Technological, phenotypic and genotypic characterisation of wild lactic acid bacteria involved in the production of Bitto PDO Italian cheese Morandi, Brasca, Lodi
To cite this version:
Morandi, Brasca, Lodi. Technological, phenotypic and genotypic characterisation of wild lactic acid bacteria involved in the production of Bitto PDO Italian cheese. Dairy Science & Technology, EDP sciences/Springer, 2011, 91 (3), pp.341-359. 10.1007/s13594-011-0016-7. hal-00930575
HAL Id: hal-00930575 https://hal.archives-ouvertes.fr/hal-00930575 Submitted on 1 Jan 2011
HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Dairy Sci. & Technol.–359 (2011) 91:341 DOI 10.1007/s13594-011-0016-7
Technological, phenotypic and genotypic characterisation of wild lactic acid bacteria involved in the production of Bitto PDO Italian cheese
Stefano Morandi & Milena Brasca & Roberta Lodi
Received: 21 May 2010 /Revised: 2 December 2010 /Accepted: 2 December 2010 / Published online: 18 March 2011 # INRA and Springer Science+Business Media B.V. 2011
Abstract Bitto is a Protected Designation of Origin raw milk cheese produced in a restricted Italian alpine area only during the summer transhumance. The indigenous microbial ecosystem of this artisanal cheese is considered a primary factor related to its typicality. The aim of the research was to investigate the dynamics of wild lactic acid bacteria (LAB) involved in Bitto production and to study the characteristics of LAB. A total of 210 LAB isolates from curd, whey and ripened cheese, were first molecularly analysed by means of randomly amplified polymorphic DNA (RAPD). After strain differentiation, LAB were identified at the species level using species- specific primers and 16S rRNA gene sequencing. Genotypic diversity and technological properties of major interest for cheese making (acidification ability, redox potential and caseinolytic activity) were also evaluated. The predominant species, in both curd and ripened cheese, was Enterococcus faecium, and there appeared a high degree of diversity in the genotypic and technological traits. By using 16S rRNA sequencing and RAPD-PCR as well as examining the phenotypic properties, the new isolates were shown to belong to a novel enterococcal species for which the name Enterococcus lactis has been proposed. Among the curd isolates, six bacteriocin producers were found belonging to E. faecium, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus species.
意大利PDO Bitto干酪中野生乳酸菌的技术、表型和基因型特性研究
摘要 Bitto 是产自意大利阿尔卑斯山部分地区,且仅在夏季放牧季节生产的 PDO(原 产地名号保护)鲜乳干酪。这种手工干酪中固有的微生物生态系统被 认为是决定 该干酪特性的主要因素。本研究目的是调查Bitto 生产过程中野
S. Morandi : M. Brasca (*) : R. Lodi Institute of Sciences of Food Production National Research Council of Italy (CNR ISPA), Via Celoria. 2, 20133 Milan, Italy e-mail: [email protected] 342 S. Morandi et. al
生乳酸菌的动态 分布以及其特性。从凝乳、乳清和成熟的干酪中分离获得了 210 株乳酸菌,利用 随机扩增多态性DNA(RAPD)方法进行了分子水平分析。在 菌株鉴别后,利用 种特异性引物和16S rRNA 基因测序分析,将乳酸菌鉴定到 种。并且评价了基因 型多态性与干酪的技术特性指标(酸化能力、氧化还原 电势和酪蛋白水解活性) 的关系。在凝乳和成熟干酪中,Enterococcus faecium 是优势菌株,其在基因型和 技术特性中呈现了高水平的遗传多态性。通过16S rRNA 测序分析和RAPD-PCR 分析以及表型特性的检测,分离菌株属于一个新的 肠球菌Enterococcus lactis。在 凝乳中分离获得了6 株细菌素产生菌,研究发现 分别属于E. faecium,Lactobacillus fermentum,Lactobacillus. delbrueckii subsp. bulgaricus 和 Streptococcus 属。
Keywords Lactic acid bacteria . Raw milk cheese . Bitto . Technological characterization . Antimicrobial activity
关键词 乳酸菌 . 鲜奶干酪 . Bitto . 技术特性 . 抗菌活性
1 Introduction
Bitto is an artisanal cheese produced at an altitude of at least 1.500 m in a restricted Italian alpine area of Lombardy (Valtellina in the province of Sondrio, and in some districts in the province of Bergamo and Lecco). It is a traditionally made, cooked, semi-hard cheese. In 1996, it was awarded the Protected Designation of Origin (PDO) certificate by the European Community (1996). Bitto is made from whole raw cow’s milk of the Italian Brown breed, but the addition of not more than 10% goat’s milk is allowable. In line with its PDO rules, it is produced only between June 1 and September 30. In this time interval, the herds move upwards, from intermediate altitudes to the highest, following the richest pastures, and then move down to former grounds where new grass has sprouted. To date, acidification is due to indigenous microflora, but some starter cultures made up from indigenous Bitto microflora could also be used according to Bitto PDO requirements. The cheeses are left to ripen for a minimum of 70 days, but the ripening period can be extended, even up to several years. It is well-known that the typicality of raw milk cheeses is linked mainly to non- starter lactic acid bacteria (NSLAB) originating from raw milk (Van Hoorde et al. 2008a). Thus, the biodiversity of the lactic acid bacteria (LAB) involved in raw milk cheese production is considered to be a fundamental factor for the features and quality of these artisanal products. Several studies focus on the characterization of cheese-associated LAB (Dolci et al. 2008; Sánchez et al. 2005; Tilsala-Timisjävi and Alatossava 1997; Van Hoorde et al. 2008b; Vernile et al. 2008) but, only one document is available on the autochthonous microflora of Bitto cheese without focusing on the technological characterisation (Colombo et al. 2010). The aim of the present research was to characterise the indigenous bacterial population of Bitto, in curd and in cheese to investigate the dynamics of wild LAB involved during the production and ripening of this PDO Italian cheese. Lactic acid bacteria in Bitto PDO cheese 343
2 Materials and methods
2.1 Sampling
Eight dairy farms were selected in the Bitto production area, and analyses were carried out on eight milk samples, eight curds, eight cheeses at 70 days of ripening and four samples of whey (product of the cutting of curd). The whey and curd samples were collected on the day of production. All the samples were transported to the laboratory under refrigeration (4 °C) no later than 24 h from collection and subjected to microbiological analysis.
2.2 Microbial counts, isolation and preliminary characterisation of bacterial strains
Milk samples were serially diluted in quarter-strength Ringer’s solution. Dilutions were plated and incubated as follows: mesophilic aerobic bacteria were enumerated on Petrifilm Aerobic Count Plates (3M, Minneapolis, MN, USA) incubated at 30 °C for 72, coliforms on Petrifilm Coliform Count Plates (3M) at 37 °C for 24 h and coagulase-positive staphylococci on Baird-Parker Rabbit Plasma Fibrinogen agar (Biolife, Milan, Italy) at 37 °C for 48 h. Ten grammes of each curd and cheese sample were homogenised in 90 mL of sterile dipotassium hydrogenphosphate solution (2% w/v; pH 7.5±0.1). Decimal dilutions were prepared in quarter-strength Ringer’s solution and were plated on different media. The following analyses were carried out: presumptive mesophilic and thermophilic cocci on M17 agar (Scharlau Microbiology, Barcelona, Spain) incubated aerobically, respectively at 30 and 45 °C for 48 h; mesophilic and thermophilic presumptive lactobacilli on de Man Rogosa and Sharpe (MRS) agar (Scharlau Microbiology) incubated anaerobically at respectively, 30 and 45 °C for 72 h; for incubation in anaerobic conditions, jars with anaerocult A (Merck KGaA, Darmstadt, Germany) were used. Enterococci were detected using KAA (Scharlau Microbiology) at 37 °C for 48 h. A total of 225 colonies were randomly picked from the countable M17, MRS and KAA plates picking up colonies of all morphologies of bacterial colonies and streaked out three times on homofermentative heterofermentative differential agar (Biolife, Milan, Italy) to check for purity. After purification, the isolates were stored at −18 °C in litmus milk. After microscopic examination, Gram and catalase reactions, the isolates were tested for their ability to grow at 15 and 45 °C in M17 broth for cocci and MRS broth for rods, salt tolerance (2%, 4% and 6.5% of NaCl in M17 or MRS broth), carbon dioxide production from glucose by subculturing the isolates in MRS broth containing inverted Durham tubes, aesculin hydrolysation and their activity in litmus milk. All these tests were performed twice.
2.3 Antimicrobial activity
Antibacterial activity in the LAB strains was detected by the standardised agar disk diffusion method (Campos et al. 2006). Briefly, brain heart infusion agar (Scharlau 344 S. Morandi et. al
Microbiology) plates were seeded with 10 μL of an overnight culture of indicator strain (Listeria monocytogenes ATCC 9525 and Staphylococcus aureus ATCC 19095). Twenty microlitres of an overnight culture of the putative bacteriocin producer was spotted onto agar. The plates were incubated at 37 °C for 24 h, the diameter (millimetres) of the growth inhibition zones was measured.
2.4 DNA extraction
Isolates were grown overnight in 10 mL of M17 or MRS broth, and DNA was extracted using the Microlysis kit (Labogen, Rho, Italy), following the manufacturer’s instructions.
2.5 Randomly amplified polymorphic DNA analysis
Randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) profiles were used to perform a first strain differentiation and to explore the genetic diversity of LAB isolated from Bitto. RAPD-PCR reactions were performed with primers M13, D11344 and D8635 (Table 1), amplification conditions, as well as electrophoresis and analysis of the amplification products, were as previously described (Andrighetto et al. 2002; Morandi et al. 2006). Grouping of the RAPD-PCR profiles was obtained with the BioNumeric 5.0 software package (Applied Maths, Kortrijk, Belgium) using the unweighted pair-group method using arithmetic averages cluster analysis. The value for the repeatability of the RAPD-PCR assay, DNA extraction and running conditions, evaluated by analysis of repeated DNA extracts of the type strains, was 95%.
2.6 Species-specific PCR and DNA sequence analysis
The DNA sequences (5′-3′) for the primers used in this study, and their corresponding specificities are listed in Table 1. PCR primers used to identify Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis subsp. lactis and Lactococcus garvieae strainsweredesignedusingthePrimer3programme(http://frodo.wi.mit.edu/primer3). They were chosen on the basis of similar melting temperatures. The primers were synthesised by Invitrogen (Minneapolis, MN, USA) and resuspended to a final concentration of 100 pmol.μL−1 in sterile double-distilled water. Each DNA amplification was performed in 200-μL microtubes using a 25-μL reaction mixture containing 50–100 ng of DNA template, Quick Load Taq 2X Master Mix (New England Biolabs, Ipswich, MA, USA), micromole per litre of the primer pair and double-distilled water, to a final volume of 25 μL. All the amplifications were carried out in a Mastercycler (Eppendorf, Hamburg, Germany) with initial denaturation at 94 °C for5minfollowedby30cyclesat94°Cfor1min,primerannealingat56°Cfor1min and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. The amplified PCR products were visualised by standard gel electrophoresis in 1% agarose gel (GellyPhor, Euroclone, Milan, Italy) stained with SYBR Safe (Invitrogen, San Giuliano Milanese, Italy). The gels were photographed under ultraviolet light using a UV transilluminator. Molecular size markers (100-bp DNA ladder) (Euroclone, Milan, atcai atrai it D hee345 cheese PDO Bitto in bacteria acid Lactic Table 1 List of PCR primers used in this study
Target Primer Sequences (5′ to 3′) Target gene Positive control strains References
Enterococcus faecalis FAE-fw CGCTAGGCTCCATTGATAGC 16S rRNA ATCC 27332 This study FAE-rev CGGTTGGGTCTTGATCACTT Enterococcus faecium FUM-fw CGGAGACTACACAATTTGTTTTT 16S rRNA DSMZ 20477 T This study FUM-rev CGGTTGGGTTTTGATCCTT Enterococcus durans DUR-fw ATTTAGATCGGGGCCTTAGC 16S rRNA DSMZ 20633 T This study DUR-rev GCGGTGTTCTCGGTTTGTAT Lactococcus lactis subsp. lactis LAC-fw TCTTGATTGTGGGGCCTTAG 16S rRNA DSMZ 20481 T This study LAC-rev TCACAGGTTTTGGTTTATTTATCG Lactococcus garvieae LGA-fw CCTTAGCTCAGCTGGGAGAG 16S rRNA DSMZ 20684 T This study LGA-rev TTCGCAGCTTTACAGAAATGTT Streptococcus thermophilus ST-fw CAC TAT GCT CAG AAT ACA lacZ gene ISPA collection (Lick et al. 1996) ST-rev CGA ACA GCA TTG ATG TTA Lactobacillus delbrueckii DelI ACGGATGGATGGAGAGCAGGCAG 16S-23S spacer DSMZ 20081 T (Van Hoorde et al. 2008a) DSMZ 20072 T DelII GCAAGTTTGTTCTTTCGAACTCAACTC Lactobacillus casei PrI CAGACTGAAAGTCTGACGG 16S-23S spacer DSMZ 2011 T (Walter et al. 2000) CasII GCGATGCGAATTTCTTTTTC Lactobacillus fermentum Lfpr GCCGCCTAAGGTGGGACAGAT 16S-23S spacer DSMZ 20052 T (Walter et al. 2000) FermII CTGATCGTAGATCAGTCAAG Lactobacillus plantarum Lfpr GCCGCCTAAGGTGGGACAGAT 16S-23S spacer DSMZ 20174 T (Walter et al. 2000) PlanII TTACCTAACGGTAAATGCGA Lactobacillus paracasei subsp. paracasei PrII CAGACTGAAAGTCTGACGGACGG 16S rRNA ATCC 25303 (Van Hoorde et al. 2008a) Pcas II GCGATGCGAATTTCTTTTTCTTTC RAPD-PCR M13 GAGGGTGGCGGTTCT (Andrighetto et al. 2002; Morandi et al. 2006) RAPD-PCR D11344 AGTGAATTCGCGGTCAGATGCCA (Andrighetto et al. 2002) RAPD-PCR D8635 GAGCGGCCAAAGGGAGCAGAC (Andrighetto et al. 2002; Morandi et al. 2006) Sequencing p8FPL AGTTTGATCCTGGCTCAG 16S rRNA (Hosseini et al. 2009) p806R GGACTACCAGGGTATCTAAT fw forward, rev reverse, T type strain; DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; ATCC American Type Culture Collection, Rockville, MD, USA; ISPA Institute of Sciences of Food Production 346 S. Morandi et. al
Italy) were included in each agarose gel. Isolates that could not be identified using species-specific PCR were subjected to partial 16S rRNA sequencing using the universal primer set p8FPL and p806R (Table 1) (Hosseini et al. 2009). Primm s.r.l. (Milan, Italy) provided the sample sequencing service. The sequences were carefully reviewed by eye using Chromas software (Griffith University, QLD, Australia). Sequence alignment was carried out using ClustalW software. The BLAST algorithm was used to determine the most related sequence relatives in the National Center for Biotechnology Information nucleotide sequence database (http://www.ncbi.nlm.nih.gov/BLAST).
2.7 Technological characteristics
Strains were subcultured in M17 or MRS broth at 37 °C for 24 h and inoculated at a level of 1% in reconstituted sterile non-fat dry milk (10% w/v) and incubated at 37 °C for 24 h. Two replicates for each bacterial strain were used to estimate the acidifying and reducing activities. During incubation, a multi-channel pH-metre (Cinac version 3 Ysebaert, Frepillon, France) was used to follow the pH values. Combined pH electrodes (InLab 51343050, Mettler-Toledo, Greifensee, Switzerland) were standardised using two buffers (pH 4.0 and pH 7.0). The acidification rate was calculated as ΔpH (ΔpH=pHzero time−pHat time). Values of ΔpH after 6 h (ΔpH6) and 24 h (ΔpH24) were used to compare the acidifying activity of the strains. An Eh metre (pH302 Hanna Instruments, Villafranca Padovana, Italy) was used to follow the redox values (Eh) in the milk during incubation. The redox electrodes (InLab 501, Mettler-Toledo) were standardised using two redox solutions (240 and 470 mV; Hanna Instruments). In order to avoid any atmospheric oxygen interference with the Eh measurement, the cultures were carried out under static conditions. The Eh values were calculated according to Brasca et al. (2007). Redox values were recorded automatically at selected intervals (30 min) for 24 h; reduction activity was evaluated over 24 h by determining the minimum Eh value. Caseinolytic activity was evaluated according to the colourimetric method, which can detect the soluble nitrogen fraction through a chromogenic product measured spectrophotometrically after sample clarification by the addition of trichloracetic acid (Hull 1947). The samples were subjected to the measurement of the absorbance at a wavelength of 650 nm using a UVIDEC320 spectophotometer (Jasco, Hachioji, Tokyo, Japan). The microbial culture was inoculated at a level of 1% in reconstituted sterile non-fat dry milk (10% w/v) and incubated at 37 °C for 15 days. The results are expressed as milligrammes of tyrosine in 5 mL of milk, using a standard curve of tyrosine over the 0–1-mg range.
3 Results and discussion
3.1 Microbial counts
The microbiological quality of the milk used for Bitto cheese making proved to be quite satisfactory. The total load of bacteria in the raw milk ranged from 3.95 to 6.00 Lactic acid bacteria in Bitto PDO cheese 347
log10 colony forming units (CFU)·per millilitre, a low level of coliforms was found −1 (mean value 2.93±0.41 log10CFU.mL ) and coagulase-positive staphylococci were only found in three of the eight milk samples, with a maximum value of 3.65 log10 CFU.mL−1. The mean counts of the different microbial groups present in curd and cheese after 70 days of ripening are summarised in Table 2. In the curd, mesophilic cocci −1 formed the most numerous group, ranging from 4.30 log10CFU.g to 6.83; also the thermophilic cocci detected on M17 agar incubated at 45 °C were numerous, with a −1 mean value of 6.11 log10CFU.g . With regard to the lactobacilli thermophilic group, this predominated the mesophilic one with a mean value of 5.98 vs 5.40 log10 CFU.g−1. The highest LAB population levels were detected in cheese after 70 days −1 of ripening with counts ranging from 7.00 log10CFU.g on M17 agar at 45 °C to −1 8.21 log10CFU.g on MRS at 30 °C. Differently from the curd, the most numerous −1 group was mesophilic lactobacilli (mean value, 8.02 log10CFU.g ). In agreement with previous studies on other raw milk cheeses, the enterococci levels increased from curd to cheese in three log units (Morandi et al. 2005). The increase in load of these bacteria during the late stages of Bitto maturation suggests that enterococci might play an important role in the ripening of this cheese. The LAB species detected in the curd and cheese samples fully concur with the microbial count data (Table 3). In the whey, there was a strong prevalence of mesophilic and thermophilic cocci (Table 2), a high concentration of mesophilic and thermophilic rods (ranging from −1 5.34 to 6.95 log10CFU.g ) and enterococci were also detected (mean value, −1 3.13 log10CFU.g ).
3.2 Identification and characterization of bacterial strains
A total of 225 bacterial isolates were collected from samples of whey, curd and 70- day-old cheese. Among them, 210 isolates (20 from whey, 97 from curd and 93 from cheese) were Gram-positive and catalase-negative and were considered for further analysis. These 210 isolates were divided into two groups according to the cell morphology: cocci and rods. The majority (82.5%) was assigned to the cocci group, and 17.5% were rods (Table 4). For isolate identification, we used a polyphasic approach. First RAPD-PCR was performed on the 210 isolates and the resulting fingerprintings were compared to a user-generated BioNumerics database for a preliminary identification; this identifi- cation was then confirmed by species-specific PCR analysis for species listed in Table 1 and partial DNA sequence analysis for the others. Table 3 shows the frequency of isolation of each LAB species in correlation with the selective isolation LAB media. Plate counts on MRS and M17 agar clearly indicated that the predominance of the enterococci counts on KAA was significantly lower than in MRS and M17 agar. It can be assumed that enterococci are undervalued when determined in KAA medium. The MRS agar displayed poor selectivity under the conditions applied here, resulting in an overestimation of the number of lactobacilli. On the other hand, MRS agar supported the growth of the largest number of LAB genera including Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Streptococcus. 348
Table 2 Enumeration and isolation data for whey, curd and cheese samples of Bitto cheese
Medium
KAA M17 30 °C M17 45 °C MRS 30 °C MRS 45 °C
− − − − − Log CFU g 1 Number Log CFU·g 1 Number Log CFU·g 1 Number Log CFU·g 1 Number Log CFU·g 1 Number
Whey 5 0 11 0 4 Mean 3.13 7.50 7.48 5.98 5.61 SD 0.80 0.16 0.26 0.67 0.20 Curd 21 13 21 18 24 Mean 4.21 6.37 6.11 5.40 5.98 SD 0.67 0.85 0.77 0.62 1.00 Cheese 18 18 18 22 17 Mean 7.14 7.79 7.66 8.02 7.46 SD 0.26 0.19 0.33 0.13 0.38 .Mrnie.al et. Morandi S. atcai atrai it D hee349 cheese PDO Bitto in bacteria acid Lactic Table 3 Frequency of isolation of LAB species on KAA, and M17 agar and MRS agar plates incubated at 30 and 45 °C
Species MRS 45 °C M17 30 °C M17 45 °C MRS 30 °C KAA Total
Whey Curd Cheese Curd Cheese Whey Curd Cheese Curd Cheese Whey Curd Cheese
Enterococcus durans 11 Enterococcus faecalis 2 31 6 Enterococcus faecium 3 6 1 7 2 12 9 4 5 18 10 77 Enterococcus lactis 33 11 210 Lactococcus garvieae 2 2 Lactococcus lactis subsp. lactis 11 Leuconostoc lactis 1 23 Leuconostoc mesenteroides 112 Pediococcus acidilactici 3 1 12 18 Pediococcus pentosaceus 2 2 13 13 12 Streptococcus bovis 1 1 Streptococcus macedonicus 1 1 Streptococcus salivarius 1 1 Streptococcus thermophilus 842111621 44 Streptococcus spp. 11 2 Lactobacillus brevis 22 Lactobacillus casei 22 Lactobacillus delbrueckii subsp. bulgaricus 231 17 Lactobacillus delbrueckii subsp. lactis 21 3 Lactobacillus fermentum 85 2 15 Lactobacillus. parabuchneri 11 Lactobacillus paracasei subsp. paracasei 77 Lactobacillus plantarum 11 Lactobacillus reuteri 11 Total 4 25 18 10 17 11 20 18 20 22 5 22 18 210 350 S. Morandi et. al − 3+ 1+ 1+ −− −− −− −− −− −− −− − −− −− −− −− L. monocytogenes S. aureus −− − −− − −− − −− − −− − −− − −− −− − − −−−− − − − − −− − ++ + ++ + ++ + + ++ + − +++ − − − − − − − ++++ − − 45 °C 15 °C 2% 4% 6.5% 11+++++ 633+++++ 22 +++++ 772+++++ 11+++++ 11+ 11 ++++ 33 28 2 2 6 1+ +32 1 + 7+11 + + + + + + 11 ++++ 11 + 11 + 22+++++ 22+++++ 72 3 2 + 7710 5 33 39 10 + + + + + + + + + + 1+ 15 10 5 + 1244 2 11 10 29 + 4 11+ + + + + lactis bulgaricus paracasei lactis subsp. subsp. subsp. subsp. spp. 2 2 + Enterococcus durans Enterococcus faecalis Enterococcus faecium Enterococcus lactis Lactococcus garvieae Lactococcus lactis Lactobacillus plantarum Lactobacillus reuteri Total 39 4 15 20 Leuconostoc lactis Lactobacillus fermentum Leuconostoc mesenteroides Pediococcus acidilactici Pediococcus pentosaceus Streptococcus thermophilus Streptococcus Lactobacillus delbrueckii Lactobacillus parabuchneri Lactobacillus paracasei Streptococcus bovis Streptococcus macedonicus Streptococcus salivarius TotalLactobacillus brevis Lactobacillus casei Lactobacillus delbrueckii 171 16 82 73 Morphological and physiological characteristics of lactic acid bacteria strains from Bitto Table 4 Morphology SpeciesCocci Total Whey Curd Cheese Growth at Growth with NaCl Antimicrobial activity Total 210 20 97 93 Rods Lactic acid bacteria in Bitto PDO cheese 351
The incubation temperatures allowed us to discriminate between mesophilic and thermophilic microflora, obtaining a marked growth differentiation among LAB species. On the contrary, the M17 agar plates were quite discriminating for cocci, and the different growth temperatures applied did not allow the recovery of different species. The KAA medium showed high selectivity towards enterococci, although pediococci were also isolated. Overall, in spite of the overlapping growth, the three selective media used in this study allowed us to isolate a high number of different LAB biotypes as evidenced by RAPD-PCR profiles. E. faecium was the predominant species, and its percentage of isolation increased from curd to cheese (Table 4). The role of enterococci as a relevant component of natural cultures involved in the fermentation of European artisanal cheeses has been described in detail (Giraffa 2003). Indeed, enterococci comprise a major part of the fresh cheese curd microflora, and in some cases they are the predominant microorganisms in the fully ripened product (Giraffa 2003; Jokovic et al. 2008). The isolation of enterococci from curd and ripened cheeses can be ascribed to their tolerance to high salt content and low pH, as well as to their adaptability to a wide range of different substances and growth conditions (Morandi et al. 2005). Moreover, the peculiar cheese-making technique used for the manufacture of Bitto, which includes heating the curd to 48–52 °C, could contribute to the selection of these microorganisms. Although the presence of enterococci in dairy samples is often associated with faecal contamination, it is supposed that they contribute positively to the development of flavour during cheese ripening as their presence is consistently reported in many cheeses (Cogan et al. 1997). Not only can enterococci influence the flavour and taste of cheeses due to their primary and secondary metabolisms, they also produce several enzymes that interact with milk components, thus promoting other important biochemical transformations (Skeie and Ardo 2000). Note, however, that our results are not in complete agreement with Colombo et al. (2010) who found E. durans as the most common species isolated from Bitto cheese. This difference in our findings could be explained by the different ripening periods of the analysed samples (120 days against 70 days), the different number of dairy farms evaluated (one against eight) and the different production areas. The next most numerous species predominant in Bitto curd was Streptococcus thermophilus (44 strains). Indeed, the frequency of isolation of these isolates decreased from curd to 70-day-old cheese. These results are in agreement with research on different European artisanal cheeses. Indeed, S. thermophilus was usually present in high concentrations in fresh cheese and curd treated with similar technology, and usually decreased during ripening, gradually being overtaken by the NSLAB (Cogan et al. 1997; Van Hoorde et al. 2008a, b). One strain could not be associated with any previously associated species. Comparative 16S rRNA gene sequence analysis recognised it as Streptococcus spp. sharing an identity of 99% (NCBI accession number GI285202051). Ten isolated strains belonged to the apparently emerging, and recently proposed, new species, Enterococcus lactis. The presence of E. lactis was detected only in the cheese of one producer, and the RAPD patterns show the presence of five different genotypes (Fig. 1). The 16S rRNA sequence analysis of these strains showed high correlation between the Bitto isolates and E. lactis sequences present in the GeneBank database (99% identity with 16S rRNA 352 S. Morandi et. al
Similarity (%) 10 20 30 40 50 60 70 80 95
Enterococcus lactis
Leuconostoc mesenteroides Streptococcus bovis Streptococcus spp. Leuconostoc lactis Leuconostoc mesenteroides Enterococcus faecium Enterococcus durans
Enterococcus faecium
Enterococcus faecalis Leuconostoc lactis Streptococcus spp.
Streptococcus thermophilus
Enterococcus faecalis Pediococcus acidilactici Lactococcus lactis subsp. lactis Lactococcus garvieae Streptococcus salivarius Streptococcus macedonicus Streptococcus spp. Pediococcus pentosaceus
Pediococcus acidilactici
Pediococcus pentosaceus
Fig. 1 Unweighted pair-group method with arithmetic averages (UPGMA)-based dendrogram derived from the combined RAPD-PCR patterns generated with primers M13, D11344 and D8635 of the 171 cocci strains isolated from Bitto samples Lactic acid bacteria in Bitto PDO cheese 353 sequence of E. lactis, GeneBank accession number FJ015055). It is important to note that E. lactis is not a validated name; this taxonomic name had still not been published at the time of submission of the corresponding sequence entries (http:// www.ncbi.nlm.nih.gov/Taxonomy). The strains belonging to this proposed new species are phenotypically close to E. faecium, but the utilisation of mannitol differentiates them from E. faecium. Indeed, E. lactis strains had already been isolated from Russian cheese and fresh milk samples from South Africa (Bauer et al. 2009; Botina and Sukhodolets 2006). Leuconostoc spp. were isolated only in curd, E. durans only in cheese, E. faecalis equally in curd and cheese, while Pediococcus spp. increased in number from curd to ripened cheese. In ripened cheese, pediococci may occur as part of the NSLAB that contribute to cheese ripening. Other cocci isolates recovered belonged to the species L. garvieae, Streptococcus salivarius, Streptococcus macedonicus and Streptococcus bovis. Among lactic acid rods, Lactobacillus fermentum was the predominant species in the curd; in 70-day-old cheese, its isolation decreased and the prevailing species became Lactobacillus paracasei subsp. paracasei, frequently isolated from artisanal cheeses (Callon et al. 2004; Dolci et al. 2008; Prashant et al. 2009). Other species were isolated in variable amounts such as Lactobacillus brevis, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus (the only rod species present in whey, curd and cheese), L. delbrueckii subsp. lactis, Lactobacillus parabuchneri, Lactobacillus plantarum and Lactobacillus reuteri. Considering the growth characteristics of the LAB isolated from the Bitto, all the isolates grew at 37 °C, the majority were able to grow at 45 °C; the selection of these thermally resistant microorganisms is probably due to the cooking of the curd at 48–52 °C.
3.3 Antimicrobial activity
According to the test of the ability to produce antimicrobial substances, L. monocytogenes wasinhibitedonlybyoneE. faecium strain (diameter of the inhibition zone, 14 mm), while five isolates (one Streptococcus spp, one L. fermentum and three L. delbrueckii subsp. bulgaricus) exhibited antagonistic activity against S. aureus (diameter of the inhibition zone, 8–11 mm) (Table 4). All the strains with antimicrobial activity were isolated from curd samples. This antimicrobial effect was always detected, even when the overnight cultures used in the test were adjusted to pH 7.0.
3.4 RAPD-PCR analysis
RAPD-PCR was carried out to explore the genetic diversity of 210 LAB isolates using primers M13, D11344 and D8635. Figure 1 shows the different banding patterns of 171 cocci. Basically, the cocci fell into three main clusters: one grouped E. faecium, E. durans and E. faecalis, one clustered S. thermophilus and one pediococci. It was found that the isolates ascribed to the same genotype clustered together, but there were a few exceptions such as one E. durans strain, which was mixed within the E. faecium group and two Pediococcus acidilactici strains that did not fall into the cluster of pediococci. There was general high biodiversity among the strains. A high degree of DNA polymorphism was detected in E. faecium and S. 354 S. Morandi et. al thermophilus where the similarity levels reached 22% and 42%, respectively. On the contrary, the E. lactis strains clustered at a high level (76%). Our results confirm earlier data reporting significant genetic heterogeneity within the species E. faecium and S. thermophilus, both of which are present in different Italian traditional cheeses (Andrighetto et al. 2002; Morandi et al. 2006). Figure 2 shows the RAPD banding patterns of 39 isolated rods. Like the cocci strains, almost all the strains were grouped according to species except for two L. delbrueckii subsp. bulgaricus strains that did not fall in the cluster of L. delbrueckii subsp. bulgaricus. The wild lactobacilli isolates showed great genetic diversity, particularly within L. fermentum (similarity level of 38%) and L. paracasei subsp. paracasei (38%). On the other hand, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains, though few, clustered at a high level (75% and 82%, respectively). For both the cocci and rods there was no correlation between the dairy farm samples from which the strains were isolated and the clustering patterns. The diversity observed in some species, such as E. faecium and S. thermophilus, could be the result of the higher number of strains used for comparison, and therefore of the increased probability of encountering more distantly related taxonomic units.
Similarity (%) 20 30 40 50 60 70 80 90 95
Lactobacillus delbrueckii subsp. bulgaricus
Lactobacillus casei Lactobacillus plantarum
Lactobacillus paracasei subsp. paracasei
Lactobacillus delbrueckii subsp. bulgaricus
Lactobacillus delbrueckii subsp. lactis
Lactobacillus parabuchneri
Lactobacillus fermentum
Lactobacillus reuteri Lactobacillus brevis
Fig. 2 Unweighted pair-group method with arithmetic averages (UPGMA)-based dendrogram derived from the combined RAPD-PCR patterns generated with primers M13, D11344 and D8635 of the 39 Lactobacillus strains isolated from Bitto samples Lactic acid bacteria in Bitto PDO cheese 355
3.5 Technological characterization
As far as concerns the acidifying activity of the 210 isolates, 6 and 24 h of fermentation led to the defining of three classes (Psoni et al. 2007) class I, high acidifying isolates showing a pH decrease over two pH units; class II, the group of medium acidifying activity, showing a pH drop ranging between 1.5 and 2.0 pH units; class III, low acidifying isolates, causing a pH decrease lower than 1.5 pH units. After 6 h of fermentation, the majority of isolates (96%) fell into class III, and only eight strains, identified as S. thermophilus, were classified as medium acidifying isolates, and could thus be considered the fastest acidifying strains (Table 5). Although the majority of isolates were initially slow, for most of them acid production became enhanced later, and after 24 h 55% of the isolates were grouped into class II, and 8% were classified as high acidifying strains. The acid production of the enterococci was generally classed as medium; in fact, after 24 h of fermentation 66% (one E. faecalis,56E. faecium and five E. lactis) fell into class II, while 34% (five E. faecalis,22E. faecium and five E. lactis) showed poor acidifying ability. Like the majority of enterococci, the lactococci strains were grouped into class II, while most of Leuconostoc and pediococci were weak acid producers. Good acidifying ability was common in the S. thermophilus isolates. In fact, 77% of these strains fell into class II, while eight isolates (18%) were grouped into class I. The Lactobacillus strains exhibited different acidifying activities; all the isolates of L. brevis, L. fermentum, and L. parabuchneri fell into class III, while L. casei, L. paracasei subsp. paracasei, L. plantarum and L. reuteri were grouped into class II. The strains identified as L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis showed the highest acidifying activity in skim milk. The most acidifying species were found among those commonly employed as dairy starter cultures. After 24 h, acid production by the isolates varied considerably. There was a notable variation in pH reduction among and within the different species, indicating that this ability depends on the strains, which is consistent with previous reports (Aquilanti et al. 2007; Cogan et al. 1997; Marino et al. 2003; Sánchez et al. 2005). Another important parameter, and one that is often not considered, is redox potential (Cachon et al. 2002). The redox potential of cheese is a major factor in determining the types of microorganisms that will grow in it. Therefore, obligate aerobes such as Pseudomonas, Brevibacterium, Bacillus and Micrococcus spp. are excluded from growth in the cheese interior (Brasca et al. 2007). However, Eh contributes to the creation of conditions necessary for balanced flavour development (Olson 1990). As far as redox potential after 24 h of fermentation is concerned, three classes were defined: class I, high reducing isolates (Eh<−102 mV); class II, the group of medium reducing activity (−102 mV
a a Species ΔpH6 ΔpH24 Eh
Class III Class II Class III Class II Class I Class III Class II Class I ΔpH<1.5 1.5<ΔpH<2 ΔpH<1.5 1.5<ΔpH<2 ΔpH>2 Eh>−2mV −102 mV Enterococcus durans 1 –– 1 – 1 (3.6) –– Enterococcus faecalis 6 – 51–– – 6 (2.4) Enterococcus faecium 77 – 22 55 – 18 (17.5) 44 (13.9) 15 (3.6) Enterococcus lactis 10 – 55–– 8 (10.1) 2 (2.3) Lactococcus garvieae 2 – 11–– – 2 (7.9) Lactococcus lactis subsp. lactis 1 –– 1 –– – 1 (6.0) Leuconostoc lactis 3 – 21– 2 (21.5) 1 (23.5) – Leuconostoc mesenteroides 2 – 2 ––– 2 (23.0) – Pediococcus acidilactici 8 – 8 ––2 (18.0) 6 (22.4) – Pediococcus pentosaceus 12 – 10 2 – 6 (23.0) 6 (21.2) – Streptococcus bovis 1 – 1 ––– – 1 (3.5) Streptococcus macedonicus 1 –– 1 – 1 (19.0) –– Streptococcus salivarius 1 –– 1 – 1 (22.0) –– Streptococcus thermophilus 36 8 2 34 8 32 (23.1) 12 (19.6) – Streptococcus spp. 2 – 2 ––– 1 (23.5) 1 (5.5) Lactobacillus brevis 2 – 2 ––– 2 (23.0) – Lactobacillus casei 2 –– 2 –– – 2 (4.3) Lactobacillus delbrueckii subsp. bulgaricus 7 –– 1 6 6 (23.2) 1 (7.5) – Lactobacillus delbrueckii subsp. lactis 3 –––3 2 (19.0) 1 (15.0) – Lactobacillus fermentum 15 – 15 ––5 (23.4) 10 (23.0) – Lactobacillus. parabuchneri 1 – 1 ––– 1 (21.0) – Lactobacillus paracasei subsp. 7 –– 7 –– – 7 (7.9) paracasei Lactobacillus plantarum 1 –– 1 –– 1 (23.0) – Lactobacillus reuteri 1 –– 1 – 1 (22.0) –– al et. Morandi S. In parentheses, the mean values of time, expressed in hours, at which the Eh minimum occurred a Activity after 6 and 24 h of incubation Lactic acid bacteria in Bitto PDO cheese 357 isolated from the cheese samples. The ten isolates of E. lactis had an Eh evolution similar to E. faecium while the strain of E. durans had low reducing power. Like E. faecalis, the lactococci strains were grouped into class I, while Leuconostoc and pediococci showed low–medium reducing power. The strains belonging to the S. thermophilus species showed low reduction power; 73% of isolates fell into class III, all the strains reached Eh min after 19 h of incubation. Among the lactobacilli, the highest reducing species were L. paracasei subsp. paracasei followed by L. casei, but these isolates reached the Eh min more slowly than the enterococci of class I. The L. fermentum strains showed medium–low reduction activity, while the greater part of strains of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis showed a low redox potential. Our results are in agreement with Brasca et al. (2007) who showed that each LAB species has a typical Eh evolution over time, and the cocci, particularly E. faecalis and E. faecium, reduced earlier, and even more than the Lactobacillus species. Moreover, within the same species the Eh can differ, and these differences are tied to the specific strain and its provenance. Protease activity is necessary for the good growth of lactic acid bacteria in milk, and for casein hydrolysis during cheese ripening. Low casein breakdown ability (<0.20 mg tyrosine·5 mL−1 of milk) was detected in 208 isolates (99%) and only two L. fermentum strains showed good proteolytic activity (>0.50 mg tyrosine·5 mL−1 of milk). 4 Conclusions Considering the limited information available on the microbiological character- ization of this traditional dairy product, the results from this study have most certainly led to a better understanding of the bacterial population involved in Bitto production, allowing the defining of dominant biotypes in curd and ripened cheese, and enabling a preliminary assessment of the technological characteristics connected with strain variability. Indeed, bacterial biotype studies provide an efficient, though limited, tool for the evaluation of complex microbial systems like those found in raw milk cheeses. Our research has shown the presence of a wide variety of LAB, including strains belonging to E. lactis that can be proposed as an emerging, possibly new species. Furthermore, it was found that the complex ecosystem of Bitto cheese includes strains belonging to different species characterised by a wide inter- and intraspecific genetic and technological variability. Indeed, bacterial biotypes with technologically interesting properties were isolated. These findings tend to corroborate the theory that the predominance of strains with particular capabilities is connected to a specific environment, therefore the autochthonous microflora of traditional cheeses represent a heritage that needs to be protected and conserved. Acknowledgements This study was partly performed within the research project VALTEC set-up and supported by the Regione Lombardia. 358 S. Morandi et. al References Andrighetto C, Borney F, Barmaz A, Stefanon B, Lombardi A (2002) Genetic diversity of Streptococcus thermophilus strains isolated from Italian traditional cheese. Int Dairy J 12:141–144 Aquilanti L, Silvestri G, Zannini E, Osimani A, Santarelli S, Clementi F (2007) Phenotypic and genotypic and technological characterization of predominant lactic acid bacteria in Pecorino cheese from central Italy. J Appl Microbiol 130:948–960 Bauer R, Bekker JP, van Wyk N, du Toit C, Dicks LMT, Kossmann J (2009) Exopolysaccharide production by lactose-hydrolyzing bacteria isolated from traditionally fermented milk. Int J Food Microbiol 131:260–264 Botina SG, Sukhodolets VV (2006) Speciation in bacteria: comparison of the 16S rRNA gene for closely related Enterococcus species. Russ J Gen 42:247–251 Brasca M, Morandi S, Lodi R, Tamburini A (2007) Redox potential to discriminate among species of lactic acid bacteria. J Appl Microbiol 103:1516–1524 Cachon R, Jeanson S, Aldarf M, Divies C (2002) Characterization of lactic starters based on acidification and reduction activities. Lait 82:281–288 Callon C, Millet L, Montel MC (2004) Diversity of lactic acid bacteria isolated from AOC Salers cheese. J Dairy Res 71:231–244 Campos AC, Rodríguez O, Calo-Mata P, Prado M, Barros-Velásquez J (2006) Preliminary characterization of bacteriocins from Lactococcus lactis, Enterococcus faecium and Enterococcus mundtii strains isolated from turbot (Psetta maxima). Food Res Int 39:356–364 Cogan TM, Barbosa M, Beuvier E, Bianchi-Salvadori B, Cocconcelli PS, Fernandes I, Gomez J, Gomez R, Kalantzopoulos G, Ledda A, Mediana M, Rea MC, Rodriguez E (1997) Characterization of the lactic acid bacteria in artisanal dairy products. J Dairy Res 64:409–421 Colombo F, Borgo F, Fortina MG (2010) Genotypic characterization of non starter lactic acid bacteria involved in the ripening of artisanal Bitto PDO cheese. J Basic Microbiol 49:521–530 Dolci P, Alessandria V, Zeppa G, Rantsiou K, Cocolin L (2008) Microbiological characterization of artisanal Raschera PDO cheese: analysis of its indigenous lactic acid bacteria. Food Microbiol 25:392–399 EU Commission Regulation EC 1107/96. Official Journal of the European Union L148 of 21, 12 June 1996 Giraffa G (2003) Functionality of enterococci in dairy products. Int J Food Microbiol 88:215–222 Hosseini SV, Arlindo S, Böhme K, Fernández-No C, Calo-Mata P, Barros-Velázquez J (2009) Molecular and probiotic characterization of bacteriocin producing Enterococcus faecium strains isolated from nonfermented animal foods. J Appl Microbiol 107:1392–1403 Hull ME (1947) Studies on milk proteins. II. Colorimetric determination of partial hydrolysis of the proteins of milk. J Dairy Sci 30:881–884 Jokovic N, Nikolic M, Begovic J, Jovcic B, Savic D, Topisirovic L (2008) A survey of the lactic acid bacteria isolated from Serbian artisanal dairy product kajmak. Int J Food Microbiol 27:305–311 Lick S, Keller M, Bockelmann W, Heller KJ (1996) Rapid identification of Streptococcus thermophilus by primer-specific PCR amplification based on its lacZ gene. Syst Appl Microbiol 19:74–77 Marino M, Maifreni M, Rondinini G (2003) Microbiological characterization of artisanal Montasio cheese: analysis of its indigenous lactic acid bacteria. FEMS Microbiol Lett 229:133–140 Morandi S, Brasca M, Alfieri P, Lodi R, Tamburini A (2005) Influence of pH and temperature on the growth of Enterococcus faecium and Enterococcus faecalis. Lait 3:181–192 Morandi S, Brasca M, Andrighetto C, Lombardi A, Lodi R (2006) Technological and molecular characterisation of enterococci isolated from north–west Italian dairy products. Int Dairy J 16:867– 875 Olson NF (1990) The impact of lactic acid bacteria on cheese flavour. FEMS Microbiol Rev 87:131–147 Prashant, Tomar SK, Singh R, Gupta SC, Arora KD, Joshi BK, Kumar D (2009) Phenotypic and genotypic characterization of lactobacilli from Churpi cheese. Dairy Sci Technol 89:531–540 Psoni L, Kotzamanides C, Yiangou M, Tzanetakis N, Litopoulou-Tzanetakis E (2007) Genotypic and phenotypic diversity of Lactococcus lactis isolates from Batzos, a Greek PDO raw goat milk cheese. Int J Food Microbiol 114:211–220 Sánchez I, Seseña S, Poveda M, Cabezas L, Palop L (2005) Phenotypic and genotypic characterization of lactobacilli isolated from Spanish goat cheeses. Int J Food Microbiol 102:355–362 Skeie S, Ardo Y (2000) Influence from raw milk flora on cheese ripening studied by different treatments of milk to model cheese. LWT Food Sci Technol 33:499–505 Lactic acid bacteria in Bitto PDO cheese 359 Tilsala-Timisjävi A, Alatossava T (1997) Development of oligonucleotide primers from the 16S-23s rRNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. Int J Food Microbiol 35:49–56 Van Hoorde K, Vandamme P, Huys G (2008a) Molecular identification and typing of lactic acid bacteria associated with the production of two artisanal raw milk cheeses. Dairy Sci Technol 88:445–455 Van Hoorde K, Verstraete T, Vandamme P, Huys G (2008b) Diversity of lactic acid bacteria in two Flemish artisan raw milk Gouda-type cheeses. Food Microbiol 25:929–935 Vernile A, Giammanco G, Spano G, Beresford TP, Fox PF, Massa S (2008) Genotypic characterization of lactic acid bacteria isolated from traditional Pecorino Siciliano cheese. Dairy Sci Technol 88:619–629 Walter JGW, Tannock GW, Tilsala-Timisjarvi A, Rodtong S, Loach DM, Munro K, Alatossava T (2000) Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers. Appl Environ Microbiol 66:297–303