abstract: It was found out that spices straight from the package are not sterile. The only way to receive sterile spices is to use radiation technology which means to irradiate spices with ionizing radiation. However, this method is quite expensive and raises great resistance of public. And this is the reason why we are interested in implementing plasma technology. The first step of the research was to choose the most appropriate spice. The range of available spices is nearly unlimited, however, we took into account the following ones: sweet paprika, basil, rosemary, saffron, marjoram, thyme and black pepper. Finally, we chose black pepper because it is most often used by butchers to make meat products. It is also called the “King of Spices” or the “Black Gold”. Black pepper is one of the most often used spices in the United States and in Europe. It is important to have sterile black pepper when we aim at ripening products for example ripening sausages or some kinds of cheeses. What is more, it was found out that black pepper has antimicrobial properties, antioxidant effects and also antipyretic and analgesic properties. The aim of the research was to receive sterile spices using low pressure cold plasma with oxygen, nitrogen, air, argon and hydrogen peroxide.
Key words: Black pepper, cold plasma, sterilization.
1. Introduction bacteria in given spices. A major problem occurs when we are dealing with foods containing a lot of
In the United States in 2010 the import of spices unknown species of microorganisms. These types of was about 608 million kilograms including black infections can be caused even by streptococci (family pepper with the amount of about 73 million kilograms. Streptococceae), bacteria of the Listeria genus, In Poland the import of pepper prevails among other Legionella, Clostridium (especially dangerous are the spices and constitutes about 70%, as well as it accounts toxins produced by them), Salmonella (cause for about 80% of the Polish spice market [1, 2]. salmonellosis), spirochetes of the Leptospira genus, In 2010 alone the spices imported into the United sticks of Brucella, bacteria of the Yersinia genus, States and probably in other countries, were infected Campylobacter and Helicobacter, a closely related with the bacteria of the genus Salmonella in up to family of Actinomycetaceae, Enterobacteriaceae 6.6% of cases. It should be noted that the survival of (including a type of Klebsiella, Proteus, Serratia), these bacteria at 25 °C and relative humidity ≤ 40 is Escherichia coli bacteria, Pseudomonas aeruginosa, equal to 400 days or more. In addition, in the spices as well as non-tuberculosis mycobacteria [3], 4]. many other types of bacteria may occur [1]. Plasma is termed as the fourth state of matter. It The above mentioned incidents concern only single displays different properties as compared with a gas,
liquid and solid phase, and is formed at the Corresponding author: Maciej Grabowski, M.S., research
temperatures at which the ionization potential value is fields: plasma and food technology. E-mail:
maciejgrabowski.mg@gmail.com. exceeded by the mean values of the particles kinetic
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
energy. The change of the physical properties of the pepper powder. No literature has been found on the gas, such as the loss of the insulation and the
application of cold plasma for black pepper appearance of the electrical conductivity properties of
sterilization, therefore, we may be regarded as the the ionized gas, is considered to be the border between
pioneers in this field, and the research is largely the state of the gas and plasma. It should be noted that
justified.
some authors do not consider the plasma the fourth
2. Materials and Methods
state of matter, but the “state of dispersion of matter” or a matter in the “potential original state”. This is
2.1 Black Pepper Powder
because the molecules are formed out of the plasma, First, black pepper powder (Piper nigrum L.) was which in turn enables to define a solid, liquid and
obtained from the local store. The next step was to gaseous state [5-10].
choose a black pepper, from one out of many foreign Plasma is neutrally ionized gas which is composed
companies, including American ones, available on the of interacting particles. These include: atoms, free
Polish market. The choice was based on the following radicals, electrons, photons, positive and negative
criteria: color, smell, and friability. The aim was to ions as well as excited and unexcited molecules. In
carry out research on available commercial spices order to generate plasma, that is ionize the gas, the
produced by significant companies, in order to work selected gas, for example oxygen, air, nitrogen or
out an alternative method to be used in the future by argon, should be subjected to the impact of an
spice companies for the spice decontamination by electromagnetic field (or a magnetic or electrical one)
means of cold plasma.
of high frequency using a direct current (or alternating
2.2 Microbiological Analyses
current) and waves (for example microwaves or radiowaves) [5, 7-9, 11-13].
All microbiological analyses that have been This process may take place under atmospheric,
conducted, were based on microorganisms existing vacuum or elevated pressure [7-9, 11, 13-16]. Plasma
naturally in black pepper powder. These operates in the temperature range which can vary from
microorganisms were divided into three groups: room temperature to the temperature within the range
non-spore forming aerobic bacteria, aerobic of energy greater than a few electron volts (1 eV =
spore-forming bacteria, and anaerobic spore-forming 1,132,685 o
C) [8, 11, 15, 17-19]. Moreover, plasma bacteria. Each sample was diluted and streaked on an can be generated either with the thermodynamic
appropriate medium. The amount of a sample applied equilibrium behavior or without it [8, 11]. In addition
on each Petri dish was 0.1 mL. Non-spore forming to plasma classification according to pressure, aerobic bacteria were streaked onto Nutrient agar, temperature and thermodynamic equilibrium, it also
with the parameters of the incubation 30 o C/24 h and divided according to the type of discharge. Each type
aerobic conditions, whereas aerobic spore-forming of the discharge in which the plasma is formed is
bacteria onto nutrient agar with manganese sulphate characterized by different parameters such as with the parameters of the incubation 37 o C/24 h and frequency, power, the distance of the electrode from
aerobic conditions, and anaerobic spore-forming the sample, voltage, current, pressure or the type of
bacteria onto Scheadler agar with the parameters of the gas used [8, 20, 21].
the incubation 37 o C/48 h and anaerobic conditions. To this day, only one example of the application of
All microbiological results were obtained in three cold plasma for microbial decontamination has been
repetitions. The total result was counted by means of found in the available literature, yet it concerns red
Farmiloe pattern.
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
2.3 Inoculation and Sample Preparation power of 400 Watts. The structure of this generator was identical to the one of Diener device.
In the case of using low pressure non-equilibrium At the beginning the sample is put into the cold plasma, all samples were dried in advance. This chamber. After that, the pressure is lowered to the was due to the fact that the process would take too most desired level. This is the reason why the long without partial drying. spices are dried in advance. If the water activity is
2.4 Low Pressure Cold Plasma System too high, the vacuum pump, which provides relatively low pressure, needs more time to lower it and thus
Diener plasma generator (Diener Electronic company) prolongs the whole process. Then the gas is generates low pressure (0.3 mbar) non-equilibrium
introduced into the chamber. The gas ionization cold plasma (Fig. 1). The plasma was produced at the
then starts after which plasma sterilization takes radio frequency of 13.56 MHz and the maximum
place.
power of 300 Watts. Plasma can be generated either in
a continuous or pulsed way. Additionally, a stationary
2.6 Temperature, Mass and Water Activity Measurements of Black Pepper Powder
(sample set on a shelf) or a rotary (sample placed in a special jar) variant may be used. The gases selected
The temperature of the process was measured by for the research included oxygen, nitrogen, air and
means of a built-in temperature sensor and an argon. The sterilization time was equal to 15, 30, 45
additional thermometer. The mass was checked with and 60 min respectively.
technical weight whereas a special meter was used in What is more, advanced Sterilization Products
order to measure water activity.
(Johnson & Johnson Company) plasma generator was
2.7 Statistical Analysis
also used. In this case, the pressure of up to 9 mbar was applied during the 60 min plasma sterilization by
The statistical significance of the low-pressure cold means of hydro peroxide. The plasma was produced at
plasma influence on the bactericidal activity of black the radio frequency of 13.56 MHz and the maximum
ground pepper was revealed by means of the U
Control panel (used for control
the process)
Chamber were plasma is generated
Vacuum pump
Gas
Fig. 1 Low pressure, cold, non-equilibrium plasma reactor.
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
Mann-Whitney nonparametric test due to In the case of hydrogen peroxide application, after non-normality of the data set (P < 0.05 in chi-square
60 min the decrease of non-spore forming aerobic and Kolmogorow-Smirnow tests). Calculations were
5 bacteria 2 was noted from 10 to 10 , of aerobic performed in Statistica 10 software.
5 spore-forming 3 bacteria from 10 to 10 , and of
3. Results and Discussion
anaerobic spore-forming bacteria from 10 to 10 . After six months’ storage, the number of each type of
3.1 Decontamination of the Microorganisms microorganisms was below a detection limit. It should To start with, it needs to be mentioned that the
be mentioned that in the case of the samples
article is based on preliminary results. The numbers of undergoing plasma sterilization by use of oxygen, microorganisms in black pepper powder after plasma
nitrogen, air and argon, the layer thickness was 4.3 sterilization with oxygen, nitrogen, air and argon are
mM, whereas 18.5 mM in the case of the sample used given in Figs. 2-5.
for plasma sterilization by use of hydrogen peroxide.
1.00E+06
1.00E+05
1.00E+04 Non-spore forming aerobic
1.00E+03
bacteria Aerobic spore-forming
1.00E+02
bacteria Anaerobic spore-forming
1.00E+01
bacteria
Number of microorganisms [cfu/g]
1.00E+00
Duration of plasma sterilization process [min]
Fig. 2 The number of microorganisms after plasma sterilization by use of oxygen.
1.00E+06
1.00E+05
1.00E+04 Non-spore forming aerobic bacteria
1.00E+03 Aerobic spore-forming bacteria
1.00E+02 Anaerobic spore-forming bacteria
1.00E+01
Number of microorganisms [cfu/g]
1.00E+00
Duration of plasma sterilization process [min]
Fig. 3 The number of microorganisms after plasma sterilization by use of nitrogen.
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
1.00E+04 Non-spore forming aerobic
1.00E+03
bacteria Aerobic spore-forming
bacteria
1.00E+02 Anaerobic spore-forming bacteria
1.00E+01
Number of microorganisms [cfu/g]
1.00E+00
Duration of plasma sterilization process [min]
Fig. 4 The number of microorganisms after plasma sterilization by use of air.
1.00E+06
1.00E+05
1.00E+04 Non-spore forming aerobic
1.00E+03
bacteria Aerobic spore-forming
1.00E+02
bacteria Anaerobic spore-forming
bacteria
1.00E+01
Number of microorganisms [cfu/g]
1.00E+00
Duration of plasma sterilization process [min]
Fig. 5 The number of microorganisms after plasma sterilization by use of argon.
After 60 min, black pepper sterilization process with the decrease in the number of microorganisms of three the use of cold rotating continuous plasma by use of
logarithms. What is more, this process did not affect oxygen, the decrease non-spore forming aerobic
the possibility of growing grains such as wheat [22].
5 bacteria was from 10 4 to 10 , of aerobic spore-forming Other researches tried to sterilize Aspergillus bacteria from 10 5 to 10 4 , and of anaerobic parasiticus from nuts’ surfaces by means of SF 6 with spore-forming bacteria was below detection limit.
five logarithm effectiveness and one logarythm loss Some researchers infected legumes and grains with
after 5 min plasma sterilization with air. The pressure Penicillum spp. and Aspergillus spp.. After this, they
was between 0.13 mbar and 0.65 mbar [17]. The tried to decontaminate the samples with low pressure
authors claim that SF 6 is non toxic gas, however, it cold plasma. The pressure was between 0.13 mbar and
can decompose, especially when discharges come to
action, for example SF4 (sulfur tetrafluoride highly hexafluoride). After 15 min, the researches obtained
0.65 mbar, and the used gas was SF 6 (sulfur
toxic), SO 2 F 2 (sulfuryl fluoride—toxic) or S 2 F 10
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
(sulfur decafluoride—highly toxic) [17, 23]. The case non-spore forming aerobic bacteria obtained after 60 of paprika powder is especially interesting. A research
min plasma sterilization with oxygen in a stationary group sterilizing the spice under the pressure varying
variant.
from 5 to 300 mbar, inoculated Aspergillus flavus
3.2 Mass and Water Activity
spores to red pepper and then lowered it by 2.5 logarithms after 20 min sterilization by means of
In the case of using low pressure non-equilibrium nitrogen [24].
cold plasma, all samples were dried in advance. This As mentioned above, in the comparative studies,
was due to the fact that the process would take too statistical results were obtained by means of a
long without drying. Thus, the initial water activity of nonparametric test, which is U Mann-Whitney test.
black pepper averaged 0.510 whereas after three hours’ Under a detection limit, significant differences drying in the laboratory drier at 50 °C and the mill set observed in each case were the findings. The time of
at 50% of the maximum power, water activity was sterilization was different for each applied gas and
0.210 on average. The loss of weight after drying was was equal to 15, 30, 45 and 60 min respectively.
approximately 0.33 g for the samples with mass of 10 Statistically significant differences (P < 0.05) were
g. Weight losses for ground black pepper are shown in attested under the following conditions shown in
Table 2 and Fig. 6.
Table 1. After 60 min black pepper sterilization with the use Importantly, both statistically and microbiologically
of cold rotating continuous plasma and with the significant findings include all those under the
application of air, the loss of the mass of the black detection limit [25] as well as the ones concerning
pepper after the trial was 2.28 g on average. The water
Table 1 Statistical significant differences in the number of microorganism occurring in black pepper powder after low pressure, cold plasma sterilization process.
Type of gas Sterilization time
Non-spore forming
Aerobic spore forming
Anaerobic spore forming
bacteria 15 x 0 0 30 0 0 x
aerobic bacteria
bacteria
Oxygen 45 x x x
60 x x 0 Oxygen rotary variant
x 15 x 0 0 30 x 0 0
60 x
Nitrogen 45 x x 0
Argon 45 x x 0
60 x x x Hydrogen peroxide
60 x
x x—statistically significant (P < 0.05, U Mann-Whitney test) 0—not statistically significant (P 0.05, U Mann-Whitney test)
Hydrogen peroxide
60 x
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
Loss of weight after sterilization with
eight
oxygen [%]
6 Loss of weight after sterilization with nitrogen [%]
Loss of
5 Loss of weight after sterilization with air
3 Loss of weight after sterilization with argon [%]
Sterilization time [min]
Fig. 6 Dependence of weight loss of black pepper on sterilization time with low pressure, non-equilibrium, cold plasma.
Table 2 The weight loss of black pepper after the sterilization process with low pressure, non-equilibrium, cold plasma, depending on the duration of the process.
Sterilization time [min] 15 30 45 60 Loss of weight after sterilization with oxygen [g]
0.89 0.96 0.90 0.91 Loss of weight after sterilization with nitrogen [g]
0.75 0.87 0.91 1.01 Loss of weight after sterilization with air [g]
0.61 0.76 1.00 1.08 Loss of weight after sterilization with argon [g]
Table 3 The temperature of black pepper after the sterilization by means of low-pressure, non-equilibrium cold plasma versus time process.
Sterilization time [min] 15 30 45 60 The temperature after sterilization in an oxygen environment [°C]
23-27 19-23 The temperature after sterilization in an nitrogen environment [°C]
28-29
27-28
23-27 27-30 The temperature after sterilization in an air environment [°C]
20-21
21-23
23-26 27-30 The temperature after sterilization in an argon environment [°C]
23-26 26-29
activity after the plasma sterilization process was from equal to 0.3, which resulted in the loss of mass [24]. about 0.055 for the duration equal to 15 min, up to
3.3 Temperature Measurement
0.020 for 60 min. During the sterilization of black pepper with hydrogen peroxide, the mass decrease and
Because of the fact that low-pressure plasma is also the water activity could not have been measured due
non-equilibrium plasma, the distribution of to the action of the electrostatic forces and the fact that
temperatures was variable (Table 3). During the the plasma sterilization packaging constitutes a barrier
sterilization by means of low pressure cold plasma in to bacteria exclusively.
a rotary variant, a similar relationship was observed. When Aspergillus flavus spores fungi were removed
In the case of plazma sterilization with hydrogen from the red pepper powder, the level of water activity
peroxide, the temperature of the samples after 60 min was also lowered. At the deepest point it was even
was about 25 °C.
Low Pressure Cold Plasma as an Alternative Method for Black Pepper Sterilization
The temperature generated during the plasma atmospheric pressure cold plasma is expected to give sterilization process in order to eliminate Penicillum
much more promising results than low pressure cold spp. and Aspergillus spp. from grains and legumes
plasma.
was within room temperature to enable their further
Acknowledgments
growth [22]. The temperature in the low pressure chamber generating cold plasma which was used for
Partly supported by the European Union within the the sterilization of nuts’ surfaces from Aspergillus
Human Capital Operational Programme, Priority VIII, parasiticus varied from 20 °C to 30 °C [17]. In the
Activity 8.2, Subactivity 8.2.2; Investment in case when low pressure cold plasma was applied on
Knowledge as Regional Development Tool, Contract sweet pepper, the temperature was from 23.8 °C to
no. WUP/142/2014 and by the Polish Ministry of
28.5 °C [24]. Science and Higher Education, Innovation Incubator Project, Contract no. DS/1558/11/W15/POIG/II/2014
3.4 Organoleptic Analysis
(internal no. 5/II/2014).
Unfortunately, no sensoric panel was used during To anyone interested in the use of plasma for spice the research, however, it was noticed that after the
sterilization, please meet me at “Natural Products application of nitrogen, air and argon, a number of
Expo East 2015” Baltimore Convention Center, lumps appeared, whereas after the use of oxygen and
Baltimore, MD USA on 16-19 September, 2015. hydrogen peroxide no lumps occured. In each case
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Journal of Life Sciences 8 (2014) 940-945
doi: 10.17265/1934-7391/2014.12.003 DAVID PUBLISHING
Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors
Ignatova-Ivanova Tsveteslava and Radoslav Ivanov Department of Biology, University of Shumen, Shumen 9712, Bulgaria
Received: November 27, 2014 / Accepted: December 12, 2014 / Published: December 30, 2014.
Abstract: Bacterial EPSs (exopolysaccharides) are believed to play an important role in the environment by promoting survival strategies such as bacterial attachment to surfaces and nutrient trapping, which facilitate processes of biofilm formation and development. These microbial biofilms have been implicated in corrosion of metals, bacterial attachment to prosthetic devices, fouling of heat exchange surfaces, toxicant immobilization, and fouling of ship hulls. In this paper, data on EPS production and the effect of EPS on corrosion of steel produced by Lactobacillus fermentum Ts are presented and discussed. The Lactobacillus fermentum Ts strain was isolated from types of Bulgarian rye flour. It was tested for its ability to produce exopolysaccharides when cultivated in a media containing 10% sucrose, 10% fructose and 10% maltose. The study of the corrosive stability of steel samples was conducted on the gravimetrique method. The rate of corrosion, the degree of protection, and coefficient of protection have been calculated. The structure of layer over steel plates was analysed by SEM (scanning electron microscopy) JSM 5510. It could be underlined that 10% sucrose and 10% maltose in the media stimulated the process of protection of corrosion.
Key words: Corrosion, inhibitor, lactic acid bacteria, SEM.
I. Introduction in polysaccharides produced by microorganisms. The physiological role of EPS depends on the ecological
EPS (exopolysaccharide) is a term first used by niches and the natural environment in which Sutherland [1] to describe high-molecular-weight microorganisms have been isolated. carbohydrate polymers produced by marine bacteria. Bacterial EPSs (exopolysaccharides) are believed to EPSs can be found as in capsular material or as play an important role in the environment by dispersed slime in the surrounding environment with promoting survival strategies such as bacterial no obvious association to any one particular cell [2]. attachment to surfaces and nutrient trapping, which Considerable progress has been made in discovering facilitate processes of biofilm formation and and developing new microbial EPSs that possess development [4]. These microbial biofilms have been novel industrial significance [3]. A vast number of implicated in corrosion of metals, bacterial attachment microbial EPSs were reported over the last decades, to prosthetic devices, fouling of heat exchange and their composition, structure, biosynthesis and surfaces, toxicant immobilization, and fouling of ship functional properties have been extensively studied. In
hulls [5-7].
recent years the increased demand for natural Corrosion of metals is a serious and challenging polymers for pharmaceutical, food, and other problem faced worldwide by industry. It has been industrial applications has led to a remarkable interest estimated that the yearly corrosion damage costs are
currently equivalent to 4.2% of the U.S. gross national Corresponding author: Ignatova-Ivanova Tsveteslava,
Ph.D., associate professor, research fields: lactic acid bacteria product. These costs could be greatly reduced by as probiotics products, prebiotics—utilization of lactic acid
better and wider use of corrosion protection techniques. bacteria, exopolysaccharides, corrosion, isolating
microorganisms from Antarctica. E-mail: radi_cvet@abv.bg. Traditional methods of corrosion protection involve
Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors
the use of organic coatings to protect metal surfaces
2.3 Study of the Corrosive Stability through barrier and passivation mechanisms. However,
The study of the corrosive stability of steel samples these coatings are not permanent and the cost of was conducted with the gravimetrique method [10]. applying organic coatings on corroding components in Before use, steel panels ( 10 × 4 × 0.2 mM) were use is extremely prohibitive. Applying coatings before treated with 70% C 2 H 5 OH, washed with water and the components are introduced into service involves dried in an oven, cooled in a desiccator, weighed on a excessive costs because they are susceptible to balance and kept in a desiccator unit used. The weight abrasions and other forms of mechanically induced of the samples was measured using analytical balances. damage. Thus, a coating that can be easily applied and The dimensions of the samples were measured with maintained on corroding parts and is cost-effective is micrometer. Three types of experimental series were an attractive alternative to the prevention methods
performed:
currently in use. Since bacteria can coat metals with a (a) cultivation of the studied strain in mMRS media regenerative biofilm, it is becoming evident that they
with 10 % of sucrose;
may be used as a means of preventing corrosion [8]. (b) in mMRS media with 10% fructose; In this paper, data on EPS production and the effect (c) in mMRS media with 10% maltose. of EPS on corrosion of steel produced by Lactobacillus Initially the steel samples were added in two fermentum Ts are presented and discussed. variants: deproteinised supernatant and free cell
2. Materials and Methods
supernatant. Then the steel samples were added in seawater as control probe and a dilution (3: 100) of the
2.1 Strain cultural media of the studied strain was added as
Strain Lactobacillus fermentum Ts was obtained inhibitor of the corrosion. The duration of the from the collection of the Department of Biology,
procedure was 120 h at 18 °C. After the treatment the Shumen University. Molecular analysis in LAB (lactic
steel samples were washed with water and dried to acid bacteria) was performed by molecular constant weight. identification (16S rRNA gene sequencing) in GeXP
The structure of layer over steel plates was analised Genetic Analysis System (Beckman Coulter, USA)
by SEM (scanning electron microscopy) JSM 5510. [9].
2.4 Parameters of Corrosion
2.2 Media After retrieval, the corrosion products were
The strain cultivated in media of MRS (de Mann removed when washed with water. They were dried in Rogosa Sharpe, Biolife 272-20128, Milano, Italia) in
an oven. After the removal of corrosion, steel plates composition, g/L: Tween 80—1; pepton from were cleaned and reweighed as above to estimate casein—10.0; meat extract—8.0; yeast extract—4.0;
weight loss.
K 2 HPO 4 —2.0; sodium acetat—5.0; amonium The rate of corrosion, the degree of protection, and
coefficient of protection were calculated. The pH of media was adjusted to 6.5 with 1 M NaOH. 2 The corrosion rate K (g/cm ·h) was presented as
citrate—2.0; MgSO 4 ·7H 2 O—0.2 and MnSO 4 —0.05.
The basic media was sterilized by autoclaving at
follows: