M. Virto

Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme

Lipase (triacylglycerol ester hydrolase, E.C.3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene. The enzyme was rapidly adsorbed on the support, and more than 60% of the soluble activity disappeared from the medium after 1 min of incubation at room temperature. A recovery of immobilized activity of 21% was obtained when the wet preparation was immediately assayed with olive oil at the end of the immobilization protocol. The activity of the immobilized enzyme drastically decreased with the loss of water of the preparation. Pretreatment of the support with organic solvents significantly increased the recovered immobilized activity. Our results strongly suggest that the soluble lipase could exist in different aggregation forms depending on the pH of the medium. At acidic pH, the relative proportion of high-molecular-weight forms of the enzyme is higher than at pH 7.0, suggesting that the lipase would be also immobilized in different aggregation forms depending on the pH used in the immobilization procedure. Crosslinking of the adsorbed enzyme with glutaraldehyde diminished its activity but increased the stability of the lipase against the washing-out effect of Triton X-100. Data on the most relevant catalytic properties of the soluble and immobilized enzyme, such as optimum pH and temperature as well as ranges of stability, kinetic parameters, and activation energy for the hydrolysis of olive oil and p-nitrophenyl acetate, are reported.

Hydrolysis of animal fats by immobilized Candida rugosa lipase

Lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) from Candida rugosa was immobilized by adsorption on a commercially available microporous polypropylene support of 200- to 400-μm particle size. A contact period of 90 min allowed the highest degrees of hydrolysis to be achieved, particularly in the second and third hydrolysis reactions. The optimal hydrolysis conditions were 0.10 kg enzyme per kilogram fat, 50% (w/v) fat, and 40°C for 24 h. The immobilized enzyme can be repeatedly used and hydrolysis degrees of 90% or higher can be achieved. Of the three animal fats studied, edible pork lard consistently yielded the highest degrees of hydrolysis (95%) in the first hydrolysis reaction and inedible beef tallow the lowest (65%). The immobilized enzyme lost its activity above 45°C. The support could be easily recovered and reused up to 5 times.

Lamb rennet paste in ovine cheese (Idiazabal) manufacture. Proteolysis and relationship between analytical and sensory parameters

Abstract Cheeses were manufactured with 2 levels of lamb rennet paste or bovine rennet, at two times of the year, to study the effect of artisanally prepared lamb rennet paste (at high and low levels) on the concentrations of proteolysis products (nitrogen fractions, casein degradation and free amino acids). Cheeses made with the lamb rennet paste presented a significantly different evolution of the αs1-I-casein fraction during ripening, as measured by urea polyacrylamide gel electrophoresis. The time of the year affected the concentrations of 39% of the individual free amino acids, as determined by reversed-phase high-pressure liquid chromatography. The relationship between some biochemical parameters (gross composition, degrees of lipolysis (Lamb rennet paste in ovine cheese manufacture. Lipolysis and flavour. International Dairy Journal (2003) in press) and proteolysis) and the sensory characteristics of the cheese (Virto et al., 2003) were studied by a principal component analysis. The effect of the technological parameters “type of rennet” and “time of the year” on these analytical variables and sensory characteristics of the cheese was described by three principal components which explained 80.8% of the total variance. The factor “rennet paste” included concentrations of short chain free fatty acids, sensory descriptors related to strong odours and flavours, an unidentified peptide A1, and αs1-I-caseins. The effect of the ‘time of the year’ was described by two other factors: the “secondary proteolysis” factor, which included the concentrations of free amino acids and could be related to the different endogenous microflora of the raw milk, and the “seasonal milk composition” factor, which included the fat content and the plasmin activity of the milk.

Enzymic hydrolysis of animal fats in organic solvents at temperatures below their melting points

Lipase fromCandida rugosa catalyzed the hydrolysis of inedible beef tallow and pork lard (edible and inedible) in the presence of organic solvents at temperatures below the melting point of the fat. Reactions were carried out at 50% substrate with 180 lipase units per gram of fat in a two-liter reactor. In the presence of isooctane (5-10%) beef tallow yielded 94% hydrolysis in 24 hr both at 37° and 31°C. Edible pork lard yielded 97% hydrolysis under these conditions and at temperatures as low as 25°C, while inedible lard gave hydrolysis intermediate between the other two fats.

Effects of seasonal changes in feeding management under part-time grazing on the evolution of the composition and coagulation properties of raw milk from ewes

Ewe raw milk composition, rennet coagulation parameters, and curd texture were monitored throughout the milk production season in 11 commercial flocks reared under a part-time grazing system. Milking season lasted from February to July. During that period, the diet of the animals shifted from indoor feeding, consisting of concentrate and forage, to an outdoor grazing diet. Lean dry matter, fat, protein, calcium, and magnesium contents increased throughout the milking season, as did rennet coagulation time, curd firmness, and curd resistance to compression. However, lean dry matter, protein content, and curd resistance to compression stabilized when sheep started to graze. Principal component analysis correlated curd resistance to compression and proteins, whereas curd firmness was highly correlated with fat content and minerals. Discriminant analysis distributed milk samples according to the feeding management. Curd firmness, fat, and magnesium turned out to be discriminant variables. Those variables reflected the evolution of the composition and coagulation parameters when fresh pasture prevailed over other feeds in the diet of the flocks. The present study shows that seasonal changes associated with feeding management influence milk technological quality and that milk of good processing quality can be obtained under part-time grazing.

Case study of a commercial sheep flock under extensive mountain grazing: Pasture derived lipid compounds in milk and cheese

Terpenoid, fat-soluble antioxidant and fatty acid (FA) composition of pasture as well as those of milk and cheese from a commercial sheep flock managed under extensive mountain grazing in the east region of the Cantabrian mountain (Northern Spain) was investigated. The grazing period lasted for 2 months and ewes were at late lactation stage. Plants, feces, bulk milk and cheese samples were collected on two sampling dates. The abundance of the dominating botanical families in the mountain pasture prevailed in the sheep diet of the commercial flock. Major terpenoids and tocols in the pasture appeared as major ones in milk and cheese, whereas C18 unsaturated FAs in milk and cheese were derived from the intake of C18 polyunsaturated FAs which were prevalent in the pasture. No carotene was detected in the dairy samples but retinol (free or esterified), derived from the intake of β-carotene present in pasture plants, was found in milk and cheese.

Influence of lamb rennet paste on the lipolytic and sensory profile of Murcia al Vino cheese

The influence of lamb rennet paste (71.1% chymosin, 177 international milk-clotting units/mL, 4.57U/g of lipase activity) during the ripening of Murcia al Vino goat cheese was studied. The aim of this study was to improve the knowledge of the effect of lamb rennet paste on the lipolytic patterns in this type of cheese by reference to the evolution of total and free fatty acids. A sensory analysis was carried out to compare cheeses made with commercial and paste rennet. The rennet paste showed higher lipolytic activity, enhancing the production of short-chain free fatty acids. In addition, the cheese produced with lamb rennet paste had a slightly more bitter and piquant taste, making it an attractive commercial alternative that can be used to develop new varieties of goat cheese.

Technological Characterization of Experimental Natural Rennets Pastes

Four types of lamb rennet pastes were characterized according to the sort of abomasa and the treatment it received: full fresh abomasa (FFA), full dried abomasa (FDA), empty dried abomasa (EDA), and empty frozen abomasa (EFA). These rennet pastes were studied by means of different technological parameters (milk clotting time, chymosin content, lipase activity, and aptitude against milk coagulation).The highest level of clotting activity (391.20 IMCU/g) corresponded to the rennet EDA whereas the lowest (172.87IMCU/g) was found in EFA. The chymosin content of the pastes classified them as rennet extracts, the highest chymosin level (80.46%) being found in the paste from FDA and the lowest (71.10%) being obtained from FFA.The highest level of lipase activity (10.57 U/g) was found in EDA whereas the lowest (1.46 U/g) was in paste FDA. Milk coagulation aptitude was studied through the use of a near infra-red radiation dispersion sensor. The highest value of Rmax (0.049 min-1) found in the rennet paste made with EDA indicated a higher level of casein hydrolysis and greater aggregation speed. For the same rennet at tmax value of 6.1 min was established lower than for other rennet preparations which reflected the fact that this rennet hydrolyzed 80% of the K-casein in a shorter period of time. It is concluded that the best natural lamb rennet paste of this work was FFA due to it combines appropriate proteolytic and lipolytic activities although it does not reach the maximum values for each technological parameters analyzed.

Short communication: Sensory profile of raw goat milk cheeses made with artisan kid rennet pastes from commercial-weight animals: Alternative to farmhouse goat cheeses

The loss of traditional kid rennet pastes in the Canary Islands (Spain), as in many other regions, is most likely due to the custom of using abomasa from very young animals killed below desirable commercial weight. In addition, the reasonable price of commercial rennets (CR) has resulted in the loss of typical sensory characteristics for most farmhouse raw goat milk cheeses, placing them at a disadvantage when local and international markets are full of different cheeses, often with aggressive marketing strategies. This paper analyzes the sensory characteristics of raw goat milk cheeses made with rennet pastes prepared from commercial kid abomasa in 2 ways: dried while full of ingested milk [full, commercial, artisan kid rennet (FCKR)], or dried after being emptied of ingested milk and refilled with raw goat milk [empty, commercial, artisan kid rennet (ECKR)]. This latter practice allows the use of empty abomasa, or abomasa with grass, soil, and so on. Sensory profiles of cheeses made with FCKR and ECKR rennets were compared with those made with CR by an expert panel (n=7). The FCKR and ECKR cheeses had similar sensory profiles. Although scores for FCKR cheeses were somewhat higher than for ECKR cheeses, they were in the range found for traditional cheeses made with rennet prepared with abomasa from very young animals. The sensory profile of CR cheeses was very different. Almost 90% of consumer panelists (n=90) preferred cheeses made with the experimental rennet pastes. These results demonstrate the possibility to prepare artisan rennet pastes from commercial-weight kids in an easy way for farmhouse cheese makers using local resources that would otherwise be destroyed in abattoirs.