Behaviour of lactic acid bacteria populations in Pecorino di Carmasciano cheese samples submitted to environmental conditions prevailing in the gastrointestinal tract: Evaluation by means of a polyphasic approach

Highlights

• Pecorino di Carmasciano cheese samples were exposed to simulated GIT conditions.

• Bacterial dynamics during simulated digestion of samples were evaluated by PCR–DGGE.

• Main species after digestion were related to the Lactobacillus plantarum and casei groups.

• LAB potentially able to arrive alive and metabolically active to the colon were isolated.

• Strains were identified and characterized by a genetic and a technological point of view.

Abstract

The survival of the autochthonous microflora, of samples collected during Pecorino di Carmasciano cheese manufacturing, was evaluated along the passage through a model mimicking the gastro-intestinal tract. The aim was the selection of lactic acid bacteria potentially able to arrive alive and metabolically active to the colon. The dynamics of lactic microbiota, throughout simulated digestion of cheese samples, were evaluated by means of an approach PCR–DGGE-based. Dominant species after cheese digestion could be related to the Lactobacillus plantarum and Lactobacillus casei groups. Sixty-three strains, which survived to simulated gastro-intestinal transit, were further evaluated for technological features and tolerance to human digestion in several experimental conditions, according to routinely used protocols. Bacterial survival appeared to be, more than strain-specific, strongly affected by experimental conditions, i.e. some strains showed an acceptable survival when resuspended in skim milk but not in ewe milk and vice versa. Nevertheless according to data, one gram of fresh Pecorino di Carmasciano cheese may convey to human colon about the same amount of viable LAB of a probiotic drink. Although it cannot be assumed that lactobacilli introduced with Pecorino have beneficial effects on the host, the healthy impact of autochthonous lactic acid bacteria of naturally fermented food has a broad consensus in the current literature.

Introduction

Probiotics are defined by the FAO/WHO as live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host by improving the intestinal microbial balance (FAO/WHO, 2002). The whole concept of probiotics is not new, and in fact they have been consumed by human beings in the form of fermented foods, for thousands of years (Cross et al., 2001, Olivares et al., 2006). Fermented milks are most frequently used to deliver probiotic bacteria (Gomes da Cruz et al., 2009), however, their low pH can hamper the survival of some probiotic strains.

In recent years, it has been suggested that cheese might hold a better potential as a carrier for probiotic microorganisms into the human intestine due to its specific chemical and physical characteristics: higher pH value and lower acidity, higher buffering capacity, fat content and nutrient availability, and lower oxygen content. Moreover, the denser matrix of the cheese texture may protect bacteria more efficiently than a fluid environment during the storage of the food and during its transit through the human gastro-intestinal (GI) tract (Karimi et al., 2011). Different cheeses have been used to deliver a variety of probiotic bacteria (Corbo et al., 2001, Vinderola et al., 2009, Karimi et al., 2011), but very few papers have dealt with the probiotic features of strains belonging to the complex microflora of traditional raw milk cheeses (Succi et al., 2005, Caldini et al., 2008, Corsetti et al., 2008, Bao et al., 2010, Zago Fornasari et al., 2011, Meira et al., 2012).

The major challenge associated with the application of probiotic cultures in the manufacture of probiotic foods is their survival during processing. In the development of a probiotic cheese, this means that probiotic strains should be cultivable to high cell density for inoculation into the cheese vat or be able to proliferate during the manufacturing and/or ripening process (Karimi et al., 2011). The selection of autochthonous probiotic strains may represent a valuable approach to overcome many of the technological hurdles that usually affect the use of cheese as a carrier for commercial probiotic strains.

In this work, attention has been focused on a traditional ewe's milk cheese. In spite of the highly nutritional value (Raynal-Ljutovac et al., 2008), mainly due to the high protein and fat content, ewe cheeses have been rarely considered as carriers for probiotic microorganisms (Corbo et al., 2001, Kalavrouzioti et al., 2005). Moreover, few studies focused on probiotic characteristics of lactic acid bacteria (LAB) isolated from this matrix (Caldini et al., 2008, Corsetti et al., 2008, Meira et al., 2012). Pecorino di Carmasciano cheese is a traditional Italian cheese produced in a small village in Alta Irpinia (Avellino, Italy). Cheese making takes place in kitchens or cellars, using raw whole ewe milk from both evening and morning milkings. Ripening lasts from 3 (semi-soft) to 15 (hard) months (Coppola et al., 2006). Ripened Carmasciano cheese is slightly piquant, with an intense smell, and a soft and elastic paste, which hardens in long-ripened cheeses. The low temperatures, used during cheese processing, allow the typical milk microflora to survive and to play an important role in the development of its sensory characteristics (Coppola et al., 2006).

The aim of this work was the selective isolation from Pecorino of Carmasciano cheese of LAB potentially able to arrive alive and metabolically active to the colon. The experimental plan included a preliminary treatment of milk and cheese samples under conditions simulating the GI tract. The dynamics of LAB microflora were evaluated by means of PCR–DGGE (Polymerase Chain Reaction–Denaturing Gradient Gel Electrophoresis). LAB strains, isolated from samples after the simulated digestion treatment, were subjected to further investigations.