Technological, Phenotypic and Genotypic Characterisation of Wild Lactic Acid Bacteria Involved in the Production of Bitto PDO Italian Cheese Morandi, Brasca, Lodi
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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