Fatty acids, vitamins and cholesterol content, and sensory properties of cheese made with milk from sheep fed rapeseed oilcake.

The aim of the present study was to evaluate the influence of rapeseed oilcake used for feeding sheep on the content of fatty acids (FA), tocopherols, retinoids, and cholesterol of milk and cheese, and on the sensory properties of cheese. Indoor animal feeding (in winter) is the highest cost of production for cheesemakers, and the inclusion of locally produced rapeseed oilcake in the concentrate feed formulation can reduce the cost of cheese production, as long as the quality of the cheese is not altered. The experiment was carried out in March (mid lactation) with 72 Latxa sheep from an experimental farm located in the Basque Country (northern Spain). Two homogeneous groups of animals (n = 36) were set to receive each a different diet based on commercial or rapeseed concentrate, respectively, and forage (Festuca hay). Animal production parameters were individually recorded for each feeding group, whereas bulk milk from each group was used for cheesemaking trials. The rapeseed concentrate had higher amounts of unsaturated FA (mainly C18:1 cis isomers, C18:2 cis-9,cis-12 and C18:3 cis-9,cis-12,cis-15) and tocopherols than the commercial concentrate. The inclusion of rapeseed oilcake in the diet of dairy sheep did not compromise animal production parameters or milk gross composition. Bulk milk and cheese from sheep fed rapeseed concentrate showed higher content of unsaturated FA and tocopherols than those from sheep fed commercial concentrate. No differences were observed in the content of retinoid in milk and cheese between feeding groups, whereas the cholesterol content was slightly lower in cheese made with milk from sheep fed rapeseed concentrate. Thus, milk and cheese from sheep fed rapeseed concentrate had a healthier lipid profile. In addition, the inclusion of rapeseed oilcake in the diet of sheep did not change the typical sensory attributes of Protected Denomination of Origin Idiazabal cheese. Therefore, rapeseed concentrate could be a good local resource for feeding sheep to improve the nutritional quality of dairy products and to provide higher returns to farms.

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 ß-carotene present in pasture plants, was found in milk and cheese.

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.

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.

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.

Very-long-chain omega-3 fatty acids as markers for habitual fish intake in a population consuming mainly lean fish: the EPIC cohort of Gipuzkoa. European Prospective Investigation into Cancer and Nutrition.

OBJECTIVE: To assess the relationship between habitual fish intake and fatty acid levels in serum as well as in the LDL fractions of serum phospholipids and cholesteryl esters. DESIGN: Cross-sectional study. SETTING: Cohort of Gipuzkoa (Basque Country, northern Spain) included in the European Prospective Investigation into Cancer and Nutrition (EPIC) project. SUBJECTS: Random sample of 120 healthy volunteers of both sexes aged 35-65 y, divided into various consumption groups according to daily fish intake. METHODS: Data on habitual intake over the previous year was collected by trained interviewers by means of a computerized questionnaire based on the diet history method. Fasting venous blood samples were drawn and fatty acids were measured by gas-liquid chromatography. RESULTS: Lean fish accounted for 78% of all fish consumption in the highest consumption group (>115 g/day) and for 60% in the lowest (<31 g/day). The mean concentrations of omega-3 polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA, C20:5, omega-3), and docosahexaenoic acid (DHA, C22:6, omega-3) in serum and in the LDL fractions of serum phospholipids and cholesteryl esters increased significantly from the lowest to the highest fish consumption categories. Fish intake showed a statistically significant relationship with omega-3 PUFA, EPA and DHA in serum and in the LDL fractions of serum phospholipids and cholesteryl esters both in the simple linear regression analysis and in a multiple regression model adjusted by age, body mass index (BMI) and vegetable intake. CONCLUSIONS: Habitual fish intake is reflected in the content of EPA and DHA in serum and in the LDL phospholipid and cholesteryl esters fractions. The concentrations of very-long-chain omega-3 fatty acids are useful biomarkers for dietary fish intake, mainly lean fish. SPONSORSHIP: Europe Against Cancer Programme of the European Union (agreement SOC 97 200302 05F02); 'Fondo de Investigaciones Sanitarias', Spanish Ministry of Health (FIS grant 99/0024-05); Government of the Basque Country; and 'Fundación Científica de la Asociación Española contra el Cáncer'.

Coagulating and lipolytic activities of artisanal lamb rennet pastes.

Lamb rennet pastes were prepared by the procedure most commonly used by Idiazabal cheese manufacturers. We studied the effects on their coagulating and lipolytic activities of the state of the stomach at the time of death (full of milk or empty), the amount of NaCl added, the origin of the lambs and paste storage time. Coagulating activities were generally between 155 and 363 units/g tissue. Pastes prepared from stomachs of lambs from slaughterhouse flocks had significantly higher coagulating activities than those of lambs from separate flocks. No significant decrease in coagulating activity was observed after 1 year storage at 4 degrees C. Chymosin represented 75-80% of the total coagulating activity with the remainder being pepsin. Rennet paste extracts with pH < 4.7 did not have increased coagulating activities when their pH was lowered to 2.0, while those with pH > 5.2 had activities 1.5-fold those before treatment. Lipase activity was higher in extracts of rennet pastes prepared using the stomachs of lambs that arrived at the slaughterhouse in the morning just prior to slaughter than in those prepared with the stomachs of lambs that had arrived on the previous evening. However, the reverse was the case for esterase activity. Activating the coagulating activity by pH cycling completely destroyed both lipolytic activities. Storage at 4 degrees C for > 1 year did not affect esterase activity but lipase activity decreased substantially after 4-5 months. Lipase, but not esterase, activity was responsible for the liberation of short-chain free fatty acids from ovine milk fat.

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.

Characterization of the delta 12 desaturase in the American cockroach, Periplaneta americana: the nature of the substrate.

The cockroach, Periplaneta americana, can convert oleic acid (18:1(n - 9], to linoleic acid, (18:2(n - 6], by a microsomal delta 12 desaturase. Most of the desaturase activity was present in the fat body tissue, with lower activity in the epidermis and no detectable activity in the thorax or gut tissue. In incubations of microsomal preparations from fat body tissues with [1-14C]18:1-CoA, increased amounts of [1-14C]18:2 were found with increasing time and protein concentration. The form of the substrate for the delta 12 desaturase was determined to be 18:1-CoA by comparing activity towards [1-14C]18:1-CoA and [1-14C]18:1 transesterified to phospholipid. Ozonolysis of the 18:2 formed from [1-14C]oleoyl-CoA followed by radio-gas-liquid chromatography gave one labeled peak, 9-oxononanoate, which showed that the product of the delta 12 desaturase is the physiologically important isomer, 18:2(n - 6).

The delta 12-desaturase from the house cricket, Acheta domesticus (Orthoptera: Gryllidae): characterization and form of the substrate.

A novel delta 12-desaturase from animals, which converts oleic acid (18:1n-9) to linoleic acid (18:2n-6), was characterized in the house cricket, Acheta domesticus. The delta 12-desaturase product, linoleic acid, was determined by silver nitrate thin-layer chromatography, radio-gas-liquid chromatography and radio-high-performance liquid chromatography with the latter being used for routine analyses. Enzyme activity was located in the microsomal fraction of whole insect homogenates. NADPH or NADH was required for activity, with NADPH being the more efficient electron donor. In short incubation times with oleoyl-CoA as substrate, the highest amount of product, linoleic acid, was found as linoleoyl-CoA. With longer incubation periods, most of the linoleic acid was recovered in the polar lipid fraction containing phospholipid. Preincubation of the microsomal preparation in the absence of NADPH, which allowed 90% of the oleoyl moiety to be transacylated into complex lipid, resulted in no detectable desaturation upon addition of NADPH. These data indicate that the oleic acid moiety used as substrate was in the form of a CoA derivative and not in the form of a phospholipid, as it is for the plant delta 12-desaturase. This is the first characterization of a delta 12-desaturase from an animal system and the first report of a delta 12-desaturase that uses oleoyl-CoA as substrate.

De novo biosynthesis of polyunsaturated fatty acids in the cockroach Periplaneta americana.

The de novo biosynthesis of 6,9,12-linolenic acid, 11,14-eicosadienoic acid, 5,11,14-eicosatrienoic acid, and arachidonic acid was demonstrated in adult female cockroaches, Periplaneta americana. These four polyunsaturated fatty acids (PUFA) were present primarily in the phospholipid (PL) fraction of both males and females. They were purified by AgNO3 thin-layer chromatography and high pressure liquid chromatography. The double bond positions of the major isomer of eicosatrienoic acid were shown to be at the delta 5,11,14 positions by gas chromatography-mass spectrometry (GC-MS) of both methoxy and epoxide derivatives and gas-liquid chromatography (GLC) and GC-MS of ozonolysis products. The other PUFAs cochromatographed with standards on both packed and capillary GLC columns. The in vivo incorporation of [1-14C]acetate into 5,11,14-eicosatrienoic acid, 11,14-eicosadienoic acid, 6,9,12-linolenic acid, and arachidonic acid was demonstrated by radio-GLC and radio-HPLC and for 5,11,14-eicosatrienoic acid by radio-GLC of ozonolysis products. The latter technique clearly demonstrated that the entire eicosatrienoic acid molecule was labeled. Thoracic tissue contained the highest amount of radiolabeled 5,11,14-eicosatrienoic acid (1.6% of total radioactivity incorporated into PL) while radiolabeled 11,14-eicosadienoic acid was found primarily in abdominal epidermal tissue (2% of total radioactivity incorporated into PL). Radiolabeled arachidonic and 6,9,12-linolenic acids comprised 0.1 and 0.02%, respectively, of the total radioactivity in the PL fraction. These data document the de novo biosynthesis of di-, tri-, and tetraunsaturated fatty acids in the American cockroach, and indicate that this animal can desaturate on both sides of the delta 9 double bond of oleic acid.

Biosynthesis of medium chain fatty acids in Drosophila melanogaster.

Adult Drosophila melanogaster synthesizes dodecanoic and tetradecanoic acids in vivo, along with the more common 16- and 18-carbon fatty acids. The radiolabeled C12 and C14 fatty acids synthesized from sodium [1-14C]acetate are found primarily in the diacylglycerol and triacylglycerol fractions. Partially purified fatty acid synthetase (FAS) synthesizes C14, C16, and C18 fatty acids (as the free acids) at 0.2 M ionic strength. Increasing the ionic strength to 2.0 M causes partially purified FAS to synthesize primarily C12 and C14 fatty acids. Addition of aliquots of the microsomal pellet and other soluble protein fractions does not alter the pattern of fatty acids synthesized by FAS. The percentage of C12 and C14 fatty acids synthesized at high ionic strength by individual fractions from the FAS peak (Sepharose 6B column) is constant across the peak. None of the soluble protein fractions is able to relieve the inhibition of FAS by phenylmethylsulfonyl fluoride. These results indicate that the FAS of D. melanogaster has the inherent capability to form C12 and C14 fatty acids and that no other soluble protein appears to be involved in their synthesis.

Monovalent cation induced reassociation of formyltetrahydrofolate synthetase monitored by Rayleigh light scattering and enzymic activity.

It has been previously established that formyltetrahydrofolate synthetase isolated from Clostridium cylindrosporum is reversibly dissociated and inactivated in the absence of certain monovalent cations. In the present paper, the reassociation of monomeric, inactive enzyme to form tetrameric, active enzyme was monitored by Rayleigh light scattering and enzymic activity. Light-scattering measurements confirmed that the active enzyme is composed of four subunits of equal weight. With the assumption that the results of analytical ultracentrifugation are correct--that monomers and tetramers are the only species ever present at appreciable levels--the amount of tetramer formed during reassociation was calculated from the light-scattering data. Evidence for the accumulation of catalytically active intermediates was obtained by comparing the rate of association of monomers (detected by light scattering) to the rate of return of enzymic activity. The accumulation of intermediates was most strikingly seen at low monovalent cation concentration at low ionic strength. Evidence is also presented that sedimentation favors reassociation of the enzyme. The reassociation data were fit to a second-order reversible rate equation. Interestingly, although the data were derived from the same experiments, the kinetic plot based on light-scattering measurements yielded a straight line function with an abrupt change in slope about 10 min after initiation of reassociation, while plots based on enzymic activity measurements gave a single slope.

Chemical cross-linking stabilizes the enzymic activity and quaternary structure of formyltetrahydrofolate synthetase.

Clostridium cylindrosporum formyltetrahydrofolate synthetase tetramers cross-linked with dimethyl suberimidate remained active in the absence of the monovalent cations normally required for enzymic activity and the tetrameric conformation. The modified enzyme was analyzed by sodium dodecyl sulfate electrophoresis, sedimentation velocity, and gel permeation chromatography. Under the experimental conditions used, the enzyme was only partially cross-linked; 74% of the enzyme was cross-linked dimer or monomer. Nonetheless, the modified enzyme is able to retain enzymic activity and the tetrameric structure under conditions where native enzyme would be completely dissociated and inactivated. The result suggests that cross-linked dimers strongly associate with each other and with monomers. Flame emission spectroscopy indicates that cross-linked enzyme contains two monovalent cations per tetramer.

Cellulases released during the germination of Dictyostelium discoideum spores.

Dormant spores of Dictyostelium discoideum contained cellulase at a specific activity of 130 to 140 U/mg of protein; when heat activated, the spores germinated, progressively releasing the cellulase activity into the extracellular medium. The cellulase release was a selective process and resulted in recovery of the cellulase activity at a specific activity of 2,000 U/mg of protein; beta-glucosidase in the spores remained completely associated with the emerging amoebae. Release of the cellulase required heat activation of the spores and occurred during the swelling stage of germination; inhibition of the emergence stage with cycloheximide had no effect on the release of the cellulase. The cellulase activity released consisted of two enzymes whose molecular weights were 136,000 and 69,000. Studies of their pH optima, heat lability, and of their sensitivity to inhibition revealed no distinctive differences between these two proteins. Analysis on diethylaminoethyl-Sephadex columns showed that the higher-molecular-weight protein could be converted into the lower-molecular-weight component in vitro.