Development of an ad hoc starter culture for the production of functional raw ewes’ milk cheeses through the addition of grape pomace powder

Abstract

The present work was carried out to select lactic acid bacteria (LAB) resistant to polyphenols in order to develop an ad hoc starter culture for the production of functional fresh ewes’ milk cheeses through the addition of grape pomace powder (GPP). To this purpose, raw ewes’ milk samples were inoculated with individual polyphenols belonging to five classes: flavanols (cathechin, epicatechin and epigallocathechin), flavonols (quercetin), flavones (rutin), hydroxyl-benzoic acid (vanillic acid and syringic acid) and hydroxyl-cinnamic acid (caffeic acid and cumaric acid). These polyphenols are commonly associated with the wine industry by-products and were added in milk to a final concentration of 0.5 mg/mL. All samples were incubated at 30°C and 44°C for 48h in order to select potential mesophilic and thermophilic starter LAB. Plate counts showed the total microbial counts (TMC) till levels of almost 109 CFU/mL and all milk samples were dominated by coccus LAB. All colonies with different morphological appearance were isolated and differentiated on the basis of their phenotypic characteristics and by Randomly Amplified Polymorphic DNA (RAPD)-PCR profiles. A total of 55 strains were collected and subjected to the genotypic analysis by means of 16S rRNA gene sequencing, which identified 5 LAB species belonging to two genera (Enterococcus and Lactococcus). The species most frequently found were Lactococcus lactis. The 55 strains were investigated in vitro for their general dairy aptitudes and four strains of L. lactis (MISE36, MISE94, MISE169 and MISE190) showed technological traits relevant to act as starter strains for cheese production.In order to produce a GPP-fortified fresh cheeses, red grape pomaces of Nero D’Avola cultivar were provided by a winemaking factory (Cantine Europa, Petrosino, Italy). Grape pomaces were dried at 54°C for 48 h and milled through a Retsch apparatus to a particle size of 250 μm. The experimental cheese making trials were carried out in a dairy pilot plant (Biopek, Gibellina, Italy) following the protocol of “pressed” cheeses. For each trial 20 L of pasteurised milk was divided into two plastic vats (10 L each) representing two different trials. Both vats were inoculated with the individual starter LAB cultures (100 mL) to reach a final cell density of 107 CFU/mL. One vat represented the control cheese (CC) production. The second vat represented the experimental cheese (EC) that, after curd extraction, was added with 1% (w/w) GPP. The cheeses were sampled after one month of ripening. All lactococci were able to perform the rapid acidification in CC and EC production, showing that polyphenols of GPP did not slow down starter development. Plate counts and RAPD analysis applied to the colonies isolated from the highest dilutions of samples confirmed the dominance of the added strains. A sensory evaluation of the resulting cheeses, indicated the cheeses processed with the addition of GPP were well appreciated by the judges. Analyses are in progress to evaluate the nutraceutical properties of the experimental cheeses. This research has been supported by the grant MISE-2017-NAZ-0228 - CUP: B78I17000260008