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Microbial diversity of the traditional Iranian cheeses Lighvan and Koozeh, as revealed by polyphasic culturing and culture-independent approaches

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Dairy Science & Technology

Abstract

The microbiota of two traditional Iranian cheeses (Lighvan and Koozeh) made of raw ewe’s milk or mixtures of ewe’s and goat’s milk without starter addition was explored by culture-independent and culture-dependent approaches. Three batches of Lighvan and one of Koozeh were subjected to culture-independent polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through manufacturing and ripening. In addition, culturing in elective media for lactic acid bacteria (M17, MRS and KAA), isolation of single colonies (n = 130), molecular identification by PCR-amplified ribosomal DNA restriction analysis and sequencing, and differentiation at the strain level by repetitive extragenic palindromic PCR was also performed. DGGE analysis showed that the dominant amplicons in all four cheese batches belonged to Lactococcus lactis and Streptococcus parauberis. In addition, Escherichia coli and Lactococcus garvieae were frequently identified in both Lighvan and Koozeh, while Streptococcus thermophilus was found occasionally. In contrast, Enterococcus faecium and Enterococcus faecalis were found to be dominant among the isolates in all batches. These species showed a high genetic diversity. The discrepancy between culturing and DGGE results suggested that dominant populations were in a nonrecoverable state in the used media. This reinforces the idea that culture-dependent and culture-independent techniques provide complementary data, ultimately affording a better description of cheese ecosystems. These data could be of help in the selection of commercial starters for industrial-scale manufacture of Lighvan and Koozeh cheeses using pasteurised milk. Alternatively, microbial analysis would allow the selection of appropriate strains for designing of specific starters for traditional cheese manufacture.

伊朗传统Lighvan和Koozeh干酪的微生物多样性

摘要采用纯培养和非培养方法研究了由生鲜羊奶或者混合羊奶(羊奶和山羊奶)自然发酵制作的伊朗传统Lighvan和Koozeh干酪的微生物区系。对3批Lighvan干酪和1批Koozeh干酪进行了非培养的PCR-DGGE分析和主要条带的测序,以此评价干酪制作和成熟过程中微生物区系的结构和动力学。此外,采用选择性培养基(M17, MRS and KAA)对乳酸菌进行了培养,采用PCR-ARDRA、基因测序以及rep-PCR方法对分离出的单个菌落(n=130)从分子水平上进行鉴定。DGGE分析结果表明,在所有4批干酪样品中优势菌群为Lactococcus lactis a和Streptococcus parauberis。此外,在Lighvan和 Koozeh干酪中Escherichia coli和Lactococcus garviea的检出频率较高,但只在几个干酪样品中检测到Streptococcus thermophilus。相反,在所有样品中Enterococcus faecium 和Enterococcus faecalis也是主导菌群。微生物菌群之间表现出较高的生物多样性。纯培养和非培养的DGGE结果之间的差异表明主导菌群在这些培养基中是不可回收的,这种结果说明在进行干酪微生物生态系统的研究中,纯培养和非培养方法获得的数据可以互补。可以从获得的菌株中筛选出具有潜在工业化生产Lighvan和Koozeh干酪的发酵剂,也可以从中筛选出特定的菌株作为发酵剂用于传统干酪的生产。

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Acknowledgements

This research was partially supported by a project from the Spanish Ministry of Science and Innovation (MICINN) to BM (Ref. AGL2007-61869-ALI). AA was awarded a scholarship of the Severo Ochoa programme from FICYT (Ref. BP08-053). The authors wish to thank the Iranian Ministry of Industries and Mines, as well as Razavi Dairy Industry (Mashhad, Iran) and the Office of Industrial Relationships (OIR) of Ferdowsi University of Mashhad (FUM).

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Authors and Affiliations

  1. Department of Food Science and Technology, Ferdowsi University of Mashhad, Mashhad, Iran

    Mohammad Reza Edalatian, Mohammad Bagher Habibi Najafi & Seyed Ali Mortazavi

  2. Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

    Mohammad Reza Nassiri

  3. Department of Clinical Science, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

    Mohammad Reza Bassami

  4. Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias, (CSIC), Carretera de Infiesto, s/n, 33300, Villaviciosa, Asturias, Spain

    Mohammad Reza Edalatian, Ángel Alegría & Baltasar Mayo

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  1. Mohammad Reza Edalatian
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  2. Mohammad Bagher Habibi Najafi
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  3. Seyed Ali Mortazavi
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  4. Ángel Alegría
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Correspondence to Baltasar Mayo.

Electronic supplementary material

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Supplementary Table 1

Microbial dynamics as determined by identification of DGGE bands through manufacture and ripening stages of the traditional Iranian cheeses Lighvan and Koozeh (DOC 93 kb)

Online Resource Fig. 1

Partial amplified ribosomal DNA restriction analysis (ARDRA) profiles of 11 species from Lighvan and Koozeh cheeses from different manufacturing and ripening stages. The 16S rRNA gene was amplified using primers 27F and 1492R and digested with the restriction enzymes HaeIII (a) and HhaI (b). M molecular weight marker GeneRuler™. After partial amplification, sequencing and sequence comparison of 16S rRNA genes, the profiles were shown to correspond to the following species: 1 E. faecium; 2 L. plantarum, 3 L. brevis, 4 L. lactis subsp. lactis, 5 E. faecalis, 6 Enterococcus durans, 7 Enterococcus casseliflavus, 8 Enterococcus italicus, 9 Micrococcus luteus; 10 S. haemolyticus, and 11 Aerococcus viridans (PPT 369 kb)

Online Resource Fig. 2

Repeatability of the REP-PCR typing assay with primer BoxA2R after analysing of three randomly selected isolates (a, b, and c) in three independent experiments (1, 2, and 3). Below, dendogram of similarity of the different typing patterns clustered by the UPGMA method using the Simple Matching coefficient. M GeneRuler™ (PPT 163 kb)

Online resource Fig. 3

Typing REP-PCR profiles obtained with primer BoxA2R among the 36 E. faecium isolates from Koozeh cheese. Below, dendogram of similarity of the different typing patterns clustered by the UPGMA method using the Simple Matching coefficient. M GeneRulerTM. The broken line denotes the arbitrary percentage of similarity used to consider isolates as different strains (PPT 895 kb)

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Edalatian, M.R., Najafi, M.B.H., Mortazavi, S.A. et al. Microbial diversity of the traditional Iranian cheeses Lighvan and Koozeh, as revealed by polyphasic culturing and culture-independent approaches. Dairy Science & Technol. 92, 75–90 (2012). https://doi.org/10.1007/s13594-011-0045-2

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  • DOI: https://doi.org/10.1007/s13594-011-0045-2

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