Biodiversity of lactic acid bacteria in Romanian dairy products

https://doi.org/10.1016/j.syapm.2005.10.002Get rights and content

Abstract

Traditionally fermented dairy products are still a very important part of the daily food in Romania, especially for people living in the countryside. To study the biodiversity of lactic acid bacterium strains of these products, 110 samples (raw and fermented milk, sour cream, and cheese) were collected from farm houses, monasteries, and local markets throughout Romania. Lactic acid bacteria (LAB) were isolated using six different cultivation conditions. All 599 isolates were tested for their Gram reaction, catalase activity, and morphology. A rep-PCR fingerprinting technique with the (GTG)5 primer and, in some cases SDS–PAGE of total cell proteins and 16S rRNA gene sequencing were used to cluster and/or identify the LAB. The biodiversity of the isolated strains was correlated with the type of product and/or technology applied. The most frequent LAB found in Romanian raw milk and fermented dairy products were Lactococcus lactis, Leuconostoc spp., and Enterococcus spp. Among the latter, a new species E. saccharominimus was found.

Introduction

Nowadays, various types of fermented milk products exist throughout the world [31], [32]. Their nature depends on the type of milk used, the pre-treatment of the milk, and the conditions of fermentation and subsequent processing. They mainly involve lactic acid bacteria (LAB), but micrococci, coryneforms, yeasts, and moulds can also occur. Historically, fermented dairy products have been produced to prolong the shelf-life of milk. These traditional foods have persisted over centuries and they often evolved from home manufacture in an artisan and traditional way to large-scale industrial production using specific starter cultures and modern equipment [7], [25]. The use of starters improved the technological quality of dairy products, but at the same time limited their biodiversity as well as the organoleptic variation of the end products [21], [38]. Therefore, an increasing demand exists for new strains that show desirable effects on the product characteristics [14]. Artisan dairy products are a candidate source of such microorganisms [21]. Fermentation of milk by specific microbiota induces changes in the taste, texture, colour, flavour, and the nutritional properties of milk, and provides a wide variety of end products [12], [22], [25]. In addition, fermented milks contribute to human health [25], [37]. They have been proposed for curing gastrointestinal disorders and they are, therefore, frequently used in developing countries [28].

The earliest studies on the microbiological composition of fermented milks date from the end of the nineteenth century [25]. At present, LAB species of the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and Bifidobacterium have been identified in fermented milks. Mainly strains of Lactococcus, Lactobacillus, Streptococcus, and Bifidobacterium are used in commercial starter cultures [7], [23]. Only limited information is available concerning the microbiota involved in artisan dairy products that are manufactured in farm houses, and cheeses in particular [4], [5], [8], [14], [27].

Traditionally fermented dairy products are still a very important part of the daily food in Romania. People living in the countryside possess their own cow(s) or sheep(s) and use the milk to produce sour cream and cheese for their own needs. In some regions, mostly located in the mountains, goats’ and buffalos’ milk is frequently used. Farmers with more animals sell the milk to small factories located in a nearby village, or sell milk and fermented products on the local market. In most cases non-pasteurized milk is used and the fermentation process relies on the natural microbiota of the milk and the environment. Backslopping, a process wherein a portion of a traditionally fermented milk or sour cream from a previous batch is used as an inoculum for the new batch, is also practiced sometimes to expedite the fermentation process. These products may have a very constant microbial content over time. In general, however, environmental conditions such as temperature, origin of the milk, processing and sanitary conditions, etc., might have a significant influence on the microbial composition of traditionally made dairy products.

The aim of this work was to identify the LAB occurring in Romanian raw milk and in traditionally prepared dairy products and that were hence involved in the natural fermentation of milk, by means of a polyphasic study of LAB isolates obtained through cultivation.

Section snippets

Sampling

As a common technique for the manufacture of Romanian artisan dairy products, the milk is left overnight to ferment in a warm place (for instance, next to the oven in the farm house). In some cases the milk is first boiled for several minutes. The second day, the upper layer of the fermented batch is collected as fermented sour cream, and the rest is used as fermented milk (sour milk) or for cheese production. For the latter, the sour milk is warmed up and rennet is added. Alternatively, rennet

Sampling and isolation of lactic acid bacteria

The viable counts on MRS agar varied mostly (68%) between 1.0×108 and 9.0×109 CFU/ml, being lower than 1.0×107 CFU/ml in ten cases. The latter samples were collected mostly from commercial yoghurts or fermented milks, but also from two milk samples, and two cheeses. For the ST agar plates, viable counts varied mostly (60%) between 1.0×106 and 9.0×108 CFU/ml, and a lower number of 1.0×104 CFU/ml for three samples. For 13 samples no viable cells could be detected after plating on ST agar. The viable

Discussion

The biodiversity of Romanian raw milk and traditional fermented dairy products is characterized by lactococci, leuconostocs, and enterococci.

Conclusions

The LAB biodiversity of Romanian raw milk and fermented dairy products is limited to lactococci, leuconostocs, and enterococci. Among the latter, a new species E. saccharominimus was found. Lactobacilli were found, but in low numbers. Studies of the biodiversity of traditional fermented food products are important as increasing information on the natural microbiota of such products can help to prevent the loss of their diversity and, in turn, the loss of a rich European fermented food

Acknowledgements

The authors acknowledge the financial support from the International Scientific and Technological Cooperation between Flanders and Romania of the Administration of Science and Innovation in Flanders (AWI-BIL01/52) and the Ministry of Education, Research and Youth in Romania.

References (38)

  • J.T.M. Wouters et al.

    Microbes from raw milk for fermented dairy products

    Int. Dairy J.

    (2002)
  • F. Ampe et al.

    Polyphasic study of the spatial distribution of microorganisms in Mexican pozol

    a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations, Appl. Environ. Microbiol.

    (1999)
  • V.K. Batish et al.

    Occurrence of enterococci in milk and milk products. II. Identification and characterization of prevalent types

    New Zealand J. Dairy Sci. Technol.

    (1984)
  • H. Beerens

    An elective and selective isolation medium for Bifidobacterium spp.

    Lett. Appl. Microbiol.

    (1990)
  • R. Bizzarro et al.

    Phenotypic and genotypic characterization of lactic acid bacteria isolated from Pecorino Toscano cheese

    Italian J. Food Sci.

    (2000)
  • A. Caridi

    Identification and first characterization of lactic acid bacteria isolated from the artisanal ovine cheese Pecorino del Poro

    Int. J. Dairy Technol.

    (2003)
  • T. Coenye et al.

    Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov.

    Int. J. Syst. Bacteriol.

    (1999)
  • T.M. Cogan et al.

    Characterization of lactic acid bacteria in artisanal dairy products

    J. Dairy Res.

    (1997)
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