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1.1 Bio-Preservation of Food With Lactic Acid Bacteria (LAB)
1.1 Bio-Preservation of Food With Lactic Acid Bacteria (LAB)
LAB has unique physiological properties like high resistance to bacteriophages, proteolytic
activity, lyophilization, freezing, production of polysaccharides and antimicrobial substances,
ability of adhesion and colonization. They are considered as GRAS (Generally Recognized
As Safe) due to their history of safe usage. The major compounds produced during bio-
preservation of food using LAB are hydrogen peroxide, carbon dioxide, organic acids and
bacteriocins.
Lactic Acids:
The most important antimicrobial effect of LAB is the secretion of lactic acid (Mobolaji &
Wuraola, 2011). Lactic acids carry out their antimicrobial effect by interfering with the cell
membrane maintenance. This is followed by reduction of active transport, intracellular pH
and several other metabolic functions of the cell (Rattanachaikunsopon & Phumkhachorn,
2010). The culture composition, growth conditions and the strain of LAB used determines
the lactic acid production and pH reduction (Olaoye & Onilude, 2011). They are capable of
restricting the actions of gram-positive and gram-negative bacteria, yeast and moulds
(Rattanachaikunsopon & Phumkhachorn, 2010). The sensitivity to lactic acid differs in
various micro-organisms. For example: At low pH, lactic acid effects the bacteria, fungi and
yeasts but at pH of 5, lactic acid is harmful only for spore-forming bacteria and not for yeasts
and moulds (Yang, 2000).
Hydrogen peroxide
Hydrogen peroxide (H2O2) is used for carrying out advanced oxidation and other biochemical
processes in various fields like food, pharmaceuticals, dental products, textiles,
environmental protection (Abbas et. al., 2010). H2O2 is secreted by LAB in the presence of
oxygen. The antimicrobial effect of H2O2 is due to the oxidation of sulfhydryl groups. It leads
to denaturing of enzymes and the effect of hydrogen peroxide on the membrane increases its
permeability. This is followed by release of bactericidal free radicals such as superoxide (O-
2) and hydroxyl (OH-) radicals that are harmful for DNA (Yang, 2000; Ammor et al., 2006;
Sunil & Narayana, 2008).
It also oxidizes cellular proteins and is produced by using enzymes like flavo protein
oxidoreductases, NADH peroxidase, NADH oxidase and α-glycerophosphate oxidase
(Rattanachaikunsopon & Phumkhachorn, 2010). It is used to control the growth of
psychotropic and pathogenic microorganisms (Yang 2000, Zalan et. al. 2005).
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Mostly LAB are classified into different genera based on morphology, glucose fermentation
mode, growth at various temperatures, configuration of the secreted lactic acid, ability to
grow at high concentration of salt, and tolerance to acid or alkaline solutions
(Rattanachaikunsopon & Phumkhachorn,2010). The two most common genera of LAB are
Lactobacillus and Carnobacterium which are used in the fermentation processes of milk, meat
and vegetable products.
Carnobacterium:
There are 10 species in the genus Carnobacterium. They are: Carnobacterium
alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C.
mobile, C. viridans, C. pleistocenium, and C. jeotgali. However, only C. divergens and C.
maltaromaticum are most repeatedly isolated from natural environment and food items. The
Carnobacterium species is an area of interesting research especially in the field of inhibition
of pathogens and microorganisms in fish. They are basically anaerobic in nature and get
energy by fermentation. However, they also grow well in the presence of oxygen. They can
tolerate freezing weather conditions and high pressure. They can grow at low temperatures
and in high CO2 concentrations, anaerobically. The shape of these cells varies from being
short to slender rod like structures, or curved. They are generally found single, or sometimes
in pairs and short chains. They may or may not move from one place to another. Some of the
other features of Carnobacterium are that they are non-spore forming and gram-stain-positive.
They predominantly produce lactic acid from glucose. One of the species of Carnobacterium
i.e. Carnobacterium pleistocenium, does not produce lactate. It produces ethanol, acetic acid,
and carbon dioxide. The production of gas from glucose is mostly negative but it also
depends on the substrate. They are also Psychrotolerant i.e. most strains of the
Carnobacterium grow at 0°C but not at 45°C. There is no growth in 8% NaCl solution, on
acetate (SL) agar or broth at pH of 5.4. But there is good growth at pH of 9. They are
Catalase- and oxidase- negative.
Culture characteristics:
The Carnobacterium generally forms white, creamy coloured, convex shaped, and shiny
colonies on agar. When it is grown on a semi-complex agar, it conical shaped colonies that
have a dense consistency in centre and a dark color in the perimeter. The surface of the
colonies is granulated and rough in texture. It has thin, irregular, torn edges with a diameter
varying from 0.5–2 mm on optimal agar (Pikuta et al., 2005).
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Antagonistic potential:
Robertson et al. (2000) conducted a study and observed the protective effect of
carnobacterium as a probiotic against other micro-organisms in fish. The Carnobacteria
species that was isolated from the Atlantic salmon exhibited in-vitro activity against several
gram-stain-negative pathogens. When the culture was applied to the Rainbow Trout and the
small Atlantic salmon, it improved the chances of survival of the fish. For example: The
Atlantic salmon exposed to pathogens like Aeromonassalmoncida, Vibrio anguillarum,
Vibrio ordalii, and Yersinia ruckerii, survived. The cultures were studied with the aim to
prevent the growth of pathogens on food especially Listeria monocytogenes. In other words,
the aim was to reduce the spoilage of food (Brillet et al., 2004; Yamazaki et al., 2003). The
strains that are used mostly form bacteriocins which are proteinaceous compounds. These
compounds were attributed to lantibiotics (group I bacteriocins) and to group II compounds
(Klaenhammer, 1993; Ouwehand, 1998; Tahiri et al., 2004).
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enzymes are very specific in nature and they may or may not require coenzymes for carrying
out the activity.
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Exopeptidases act on the peptide bonds at the amino or carboxyl ends of the polypeptide
chain, whereas endopeptidases act on the internal peptide bonds (Sternlicht et al., 2001).
They may also be classified on the basis of their sensitivity to pH, as acidic, neutral or
alkaline proteases. They are also often classified according to their substrate specificity, the
response to inhibitors or by their mode of catalysis into four groups (Simpson, 2000). They
are:
Serine
Cysteine
Aspartic and
Metallo proteases
Irrespective of the source, proteases can also be classified on the basis of their similarity to
other well-characterized proteases, such as trypsin-like, chymotrypsin-like, chymosin-like or
cathepsin-like (Klomklao, 2008).
Cathepsins are acid proteases which are usually located in lysosomes. These are mostly
inactive in a living tissue, but are released at sites of injury or upon freezing and thawing of
postmortem muscle. Lysosomes are possess a minimum of 13 cathepsins (Kolodziejska and
Sikorski, 1995). Among these 13, B, D, L, L-like have already been purified from fish.
Cathepsins B, D, L, and H are the major cathepsins that are found in the fish muscles’
lysosomes (Aoki et al., 2000). Cathepsin B is the first described member of the large family
of lysosomal cysteine peptidases. Cathepsins B is the major lysosomal protease in post
mortem fish muscle (Yamashita & Konagaya, 1990a).
The effects of temperature on bio-preservation of LAB:
Bio-preservation by using LAB is hampered by various factors. The previous studies in this
area have shown that the antimicrobial activity of LAB is affected by several factors like
temperature, pH, composition, structure, and natural microbiota of food (Zhou et al., 2014).
Bacterial growth is strongly influenced by temperature. Most Carnobacteria species are
psychrophilic and psychrotolerant in nature i.e. they are able to grow and reproduce at
temperatures ranging between -10 to 20 °C. According to Qin, et al., (2012), the specific
growth rate of LAB increases with increase in temperature up to a certain extent and then
decreases with further increase in temperature. According to Sumathi V., & Reetha D.
(2012), the activity of bacteriocin decreases with increase in the storage period. Moreover
Ohenhen (2015), found in his study that bacteriocin extract exhibits maximum antibacterial
activity against E. coli when stored at -20ºC for 7 days, while at the ambient temperature of
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(28±2ºC) the extract exhibits minimum antibacterial activity. It indicated that cold storage
temperature may be the most appropriate preservation method for the extract against E. coli.
In addition, Brillet-Viel (2016), described that activity of bacteriocins’ depends mainly on pH
and temperature. Maximum bacteriocins were obtained at low pH and low temperature.
Similarly, Buchanan and (Klawitter, 1992a) observed an increase in effectiveness of
bacteriocin production at refrigeration temperatures and decrease in bacteriocin production at
higher temperatures. Moreover, Ananthanarayanan (2013), pointed out that a storage
temperature of 0 to 4ºC is suitable for the preservation of most types of fresh food for short
term storage.
Temperature can affect lactic acid production of LAB. Taleghani et al., (2016) studied the
effect of temperature at 32ºC, 37ºC, 42ºC and 47°C. Results showed that the concentration of
cell dry weight increased with increase in temperature from 32ºC to 42°C. The maximum cell
and lactic acid concentration was obtained at 42°C for Lactobacillus species. A slight acidic
pH can increase the activity of cathepsins. According Taylor et al., (2002) increase in
temperature can speed up movement of molecules and microbial activities. Consequently,
fish muscle degrades faster in higher storage temperature.
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References:
Abbas, M. E., Luo, W., Zhu, L., Zou, J. & Tang, H., (2010), ‘Fluorometric determination of
hydrogen peroxide in milk by using a Fenton reaction system’, Food Chemistry, vol.120,
pp.327-331.
Ammor, S., Tauveron, G., Dufour, E. & Chevallier, I., (2006), ‘Antibacterial activity of lactic
acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-
scale facility 1- Screening and caharacterization of the antimicrobial compounds’, Food
Control, Vol.17, pp.454-461.
Mobolaji, O. A., & Wuraola, F. O., (2011), ‘Assesment of the antimicrobial activity of lactic
acid bacteria isolated from two fermented maize products-ogi and kunni-zaki’, Malaysian
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Olaoye, O. A., Ade-Omowaye (2011). Composite flours and breads: potential of local crops
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Robertson P. A. W., (2000). Use of Carnobacterium sp. as a probiotic for Atlantic salmon
(Salmo salar L.) and rainbow trout (Oncorhynchus mykiss, Walbaum) Aquaculture. Vol. 185.
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Sunil, K., & Narayana, B., (2008),’ Spectrophotometric determination of hydrogen peroxide
in water and cream samples’, Bulletin of Environmental Contamination and Toxicology,
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Tahiri I., (2004). Purification, characterization and amino acid sequencing of divergicin
M35: a novel class IIa bacteriocin produced by Carnobacterium divergens M35. Int J Food
Microbiol. Vol. 97. pp. 123–136
Yamazaki K., (2005). Purification and characterization of a novel class IIa bacteriocin,
piscicocin CS526, from surimi-associated Carnobacterium piscicola CS526. Appl Environ
Microbiol. Vol. 71. pp. 554–557.
Zalan, Z., Nemeth, E., Barath, A. & Halasz, A., (2005), ‘Influence of growth medium on
hydrogen peroxide and bactteriocin production of Lactobacillus strains’, Food Technology
and Biotechnology, Vol.43(3), pp.219-225.