Discrepancies between the phenotypic and genotypic characterization of Lactococcus lactis cheese isolates

Plaza, Marta; Rodrı́guez, Ana; Palencia, Pilar Fernández de; Martínez-Cuesta, M. Carmen; Peláez, Carmen; Requena, Teresa

doi:10.1111/j.1472-765x.2006.02012.x

Cited by 20 publications

(16 citation statements)

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“…lactis genotype have been reported (Fernández et al 2011). Phenotypic differences between genotypically similar strains of L. lactis have also been found, probably linked to differences in enzymatic expression (de la Plaza et al 2006). This preliminary study of L. lactis microbiota revealed its complexity and suggests that a deeper knowledge is needed to select strains with the best properties for use in starter cultures in the manufacture of Ossau-Iraty cheese and to understand the impact of this microbiota on cheese characteristics.…”

Section: Discussionmentioning

confidence: 95%

“…These studies have highlighted the wide diversity which exists among wild strains, much greater than among commercial or reference strains (Sánchez et al 2000). Strain diversity can produce a wide range of genotypic profiles Fernández et al 2011) which are not always consistent with phenotypic profiles at subspecies or strain levels (de la Plaza et al 2006;Fernández et al 2011). This diversity can lead to a wide range of technological abilities of individual strains including flavor formation, acidifying activity, production of antimicrobial compounds, and phagic resistance.…”

Section: Introductionmentioning

confidence: 99%

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Lactococcus lactis strains from raw ewe’s milk samples from the PDO Ossau-Iraty cheese area: levels, genotypic and technological diversity

Feutry¹,

Torre

Arana

et al. 2012

Dairy Sci. & Technol.

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The aims of this work were to assess the levels of Lactococcus lactis (L. lactis) in ewe's milk produced in three Ossau-Iraty cheese sub-areas and to investigate the genotypic and technological diversity of isolated wild strains of L. lactis in order to assess their suitability for use as components of starter formulations. Thirty-two milk samples from 32 farms were collected. Strains of L. lactis were identified and quantified using a combination of species and subspecies-specific polymerase chain reaction (PCR) and PCR amplification of repetitive bacterial DNA elements (Rep-PCR). The genotypic and technological diversity of the indigenous strains was compared to that of 12 commercial strains. L. lactis was detected in milk samples from only 20 farms. The levels detected were below 4 log 10 cfu.mL −1 in 75% of the milks. L. lactis subsp. lactis dominated in 66% of the samples. Forty-three genotypic profiles of wild L. lactis strains were detected and showed greater diversity than those of the commercial strains. Milks containing L. lactis contained one to four distinct strains. With the exception of two strains, each strain was found in milk from only one farm. The Prt + strains were the most acidifying. Sensitivity to phages collected from wheys differed widely between the commercial (60%) and indigenous strains (5%). Wild strains of L. lactis displayed a wide genotypic and technological diversity. Genotypic diversity seemed to be linked to the farm of origin. This study addresses questions regarding the environmental factors which influence such natural diversity. A deeper knowledge of the strain-dependent technological properties would be useful in selecting strains for use in starter blends.

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Section: Discussionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Lactococcus lactis strains from raw ewe’s milk samples from the PDO Ossau-Iraty cheese area: levels, genotypic and technological diversity

Feutry¹,

Torre

Arana

et al. 2012

Dairy Sci. & Technol.

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“…In order to optimize the degradation of ␣-keto acids into volatile compounds, we have inactivated the panE gene in L. lactis IFPL953. The strain was isolated from raw milk cheese and was selected because it possesses glutamate dehydrogenase (GDH) activity, to produce the ␣-ketoglutarate required for AraT and BcaT transamination reactions, and KivD activity, which is needed for the transformation of ␣-keto acids into aldehydes (8).…”

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confidence: 99%

Inactivation of the panE Gene in Lactococcus lactis Enhances Formation of Cheese Aroma Compounds

Cadiñanos

García‐Cayuela

Yvon

et al. 2013

Appl Environ Microbiol

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bHydroxyacid dehydrogenases limit the conversion of ␣-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the ␣-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953⌬panE was an efficient strain producing volatile compounds related to cheese aroma. Flavor formation in dairy products is directly related to lactic acid bacteria (LAB), which are equipped with a large number of key intracellular enzymes responsible for the formation of volatile compounds from amino acid catabolism (1, 2). In Lactococcus lactis, amino acid degradation is initiated by a transamination reaction, catalyzed mostly by AraT and BcaT aminotransferases, that produces the corresponding ␣-keto acid (3, 4). The ␣-keto acids are subsequently degraded either into aldehydes, which are further converted into alcohols, carboxylic acids, and esters, or into ␣-hydroxy acids, which are not aroma compounds (1). Two key enzymes are involved in these reactions, an ␣-ketoacid decarboxylase (KivD) responsible for the transformation of the ␣-keto acids into aldehydes (5) and an ␣-hydroxyacid dehydrogenase (PanE) responsible for the conversion of the ␣-keto acids into the nonaromatic ␣-hydroxy acids (6). Hydroxyacid dehydrogenases (HA-DHs) negatively affect aroma production by competing with decarboxylases for ␣-keto acids (7). In order to optimize the degradation of ␣-keto acids into volatile compounds, we have inactivated the panE gene in L. lactis IFPL953. The strain was isolated from raw milk cheese and was selected because it possesses glutamate dehydrogenase (GDH) activity, to produce the ␣-ketoglutarate required for AraT and BcaT transamination reactions, and KivD activity, which is needed for the transformation of ␣-keto acids into aldehydes (8).The HA-DH activity in L. lactis IFPL953 was inactivated by double-crossover mutation using the thermosensitive pG ϩ host9 vector (9) containing a 408-bp deletion in the panE gene. All primers used for PCR amplification and DNA sequencing during the inactivation of the panE gene are shown in Table 1. The plasmid pGϩ9::⌬panE was produced in Escherichia coli TIL206 (10), selected for erythromycin resistance (150 g/ml), and transformed into L. lactis IFPL953 by electroporation (11). L. lactis IFPL953⌬panE was achieved by chromosome integration of pGϩ9::⌬panE at 37°C and excision at 28°C. The mutation was verified by sequencing the 1,645-bp PCR product ⌬panE (Table 1). L. lactis IFPL953⌬panE and the wild-type strain were analyzed for HA-DH activity using ␣-ketoisocaproic acid (KIC) as the substrate (6), showing L. lactis IFPL953 values of 5.78 U/mg (1 U is defined as the quantity of enzyme that oxidizes 1 mol NADH per minute at 37°C), whereas the mutant strain had lost the HA-DH activity. The effect of the mutation on L. lactis IFPL953⌬panE growth was tested by comparing the growth to that of L. lactis IFPL953 grown in a buffered chemically defined medium (CDM) (12) for 24 h (Fig. 1A) and i...

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“…However, molecular characterization of field isolates often does not match with their subspecies designation based on the phenotypes (2,13,23,25 (8,11,14,16,26), and the MG1363 L. lactis subsp. cremoris lab strain is the well-known representative for these frequently encountered diverging strains.…”

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confidence: 99%

“…However, molecular characterization of field isolates often does not match with their subspecies designation based on the phenotypes (2,13,23,25). Fingerprinting studies reveal two main genomic lineages: the L. lactis subsp.…”

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High-Resolution Amplified Fragment Length Polymorphism Typing of Lactococcus lactis Strains Enables Identification of Genetic Markers for Subspecies-Related Phenotypes

Kutahya

Starrenburg

Rademaker

et al. 2011

Appl Environ Microbiol

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A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primernucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers.Lactococcus lactis is commonly found in defined and artisan cheese starter cultures as blends of strains with differential functionality during the cheese production process. L. lactis accommodates three subspecies: L. lactis subsp. lactis, L. lactis subsp. cremoris, and L. lactis subsp. hordniae, with the first two having significant economic importance to dairy fermentations. The subspecies differentiation and nomenclature are traditionally based on phenotypes according to the taxonomic reclassification published by Schleifer et al. (20). The so-called L. lactis subsp. lactis phenotype is differentiated from the L. lactis subsp. cremoris phenotype by its growth at 40°C, resistance to 4% salt, and utilization of arginine and maltose (4,20). L. lactis subsp. lactis also includes the biovariety diacteylactis, which is a citrate-fermenting variant of the L. lactis subsp. lactis phenotype, and the corresponding metabolic pathway is generally encoded on a plasmid (6,22).Phenotypes are of primary importance for screening of potential starter cultures because of their postulated linkage to performance in the fermentation. However, molecular characterization of field isolates often does not match with their subspecies designation based on the phenotypes (2,13,23,25 (8,11,14,16,26), and the MG1363 L. lactis subsp. cremoris lab strain is the well-known representative for these frequently encountered diverging strains.Current genomic fingerprinting studies are far from the resolution level required to reveal a phenotype-specific genetic signature. High-resolution fingerprinting, optimized for the species of interest, may deliver phenotype-specific genetic markers that could be employed as an effective alternative to the labor-intensive phenotypic scree...

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Discrepancies between the phenotypic and genotypic characterization of Lactococcus lactis cheese isolates

Cited by 20 publications

References 36 publications

Lactococcus lactis strains from raw ewe’s milk samples from the PDO Ossau-Iraty cheese area: levels, genotypic and technological diversity

Lactococcus lactis strains from raw ewe’s milk samples from the PDO Ossau-Iraty cheese area: levels, genotypic and technological diversity

Inactivation of the panE Gene in Lactococcus lactis Enhances Formation of Cheese Aroma Compounds

High-Resolution Amplified Fragment Length Polymorphism Typing of Lactococcus lactis Strains Enables Identification of Genetic Markers for Subspecies-Related Phenotypes

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