Proteolytic degradation of ewe milk proteins during fermentation of yoghurts and storage Khaled El-Zahar, Jean-Marc Chobert, Mahmoud Sitohy*, Michle Dalgalarrondo and Thomas HaertlØ 1 Introduction Yoghurts are fermented milk products produced by the action of two symbiotic microorganisms, Streptococcus thermophilus and Lactobacillus bulgaricus , populations of which and activity change constantly during yoghurt manufacture. At the start of fermentation, S. thermophilus grows quickly using essential amino acids produced by L. bulgaricus . In addition to small amounts of formic acid, L. bulgaricus produces lactic acid low- ering the pH to a level stimulating even more its growth. As a consequence, L. bulgaricus growth and activity are consider- ably enhanced, producing still more lactic acid and further drop- ping pH. This decreases the growth of S. thermophilus [1]. The biochemical activity of L. bulgaricus is not limited to lactic acid production only since these lactic acid bacteria are responsible also for protein hydrolysis and for the formation of flavor compounds. Lactic acid bacteria secrete a complex sys- tem of proteinases and peptidases, enabling them to use milk casein as a source of amino acids and nitrogen. The first step in casein degradation is mediated by the proteases located in cell wall, which cleave casein into oligopeptides. Several endo- and aminopeptidases have been purified from L. bulgar- icus and S. thermophilus. These enzymes are also very impor- tant for the development of flavors in fermented milk products [2, 3]. Both milk type and process conditions can influence the biochemical activity of the fermenting microorganisms and the final characteristics of the produced yoghurt. Yoghurts are mostly produced from cow milk and to a very limited extent from ewe milk. Ovine milk contains higher amounts of pro- teins than bovine milk (58 and 33 g/kg, respectively) and does not require fortification of the milk in non-fat solids during the production of yoghurt [4]. Heat pretreatment of milk is one of the most important pro- cesses affecting the texture and consistency of yoghurt. Heat treatment at a temperature A 70 8C causes unfolding and dena- turation of whey proteins, it promotes also their interactions with casein micelles via hydrophobic interactions and the for- mation of intermolecular disulfide bonds [5] involving whey proteins and some caseins (j and a S2 ). This affects the bio- chemical activity of the fermenting microorganisms and the physicochemical properties of the resulting yoghurts. Heat treatment of ovine milk at 91 8C for 2–3 min reduces its fer- mentation time as compared with bovine milk [6]. Caseins are not denatured by heat as are the whey proteins, mainly b-lacto- globulin and a-lactalbumin, which are denatured at the tem- peratures used during the yoghurt production. Upon denatura- tion, b-lactoglobulin reacts with other milk components being also able to oligomerize itself while a-lactalbumin undergoes heat-induced interactions only after severe heat treatment [7–11]. The observed improvements in the rate of gel forma- tion are mainly due to interactions between b-lactoglobulin and caseins, since heating milk at 80 8C for 30 min denatures more than 90% of b-lactoglobulin as compared with only 60% of a-lactalbumin. Consequently, the heat pretreatment of milk before the production of yoghurt should reach temperatures between 85 and 95 8C. Mottar et al. [12] have proposed a slightly modified model in which the denatured b-lactoglobu- lin becomes associated with j-casein of the casein micelles. This results in the formation of “hairy” casein micelles, irregu- lar in structure, the surface of which would become more hydrophobic. At higher temperatures when a-lactalbumin starts to be denatured, it interacts also with b-lactoglobulin forming disulfide bonds. The amount of a-lactalbumin present on the micellar surface is dependent on the temperature and the intensity of the heating process. Thermally induced aggre- gation of a-lactalbumin depends on the concentration of free- SH groups present in other whey proteins (mostly b-lactoglo- bulin) issued from the disruption of the intramolecular S1S bonds in a-lactalbumin. Nahrung/Food 47 (2003) No. 3, pp. 199 – 206 i 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0027-769X/2003/0306-0199$17.50+.50/0 199 Yoghurts are mostly produced from cow milk and to avery limited extent from ewe milk. The evolution of caseins and whey proteins in ovine milk submitted to different thermal treatments (63 8C/30 min; 73 8C/15 min; 85 8C/10 min or 96 8C/5 min) was followed during fer- mentation of yoghurts and during their storage up to 14 days, using two different sets of starters. One set of starter LAB was a “ropy” culture (YC-191), which is a well-defined mixed strain culture containing Streptococcus thermophilus ST-143 and Lactobacillus delbrueckii subsp. bulgaricus (LB-18 and LB-CH2). The other set of starter bacteria (YC-460) was a standard yoghurt culture (“non-ropy”) containing mixed strain culture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Contents of free amino groups in pro- duced yoghurts increased gradually during the fermentation, up to a maximal value obtained after 4 h fermentation, then they did not change significantly during storage of yoghurt produced with YC-191 starter. In contrary, a large drop in the amount of free amino groups was observed in the first 24 h of storage in the case of yoghurt made with YC-460 indicating that microorganisms continue still to grow in low temperatures. During fermentation and storage of both yoghurt types, a-lactalbumin was hydrolyzed to a slightly bigger extent than b-lacto- globulin. During fermentation, b-casein was slightly more degraded than a S -caseins; however, the opposite was observed during storage up to 14 days. Generally, a more intense heat pretreatment led to a higher degradation of whey proteins and caseins during fermentation and stor- age. Differences in proteolytic activity between the two starters used (whey proteins more degraded by YC-191; caseins more degraded by YC-460) may lead to improvement in production and formulation of yoghurts differing in their physicochemical and rheological properties. Correspondence: Dr. J.-M. Chobert, Institut National de la Recherche Agronomique, Laboratoire d’Étude des Interactions des MolØcules Alimentaires, BP 71627, F-44316 Nantes Cedex 3, France E-mail: chobert@nantes.inra.fr Fax: +33-2-4467-5084 Keywords: Ewe milk proteins / Fermentation of yoghurt / Lactobacil- lus delbrueckii / Streptococcus thermophilus / * Permanent address: Zagazig University, Faculty of Agriculture, Biochemistry Department, Zagazig, Egypt