International Dairy Journal 13 (2003) 169–178 Preliminary observations on proteolysis in Manchego cheese made with a defined-strain starter culture and adjunct starter (Lactobacillus plantarum) or a commercial starter J.M. Poveda a, *, M.J. Sousa b , L. Cabezas a , P.L.H. McSweeney b a Departamento de Qu ! ımica Anal ! ıtica y Tecnolog ! ıa de Alimentos, Faculdad de Quimicas, Universidad de Castilla-La Mancha, Campus Universitario s/n, Ciudad Real 13071, Spain b Department of Food Science, Food Technology and Nutrition (Food Chemistry), University College, Cork, Ireland Received 8 October 2001; accepted 11 September 2002 Abstract Different authors have demonstrated the potential of adding lactobacilli as adjunct cultures to pasteurized milk used in cheese manufacture. The aim of this work was to observe the effect of the use of a defined-strain starter culture and the addition of an adjunct culture (Lactobacillus plantarum) to cheesemilk in order to determine their effect on the ripening of Manchego cheese. Manchego cheeses were manufactured using one of the following starter culture systems: a defined starter consisting of Lactococcus lactis ssp. lactis and Leuconostoc mesenteroides ssp. dextranicum; a defined starter, as described above, and Lb. plantarum, which were isolated from a good quality Manchego cheese made from raw milk, or a commercial starter comprised of two strains of Lc. lactis. The cheeses were sampled at 15, 45, 90 and 150 d of ripening. Principal component analysis of peak heights of reversed-phase HPLC chromatograms of 70% (v/v) ethanol-insoluble and -soluble fractions distributed the samples according to the starter used in their manufacture. Quantitative differences in several peptides were evident between the three cheeses. Cheeses made with the defined-strain starters received higher scores for the flavour quality and intensity and for overall impression than the cheeses made with the commercial starter. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Manchego cheese; Proteolysis; Peptides; RP-HPLC; Defined-strain starter; Adjunct starter (Lb. plantarum) 1. Introduction Proteolysis is an important biochemical event in the ripening of most cheese varieties. Proteolysis, which contributes to the development of cheese texture and flavour, is due to the action of enzymes from (1) coagulant, (2) milk (generally plasmin), (3) starter bacteria, (4) the adventitious non-starter microflora, (5) secondary starter (in some cheese varieties) and (6) exogenous proteinases sometimes added for accelerating ripening or accentuating flavour. Proteolysis involves the hydrolysis of caseins to peptides and free amino acids, which are involved directly in cheese flavour or serve as precursors for flavour development (Fox, 1989). Manchego cheese, the most popular ewe’s milk cheese produced and consumed in Spain, is a cured, semi-hard, enzymatically coagulated, uncooked, pressed, high fat cheese. Artisanal cheeses can be made from raw milk, whereas industrial cheeses are made from pasteurized milk. Milk is generally inoculated with commercial mixed-strain starter cultures, which comprise strains of Lc. lactis ssp. lactis, Lc. lactis ssp. cremoris and L. mesenteroides ssp. cremoris (N ! u * nez, Medina, & Gaya, 1989). Pasteurization of milk for cheesemaking ensures a higher uniformity of the product and improves the sanitary conditions of cheesemaking, but eliminates some of the indigenous microflora of the milk which are partly responsible for the development of typical cheese flavour. The observation that raw milk cheese develops more intense flavour than pasteurized milk cheese (Gaya, Medina, Rodr ! ıguez-Mar ! ın, & N ! u * nez, 1990; Lau, Barbano, & Rasmussen, 1991) has stimulated interest in adding non-starter lactic acid bacteria *Corresponding author. Tel.: +34-926-295300; fax: +34-926- 295318. E-mail address: justamaria.poveda@uclm.es (J.M. Poveda). 0958-6946/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. PII:S0958-6946(02)00150-4