A review on industrially important Lactococcus lactis
General information, metabolism and genotypic identification tools
©2016
Academic Paper
27 Pages
Summary
Lactococcus lactis is one of the most important starter cultures in the dairy industry for the manufacture of a variety of fermented probiotic dairy products. Presently, four subspecies of L. lactis have been recognized, i.e. L. lactis ssp. cremoris, ssp. hordniae, ssp. lactis (including biovar diacetylactis) and ssp. tructae. Amongst them, the two subspecies lactis and cremoris are industrially important. Development of new strains of these two subspecies as starter culture with distinct characteristics generally requires the identification up to sub-species level. Classical identification methods such as physiological and biochemical tests can not differentiate organisms at species and subspecies levels. Moreover, these traditional phenotype-based methods encounter many challenges and shortcomings which limit their applicability. New approaches such as molecular characterization have been developed in the recent past. The molecular techniques provide a new perspective for characterization of the “new lactococcal” strains. However, there is no unique approach suggested for molecular characterization of the indigenous strains. Continuous research is going on around the world to improve the methodology and applicability of these methods as well as to make them economic in use. The objective of this review is to provide an insight into all genotypic approaches for the identification of L. lactis.
Excerpt
Table Of Contents
4
are common components of L. lactis genomes, which are diverse in size, copy number and
distribution. Plasmids often carry some significant characteristics, such as carbohydrate
fermentation, proteolysis, bacteriophage resistance, polysaccharide production, lactose
utilization, sucrose degradation, metabolism of sucrose, galactose, mannose, xylose, glucose, or
citrate utilization, phage resistance and DNA restriction and modification, cell aggregation,
production of bacteriocines, mucoidness and resistance to inorganic ions (Von Wright and
Sibakov, 1998). Many of these traits of plasmids construct L. lactis useful in manufacturing the
fermented food products. As plasmids are not necessary for cell survival, the lactococcal cell
may have lost one or more plasmids spontaneously by protoplast regeneration, transduction,
conjugation and transformation, as a consequence of the function encoded by this plasmid
(Chopin, 1993). It was also reported that frequently repeated cultivations in milk or differed
culture conditions resulting in the loss of plasmid (Foucaud et al., 1990).
The remarkable taxonomic structure of L. lactis is also evident from the genome
sequences of three different strains (Bolotin et al., 2001, Wegmann et al., 2007). These strains
include an important model strains of dairy origin such as strain IL-1403 (L. lactis ssp. lactis
type-strain-like genotype and L. lactis ssp. lactis phenotype), strain MG1363 (L. lactis ssp.
cremoris type-strain-like genotype and L. lactis ssp. lactis phenotype) and strain SK11 (L. lactis
ssp. cremoris type-strain-like genotype and L. lactis ssp. cremoris phenotype). The strain IL-
1403 is actually a plasmid-cured derivative of the biovar diacetylactis strain CNRZ157 making it
cit negative.
The first lactic acid bacterium genome to be sequenced was L. lactis ssp. lactis IL1403
(Bolotin et al., 2001). In addition, the genome of two L. lactis ssp. cremoris has also been
sequenced: L. lactis ssp. cremoris MG1363 (Wegmann et al., 2007) and L. lactis ssp. cremoris
SK11 (Makarova et al., 2006). The genome of L. lactis is a circular chromosome with 2,365,589
base pair, in which 86% of genome code for protein, 1.4% for RNA, 12.6% for non-coding
region, 64.2% of genes code for known functional proteins and 20.1% of genes for known
protein with unknown functions. The remaining 15.7% of genes are unidentified proteins may be
unique to Lactococcus. The genome of L. lactis ssp. cremoris MG1363 is 160 kbps and 90 kbps
larger than L. lactis ssp. lactis IL1403 and L. lactis ssp. cremoris SK11, respectively. L. lactis
5
ssp. cremoris MG1363 has 465 and 346 genes that are lacking in L. lactis ssp. lactis IL1403 and
L. lactis ssp. cremoris SK11, respectively (Wegmann et al., 2007).
L. lactis is widely distributed in different ecosystems and is commonly found in variety
of foods (dairy products, fermented meats and vegetables, sourdough, silage, beverages),
sewage, on plant materials mainly grasses and also in the genital, intestinal and respiratory tracts
of human beings and animals. The lactococci from plant materials are easily inoculated into milk
therefore, they normally found in milk. Due to PEP-PTS, which ensure their efficient uptake and
fermentation of lactose, some of these organisms have been adapted well for growth in milk and
today the most recognized habitat for lactococci are untreated milk, fermented milk, cheeses and
other dairy products (Axelsson, 1998). Heikkila and Saris have isolated L. lactis from human
milk (34). The maternal skin has also been reported as a source of lactococci (50). The
abundance of L. lactis ssp. lactis in many cheese varieties has been reported (Lopez-Diaz et al.,
2000; Prodromou et al., 2001).
L. lactis has also been reported within the digestive tract of cows. It is believed that in
nature, L. lactis remains dormant on plant surfaces from where they are ingested along with the
plants into animals' gastrointestinal tract, where it becomes active and multiplies intensively
(10). Its presence in human beings or animals is accidental because they are not normally found
in significant numbers in excrement or soil (Roissart, 1994).The sub-species of L. lactis have
been isolated mainly from fresh vegetables and skin of animals. Liliana Serna Cock isolated L.
lactis ssp. lactis strain from the leaves of sugar cane plants (Liliana Serna Cock 2006).This
indicated that fodder may be also one of the principal contamination sources.
6
METABOLISM
L. lactis is used extensively as a model organism for facultative anaerobic and low GC gram-
positive bacteria since this food-grade bacterium is easy to culture in vitro and has a relatively
simple metabolism. The metabolic properties of L. lactis species have a direct or indirect
influence on organoleptic, nutritional and hygienic qualities of fermented dairy products, which
makes knowledge of their characteristics extremely important from economic point of view. The
metabolic pathway of L. lactis can function through aerobic and anaerobic reactions. It consists
of 621 reactions and 509 metabolites and requires minimally glucose, arginine, methionine,
glutamate and valine for growth. The main metabolism of L. lactis is through the anaerobic
pathway which produces lactic acid from the available carbohydrates. The carbon sources
include fructose, galactose, glucosamine, glucose, lactose, maltose, mannitol, mannose, ribose,
sucrose and trehalose. However, the growth rate with the intake of each carbon source is
different. The growth rate with carbon sources as glucose, mannose, galactose, sucrose, lactose
and glucosamine was the same, whereas the growth rate with fructose and mannitol was at lower
magnitude (Oliveira et al., 2005).
L. lactis can import various sugars for its metabolism, all of which are degraded to
pyruvate via the EMP pathway. The produced pyruvate has several alternative fates depending
on the environmental and intracellular conditions. The first step in the metabolism of glucose,
galactose and lactose lead to the production of glucose-6-phosphate (G6P) which is then
converted to fructose-1, 6- bisphosphate (FBP) by phosphoglucose isomerase and 6-
phosphofructo-1-kinase enzyme activity. The FBP is converted to triosephosphates,
dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) by fructose-
bisphosphate aldolase. The triosephosphates can be interconverted by triosephosphate isomerase.
The 3- phosphoglycerate (3-PGA) is produced from GAP by glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) followed by 3-PGA is converted to PEP and subsequently to pyruvate
by an enolase and pyruvate kinase (PK), respectively. At the level of pyruvate, the metabolic
pathway gets branched. Although L. lactis is generally grown homofermentatively under less
favourable growth conditions, it can perform mixed-acid fermentation in which acetate, ethanol,
formate and 2, 3-butanediol is also produced along with the lactic acid (Jensen et al., 2001;
Melchiorsen et al., 2001; Cocaign et al., 2002).
7
When growth conditions of L. lactis are not optimal or when glucose is limited or when
cells are grown on a less favorable sugar, pyruvate serves as substrate for pyruvate formate lyase
(PFL) which leads to the production of formate, acetate and ethanol. When L. lactis is grown in
the presence of low oxygen; pyruvate can be used as substrate for pyruvate dehydrogenase
(PDH) and in this condition carbon dioxide is produced along with acetate and ethanol. When
pyruvate accumulates, it can also be converted to 2, 3-butanediol or when small amounts of
oxygen are present, to diacetyl, since the affinity of -acetolactate synthase for pyruvate is very
low (Snoep et al., 1992).
Under anaerobic reaction, glycolysis breaks down extracellular carbohydrates to pyruvate
which yields lactic acid in the presence of lactate dehydrogenase. The synthesis of lactic acid is
the basis of many important food fermentations. Lactate helps protons to be out with membrane
protein creating the membrane potential necessary for energy production (Bolotin et al., 2001).
NADH, the cofactor for the lactate dehydrogenase is regenerated into NAD+ to be reused for
glycolysis. Due to relatively short and simple metabolic pathway, less energy is produced from
one sugar molecule. The flux of the metabolic pathway is high to obtain enough energy which
results in the fast acidification of the dairy products. This is a competitive advantage over other
micro-organisms.
Besides the anaerobic pathway, L. lactis has also an aerobic system. Normally, the uptake
of oxygen would affect the fermentation process or even interferes with oxygen reactive
substances. However under low oxygen consumption, aerobic pathway is limited due to low
recycle rate of NAD from NADH oxidase. These damages can be fixed when cells are grown
with oxygen and heme source. Cells increase their growth, resistance to oxidation and improve
survival at low temperature.
Among lactococci, only L. lactis ssp. lactis biovar diacetylactis has the ability to
metabolize the citrate of milk. The metabolic end products of citrate metabolism include
diacetyl, acetoin, 2, 3-butanediol, acetic acid and carbon dioxide (Cogan, 1995; Steele, 1998)
which are responsible for flavor development in fermented dairy products. Diacetyl is an
important flavor component and produced in low amounts from breakdown of citric acid during
milk fermentations. During citrate metabolism, three decarboxylation reactions take place:
oxalacetate to pyruvate, pyruvate to acetaldehyde-thiaminopyrophosphate (TPP) and -
8
acetolactate to acetoin. Quantitatively, acetoin is the most important product of citrate
metabolism and occurs as a mechanism of preventing accumulation of pyruvate. Diacetyl and
CO
2
are produced in small quantities, but their production has immense significance for
developing both texture and flavor of fermented products (Cogan, 1995). Diacetyl is produced
intracellularly by non-enzymatic chemical decomposition of -acetolacete. This reaction is
accelerated by aeration and low pH (Axelsson, 1998; Rondags et al., 1998).
Lactococcal growth also requires proteins, peptides, specific amino acids, derivatives of
nucleic acids and vitamins for the synthesis of cell compounds. L. lactis is not able to produce all
essential amino acids. Typically industrial L. lactis ssp. cremoris strains require more amino
acids for growth than dairy and non-dairy strains (Ayad et al., 1999). The absence of some amino
acid biosynthetic pathways in dairy lactococci might be a consequence of their adaptation to milk
since amino acids are readily available by the proteolysis of caseins and as a result amino acid
auxotroph will not have a negative influence on their growth and survival. Strains isolated from
natural niches are usually not associated with a rich environment such as milk, which makes
them more dependent on their own synthesis of amino acids compared to dairy strains (Engel et
al., 2003).
9
IDENTIFICATION
Classical identification
Classical microbiological taxonomy has traditionally been used to discriminate morphological
and physiological differences among species and sub-species. The morphological tests only
discriminate at the species level and physiological methods would not be able to distinguish the
species up to sub-species or strain level. At the genus level, several characteristics can contribute
to the differentiation. The two sub-species and one bio-variety of L. lactis have been
taxonomically differentiated by few phenotypic characteristics. The identification of L. lactis up
to sub-species level has traditionally been performed by studying some physiological and
chemotaxonomic properties such as growth at temperature of 10, 40 and 45
o
C, pH 9.2, 4% (w/v)
NaCl, (Salama et al., 1995; Batt, 2000), production of L (+) lactic acid from glucose (Samarzija
et al., 2001), homo-fermentative activity, litmus reduction before milk curdling (Salama et al.,
1995), fermentative capability of wide range of carbohydrates (Kimoto et al., 2004) and arginine
deiminase activity (Mundt 1986). L. lactis ssp. cremoris is characterized by the inability to
produce ammonia from arginine and by low tolerance to elevated temperatures and salt
concentrations. L. lactis ssp. lactis produces ammonia from arginine and is tolerant to 40°C and
4% NaCl. Some L. lactis ssp. lactis strains are able to ferment citrate and to produce the flavor
compound diacetyl and these are referred to as L. lactis ssp. lactis biovar diacetylactis strains
(Schleifer, et al., 1985).
L. lactis can initially be distinguished by its ability to grow at temperatures above 40
o
C
and in >4 % NaCl (Batt, 2000). This is most important in the production of foodstuffs such as
yoghurts and cheese. Some L. lactis ssp. lactis showed atypical properties such as growth at
45°C and growth in 6.5% NaCl. These atypical properties can be an advantage in dairy industry.
Similarly, in a recent work lactococci isolates which grow in the presence of 6.5% NaCl and/or
at 10°C has been reported (Fortina et al., 2003).
Species and subspecies level molecular identification
The main objective of microbial classification is to identify the isolates up to genus to species to
sub-species to strain level. Discriminating or typing the strains of a species is nowadays gaining
increasing importance from the industrial point of view. As L. lactis has wide industrial
importance, it has been the subject of numerous studies which has resulted in detailed knowledge
10
of its physiology and molecular biology. Moreover, due to the availability of a vast molecular
toolbox and three whole genome sequences of L. lactis strains IL-1403, SK11 and MG1363
(Bolotin et al., 2001; Makarova et al., 2006; Wegmann et al., 2007), L. lactis has gained a strong
position as a model organism for low-GC gram-positive bacteria. Whole genome sequence
analysis of L. lactis has shown the genetic differences in the sub-species lactis and cremoris. It
has been postulated on the basis of DNA hybridization stringency that 20-30% divergence
occurred between ssp. lactis and cremoris (Godon et al., 1992). The identification of bacterial
species is usually based on the combination of phenotypic and genotypic techniques
(Temmerman et al., 2004, Aquilanti et al., 2007).The phenotypic tests however are insufficient
for accurate species identification due to great number of species of lactic acid bacteria with
similar phenotypic characteristics. Thus, new methods have been developed depending on the
genotypical features and used effectively for the identification of the bacteria (Conter et al.,
2005; Ammor et al., 2007). However, the DNA-based techniques offer much greater
discriminatory power to differentiate the individual sub-species and strains of bacteria. The use
of molecular tools has revolutionized the identification of many bacterial species, including
LAB. Many of these techniques are based on the PCR using oligonucleotide primers to amplify
targeted DNA fragments. These PCR primers may be designed to differentiate the bacteria from
genus to species to subspecies level.
The methods used for the genotypic study of Lactococcus such as 16S rDNA sequencing,
ribotyping, protein profiling and PFGE are too laborious and limited in their resolving power or
require a species-specific methodology. Therefore, a method that is universally suitable for the
Lactococcus with a high resolving power both on the species and intra-species level would be a
highly valuable tool. In this regard, PCR based genomic fingerprinting techniques are believed to
have the most potential and are easy to perform (Gevers et al., 2001). Highly conserved
repetitive DNA elements such as PCR amplification of repetitive bacterial DNA elements (rep-
PCR) using the REP sequences such as PCR amplification using (GTG)
5
primer, referred to as
(GTG)
5
-PCR fingerprinting (Rademaker et al., 2007), 124-127 bp Enterobacterial Repetitive
Intragenic Consensus (ERIC) elements (Nanda et al., 2001; Gonzalez et al., 2004) and 154 bp
BOX elements (Adiguzel and Atasever, 2008) are widely distributed in the genomes of various
bacterial groups. PCR amplification of 35-40 bp repetitive bacterial DNA elements (rep-PCR)
has been known as a simple PCR-based technique with the following characteristics: (1) low cost
11
(2) a high discriminatory power (3) suitability for a high-throughput of strains and (4) a reliable
tool for classifying and typing a wide range of gram-negative and some gram-positive bacteria
(De Vuyst et al., 2008).Some molecular approaches used so far have been discussed below.
PCR analysis
A highly efficient, rapid and reliable PCR-based method for distinguishing L. lactis ssp. lactis
and ssp. cremoris has been described by targeting a portion of glutamate decarboxylase gene.
Primers complementary to positions in the glutamate decarboxylase gene have been constructed.
Observation of glutamate decarboxylase (GAD) can be found in the L. lactis ssp. lactis, but not
in L. lactis ssp. cremoris. GAD catalyzes the irreversible decarboxylation of glutamate to gamma
aminobutyric acid with the glutamate-GABA antiporter (GABA) (Nomura et al., 2002). The
gene that encodes GAD in L. lactis ssp. cremoris is inactivated by a frameshift mutation
resulting in a nonfunctioning protein. The specific strain typing based on PCR amplification of
conserved DNA sequences of acmA gene for lactococcal N-acetylmuramidase (Garde et al.,
1999; Prodevalova et al., 2005) Histidine biosynthesis operon (Corroler et al., 1999) has also
been carried out for the identification of L. lactis.
The gene encoding nisin was amplified with specific primer for the identification of L.
lactis characterized with nisin gene (Ulhman et al., 1992; Klijn et al., 1995; Cardinal et al., 1997;
Noonpadkee et al., 2003; Prodevalova et al., 2005; Bravo et al., 2009; Sanlibaba et al., 2009).
The nisin producing strains L. lactis EN3a, EN14a, and EN15b were isolated and identified by
the amplification of nisin structural gene from mayonnaise-based commercial products and from
the raw materials used in their production (Miller et al., 2010). Mitra et al., (2007) and recently
Khemariya et al. (2013) also identified nisin producing L. lactis from dairy and non-dairy
samples by PCR amplification of a part of nisin Z gene.
16S rDNA gene is a favorable PCR amplification target for identification and phylogenic
purposes since it is universally distributed among bacteria and it contains enough variations
amongst strains and species within the DNA sequence (Weisburg et al., 1991). The availability
of whole genome, small subunit ribosomal RNA gene sequences such as 16S rDNA data is
constantly increasing and public-domain databases have been established such as Ribosomal
Data Base Project (http://rdp.cme.msu.edu/) and NCBI Blast Library
(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). These database libraries can be applied for the
Details
- Pages
- Type of Edition
- Erstausgabe
- Year
- 2016
- ISBN (PDF)
- 9783960675389
- File size
- 346 KB
- Language
- English
- Institution / College
- Indian Institute Of Vegetable Research
- Publication date
- 2016 (April)
- Grade
- No grade
- Keywords
- Lactococcus lactis ssp. lactis Lactococcus lactis ssp. cremoris Identification Molecular approach L. lactis Lactococcal strain Dairy production Dairy industry Starter culture
