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).

1
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).

2
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).

2.2 Protein and amino acidin fish:

Due to the rapid increase in human population, there is a need to increase the amount of food
available. This is more important for foods with necessary ingredients like protein rich foods
for a healthy diet. One of the major sources of protein is fish. It is made up of approximately
20% protein and 80% water and lipids. The proteins in the fish muscle are divided into three
groups. They are: the myofibrillar proteins which account for about 70-80%, the sarcoplasmic
proteins which account for about 25-30 % and the stroma proteins (collagen) that account for
3% in bony fish and 10% in cartilaginous fish (Shenouda, 1980; Careche et al., 1999).
Moreover, proteins are the basis of structure and function of our body. It is made up of twenty
amino acids. They are the building blocks of human body. They are organized into primary,
secondary, tertiary, quaternary structures. They are also classified as simple, conjugated and
derived proteins. The peptides and proteins are made up of amino acids. Two or more amino
acid molecules join by a covalent bond and form either dipeptides, or oligopeptides or
polypeptides. Proteins are a group of linear chains of amino acids which are linked together
by peptide bonds.
2.4 Proteolytic enzyme:
Proteases are the enzymes that catalyze the breakdown of proteins. In other words, enzymes
are proteins that fasten the rate of reactions in living cells. The catalysts are prepared by the
cells in very small quantities and they are not consumed during a chemical reaction. These

3
enzymes are very specific in nature and they may or may not require coenzymes for carrying
out the activity.

Proteolysis is a biochemical phenomenon. In this, the degradation of proteins and the

generation of small peptides and free amino acids take place. The muscle proteases like
mainly cathepsins B and L, are able to break down the structural myofibrillar proteins of the
fish and generate large peptides and protein fragments. It leads to softening of the texture of
the fish. Texture is one of the major food quality attributes that affects the consumer's
acceptability of the fish and its market value.
Further problems may arise when the large peptides become hydrolyzed by muscle peptidases
and generate small peptides and free amino acids. These can be used as substrates by
microorganisms for growth. It may also transform the amino acids into other compounds like
biogenic amines or offflavors like ammonia (Kilcast, and Subramaniam, 2011). In general,
proteolysis has many steps (Nielsen and Nielsen, 2006). They are as follows:
1. Calpains and cathepsins act on major myofibrillar proteins to generate protein
fragments and intermediate-size polypeptides.
2. The fragments and polypeptides are then hydrolyzed to small peptides by di- and tri-
peptidylpeptidases.
3. The dipeptidases, amino peptidases and carboxy peptidases act on the small peptides
to generate free amino acids.
The progress of proteolysis varies depending on factors like
 The environmental conditions while processing
 The type of muscles and
 The amount of endogenous proteolytic enzymes.
For instance, an increase in temperature will lead to enzymatic action and a slightly acidic pH
would speed up the action of lysosomal cathepsins. The connection between the myofibrils
and connective tissue is also hydrolyzed by proteases (Taylor et al., 2002). Collagen fibrescan
can also be degraded by proteases and this will affect the texture of the fish (Sato et al.,
2002).
Enzymes that hydrolyze the peptide bonds can be roughly grouped into two subclasses based
on where the reaction takes place (Sternlicht and Werb, 2001). They are:
 exopeptidases and
 endopeptidases.

4
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 (28±2ºC)
the extract exhibits minimum antibacterial activity. It indicated that cold storage temperature

5
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.

References:

6
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.

Brillet A., (2004). Biodiversity of Listeria monocytogenes sensitivity to bacteriocin

producing Carnobacterium strains and application in sterile cold-smoked salmon. J Appl
Microbiol. Vol. 97, pp. 1029–1037.

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
Journal of Microbiology, Vol.7(3), pp.124-128.

Olaoye, O. A., Ade-Omowaye (2011). Composite flours and breads: potential of local crops
in developing countries. In V.R. Preedy RR, Watson, Patel VB (Eds.), Flour and breads and
their fortification in health and disease prevention, pp. 183 – 192. London, Burlington, San
Diego: Academic Press, Elsevier. ISBN: 9780123808868.

Pikuta E. V., (2005) Carnobacterium pleistocenium sp. nov., a novel psychrotolerant,

facultative anerobe isolated from permafrost of the Fox tunnel in Alaska. Int J System Evol
Microbiol. Vol. 55. pp. 473–478.

Rattanachaikunsopon, P., & Phumkhachorn, P., (2010), ‘Lactic acid bacteria: their
antimicrobial compounds and their uses in food production’, Annals of Biological Research,
Vol.1(4), pp.218-228.

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.
pp. 235–243.

Sunil, K., & Narayana, B., (2008),’ Spectrophotometric determination of hydrogen peroxide
in water and cream samples’, Bulletin of Environmental Contamination and Toxicology,
Vol.81, pp.422-426.

7
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.

Yang, Z., (2000), ‘Antimicrobial compounds and extracellular polysaaharides produced by

lactic acid bacteria: Structures and properties’, MSc Thesis, Faculty of Agriculture and
Forestry, Department of Food Technology University, University of Helsinki, pp. 61.

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.