Phenotypic and genotypic characterization of lactic acid bacteria isolated from cow, ewe and goat dairy artisanal farmhouses

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Phenotypic and genotypic characterization of lactic acid bacteria isolated from cow, ewe and goat dairy artisanal farmhouses

Stella M. Reginensi; Marcela J. González; Jorge Bermúdez

Unidad de Tecnología de Alimentos, Facultad de Agronomía, Universidad de la República, Montevideo, Uruguay

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ABSTRACT

Lactic acid bacteria collected from artisanal farmhouses were characterized using a polyphasic approach. Phenotypic methods including biochemical assays, ribosomal DNA restriction analysis and 16S rDNA sequence analysis were performed. This approach provides accuracy for identification, and helps to avoid the loss of natural biodiversity including potentially valuable strains.

Key words: Native lactic acid bacteria, polyphasic identification, artisanal dairy products.

Lactic acid bacteria (LAB) are microorganisms of great economic importance for artisanal and industrial dairy fermentation processes. There is an ongoing need for improving useful properties of starter cultures, and natural microbial populations present in artisanal dairy environments provide a resource of genetic diversity. This is particularly evident in environments where cheeses are produced without pasteurization or commercial starters (Coppola et al., 2001). LAB isolated from natural habitats may possess unusual characteristics including phenotypic differences and intraspecies variability compared with standard type strains (Fortina et al., 1998). Identification of suitable strains as starter cultures for production of fermented dairy-based foods requires identification and consideration of some important technological properties, including acidification rate, exopolysaccharide production, aminopeptidase activity, antibacterial activity against pathogens and antibiotics resistance (Marroki et al., 2011). The occurrence of antibiotic resistance should be taken into account as potentially adverse property that would prohibit their application in food and feed.

Conventional phenotypic identification of LAB depends mainly on morphological, physiological and biochemical criteria. A broad range of DNA-based methods are available for identification and typing of LAB isolates (Temmerman et al., 2004), and current taxonomic polyphasic methods include both phenotypic and genotypic analysis. 16S rRNA gene sequencing is one of the most commonly used techniques for identification of LAB species, but the method often fails to discriminate between phylogenetically closely related LAB species or between subspecies (Temmerman et al., 2004). Although, phenotypic tests provide evidence for different metabolic capabilities among isolates, the complex and sometimes graded differences make distinctions difficult. Designation of certain new type strains based solely on phenotypic characteristics has resulted in ambiguities which have ultimately been resolved using molecular techniques (Van Hoorde et al., 2008). The aim of the present study is to use a polyphasic approach to identify selected strains of LAB isolated from raw milk, cheese and cheese whey starter by characterization of phenotype, amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequence analysis.

Twelve cocci LAB isolates from cow (8), ewe (1) and goat (1) raw milk, artisanal cheese (1) and cheese whey starter (1) from our laboratory culture collection and three type strains of the American Type Culture Collection (ATCC) were analyzed in this study. The strains to be identified were isolated within the last ten years from samples collected at different farmhouses which process cow, goat or ewe raw milk to cheese without adding commercial starters. The main technological properties of the isolates selected were fast acidification rate, aroma compounds production or bio-protective activity. Working cultures were prepared from isolate and type strain frozen stocks after growth and transfer twice in fresh MRS broth (Oxoid, UK) at 32 ºC or 45 ºC. Selected strains were assigned to the genus level based on phenotype including cell morphology, Gram staining, catalase reaction, CO2 production from glucose in modified MRS broth (excluding ammonium citrate) containing inverted Durham tubes, arginine hydrolysis, growth at different temperatures (10 ºC and 45 ºC), medium pH (4.4 and 9.6), and salt content (6.5% NaCl), according to Sharpe (1979). The Voges-Proskauer test was used for further discrimination within the Lactococcus genus.

Acid production after fermentative growth of isolates/type strains on selected carbohydrates (ribose, esculin, melezitose, salicin, maltose, lactose, fructose, galactose, arbutin, raffinose, glucose, sucrose, arabinose, xylose, mannose, melibiose, rhamnose, mannitol, sorbitol and trehalose) was evaluated using a miniaturized microplate assay. Each 96-well microplate was prepared by addition to individual wells 10 µL of each filter sterilized sugar solution (20%, w/v) (Marroki et al., 2011), 100 µL LAPTg media (peptone 15 g/L, tryptone 10 g/L, glucose 10 g/L, yeast extract 10 g/L , Tween 80 1 mL/L, and phenol red 2 mg/L) without glucose (Petrov et al., 2008), and inoculated with a type strain or isolate to be identified. Microaerophilic conditions were generated by sealing the well with 50 µL sterile vaseline. Isolates analyzed by biochemical assays were grown overnight, harvested and dispensed to microplates as in Gussils (2001). Microplates were incubated at 34 ºC for 48 h, and sugar fermentation was considered as positive by a color change to yellow in medium of the corresponding well. A weak fermentation was associated with a color change to orange.

LAB isolates and type strains were grown in MRS broth at 34 ºC and 42 ºC to an OD_{600 nm} of 1.0. Cultures were harvested, washed twice, and suspended in 200 µLTE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA was purified using a Genomic DNA purification kit (Fermentas International Inc., USA) following manufacturers instructions. DNA was used as template in amplification reactions and concentrations were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific Inc., Wilmington, MA). 16S rRNA gene amplification was performed as described by Weisburg et al. (1991). PCR was carried out in a Corbett CG1-96 thermal cycler (Corbett Research Ltd., Cambridge, UK).

A 20 µL sample of purified PCR amplicon was digested with individual restriction enzymes in 40 µLat 37 ºC, using 2U HhaI, 1U Hinf I (New England Biolabs, USA), or 10U AluI (Fermentas, USA) following conditions recommended by the manufacturer. Digestions using MspI (BIORON, GmbH, Ludwigshafen, Germany) were done at 55 ºC. Restriction patterns were analysed on 2% agarose gels using 0.5 x TBE (45 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA, pH 8.0) as running buffer at 10 V/cm for 2 h, stained with 0.5 µg/mL ethidium bromide and visualized and photographed on a UV transilluminator. The amplified DNA fragment of the16S rRNA gene of each isolate was purified and then sequenced by Macrogen Sequencing Service, Korea. DNA sequences were compared by Blast analysis using non-redundant databases at the NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine which type strains had highest similarity.

Isolates under study were initially classified based on phenotypic characteristics and acid production from carbohydrates. All LAB isolates were homofermentative, Gram positive and catalase-negative cocci. Growth at 45 ºC, pH 9.6 or with 6.5% NaCl was tested for all isolates and type strains. Five isolates (F-11, F-22, F-130, F-131, and F-B2) were not able to grow under these conditions but were able to growth at 10 ºC as described by Axelsson (2004) for Lactococcus sp. Other studies, however, found variation in salt resistance among wild L. lactis spp lactis isolates in that a high proportion could grow with 6.5% NaCl (Aquilanti et al., 2007, Nomura et al., 2006). Four isolates (F-53, F-57, F-B1, and F-B3) were classified as Streptococcus sp. in accordance with the criteria established by Devriese and Pot (1995). These isolates grew at 45 ºC but were unable to grow at 10 ºC, extreme pH (4.4 or 9.6) or with 6.5% NaCl. Three other isolates (F-45, F-60, and F-63) having a characteristic cocci-tetrad cell arrangement were identified as Pediococcus sp. These were able to grow at 45 ºC, in 6.5% NaCl and at pH 4.4, but did not grow at pH 9.6 or 10 ºC.

Carbohydrate fermentation using the microplate assay confirmed differences among isolates at the species level (Axelsson, 2004). All isolates and type strains were able to ferment glucose, fructose, mannose and ribose, but unable to ferment melezitose, raffinose, melibiose and sorbitol. The carbohydrate utilization of wild isolates matched those of type strains, except for strains F-63 and F-60 whose carbohydrate fermentation patterns differed from the corresponding type strain, P. pentosaceus (ATCC 25745). Isolates F-63 and F-60 did not ferment salicin, maltose, arbutin, rhamnose, and threalose, while F-45 and the P. pentosaceus type strain produced acid from these carbohydrates. Bhunia and Johnson (1992) found that isolates unable to ferment rhamnose, arbutin and maltose could be identified as P. acidilactici. These are key sugars used for phenotypic identification of these species, and were consistent with 16S rDNA sequencing. Phenotypic and biochemical characterization of the isolates enabled grouping them into four species. Four isolates (F-53, F-57, F-B1, and F-B3) and S. salivarus subsp. thermophilus (ATCC 19258) were able to ferment lactose, sucrose and glucose, but unable to ferment galactose. However, Giraffa et al. (2001) found that acid production from these sugars was inconsistent within wild isolates of S. thermophilus. Five isolates (F-11, F-22, F-130, F-131, and F-B2) and L. lactis subsp lactis biovar. diacetylactis (ATCC 13675) were able to ferment all sugars tested except melezitose, raffinose, xilose, melibiose, rhamnose and sorbitol. All isolates of this group were assigned to biovar diacetylactis because of the ability to produce acetoin in the Voges-Poskauer reaction.

Digestion of 16S rDNA amplicons with restriction enzymes HinfI, AluI or MspI resulted in clear banding patterns but these could not be used to discriminate isolates at the species level. Aquilanti et al. (2007) working with AluI double digestions (using either HaeIII or FokI as the second enzyme), however, could discriminate subgroups among 112 isolates from Pecorino cheese. In our study, use of HhaI allowed for generation of five banding patterns found within four species (Figure 1). Isolates were classified in the following groups: Group 1, L. lactis subsp. lactis corresponding to isolates F-22, F-11, F-130 and F-131; Groups 2 and 3, S. salivarius subsp. thermophilus corresponding to isolates F-53 and F-57, and F-B1 and F-B3, respectively, Group 4, P. pentosaceus corresponding to isolate F-45 and Group 5, P. acidilactici corresponding to isolates F-60 and F-63. The different profiles found for S. salivarius subsp thermophilus could be associated with intra-subspecies variation as previously reported by Schlegel et al. (2003), however other differences between these isolates were not found in this study. Our results are in accordance with Petrov et al. (2008) who found that HhaI alone could be used to discriminate L. lactis subsp. lactis, L. lactis subsp cremoris, E. faecium, E. faecalis, Leuconostoc mesenteroides subsp. mesenteroides, L. lactis and S. thermophilus. However, Yousif et al. (2010) required HhaI and HinfI double digestions to discriminate P. acidilactici, P. pentosaceus and other Pediococcus sp.

Species designations of LAB isolated from milk and dairy products by phenotypic and genotypic characterization was confirmed by 16S rRNA gene sequencing (Table 1). All the isolates, except F-131, identified by partial 16S rRNA gene sequence analysis had an alignment identity of 99-100%. Since, isolate F-131 had an alignment identity of 96%, which is below species demarcating criteria, additional criteria for identification were established by phenotypic and ARDRA analysis.

Wild strain selection and accurate identification of LAB is of great artisanal and industrial interest and promises to provide resources for development of innovative processes and products. New strain availability is needed for development of customer-designed starters with the objective of providing "uniqueness" to value added products. Additionally, wild strains provide phenotypic and genetic variation resources for improvement of current commercial starter strains. The polyphasic approach for strain identification provides accuracy that will greatly facilitate developing and improving new starter systems. LAB identification using a polyphasic strategy clearly helps to overcome problems with inherent variation in wild isolates from dairy sources using either phenotypic tests including biochemical analyses (Moschetti et al., 1998) or genetic heterogeneity (Giraffa et al., 2001).

Acknowledgments

We gratefully acknowledge INIA, Uruguay, for partially supporting this study.

Submitted: June 27, 2011

Approved: September 10, 2012

All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License CC BY-NC.

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Publication Dates

• Publication in this collection
  09 Oct 2013
• Date of issue
  2013

History

• Received
  27 June 2011
• Accepted
  10 Sept 2012