Starter bacteria are the prime agents of lipolysis in cheddar cheese

To assess the contribution of starter lactic acid bacteria (LAB) to lipolysis in Cheddar cheese, the evolution of free fatty acids (FFAs) was monitored in Cheddar cheeses manufactured from pasteurized milks with or without starter. Starter-free cheeses were acidified by a combination of lactic acid and glucono-delta-lactone. Starter cultures were found to actively produce FFAs in the cheese vat, and mean levels of FFAs were significantly higher in starter cheeses over ripening. The contribution of nonstarter LAB toward lipolysis appears minimal, especially in starter-acidified cheeses. It is postulated that the moderate increases in FFAs in Cheddar cheese are primarily due to lack of access of esterase of LAB to suitable lipid substrate. The results of this study indicate that starter esterases are the primary contributors to lipolysis in Cheddar cheese made from good quality pasteurized milk.     

Ethyl ester formation is enhanced by ethanol addition in mini Swiss cheese with and without added propionibacteria

Esters are important contributors to cheese flavor, but their mechanisms of synthesis in cheese are largely unknown. This study aimed to determine whether ethanol concentration limits the formation of ethyl esters in cheese. Mini Swiss cheeses were manufactured with (E) or without (C) the addition of ethanol to cheese milk. Ethanol concentrations (enzymatic analysis) were 64 +/- 17 and 330 +/- 82 microg g(-1), respectively, in C and E cheeses. E cheeses also contained 5.4 +/- 2.3 times more of the five ethyl esters quantified than C cheeses, regardless of the concentrations of esters in C cheeses (range 1-128 ng g(-1)). Furthermore, the presence of propionibacteria added as acid-producing secondary starters was associated with greater concentrations of esters, due to the increase in acid concentrations that propionibacteria induced and/or to an involvement of propionibacteria enzymes in ester synthesis. This study demonstrates that ethanol is the limiting factor of ethyl ester synthesis in Swiss cheese.     

Spectrometric and liquid chromatographic determination of natamycin in cheese and cheese rind

Methods for determining natamycin content of cheese rind and cheese are presented. Cheese and rind samples are extracted with methanol and the fat precipitated by cooling the sample solution in methanol-water at -15 to -20 degrees C for ca 1 h. Natamycin levels are measured by UV spectrometric detection at absorbance minimum 311 nm, maximum 317 nm, and at exactly 329 nm, or by LC separation over Lichrosorb RP-8 column with detection at 303 nm. For measuring low levels, a concentration step is provided. The method is applicable to natamycin in cheese rind and in the interior of the cheese. Detection limit is 0.5 mg/kg. The method is suitable for controlling a maximum tolerance of natamycin on the cheese rind, at a level of 1 mg/dm2, and for detecting migration of natamycin into the cheese.     

Determination of natamycin in cheese and cheese rind: interlaboratory collaborative study

A collaborative test on the determination of natamycin in cheese and cheese rind was conducted. Participants were from 37 laboratories in 13 countries. Eight samples, consisting of 4 duplicates, were investigated by a spectrometric method and a liquid chromatographic (LC) method. The spectrometric method gave good results (coefficient of variation [CV] = 12%) and the LC method with ultraviolet detection gave reasonable results (CV = 25%) for levels down to 15 mg/kg (0.9 mg/dm2). For very low levels, a preconcentration step is necessary, but even then quantitation is poor (CV = 35-37%) for both methods at 1.7 mg/kg, although the presence of natamycin can be detected qualitatively. For a level of 0.3 mg/kg, quantitation is poor (CV = 39%) for the LC method and impossible (CV = 60%) for the spectrometric method.     

Identification of allyl esters in garlic cheese

This study describes the identification of six allyl esters in a garlic cheese preparation and in a commercial cream cheese. The extracts were prepared by liquid/liquid extraction and concentrated by the SAFE process. The identification of the allyl esters of acetic, butyric, hexanoic, heptanoic, octanoic, and decanoic acids is based on the correlation of their mass spectrometric data and chromatographic retention time data obtained from the extracts with those of authentic standards. In addition to the gas chromatography (GC)/mass spectrometry analysis, the flavor ingredients were characterized by GC sniffing by a trained flavorist. Some of the esters were isolated by preparative GC.     

Time course of N<Subscript>2</Subscript> fixation and growth of chickpea

The ¹⁵N isotopic dilution technique was used to assess N₂ fixation in desi chickpea (Cicer arientinum L.) cv. Myles at different growth stages as influenced by inoculation method. In this growth chamber study, no significant differences in nodule dry weight, amount of N₂ fixed and plant dry matter were observed between seed inoculation with seed-applied peat inoculant and soil-applied granular inoculant placed 2.5 cm below the seed. However, seed inoculation with liquid inoculant was inferior to the seed-applied peat or the granular inoculant for all parameters measured at all sampling dates. The seed-applied peat and granular inoculant treatments fixed 4.8 and 4.1 mg N plant^-1, respectively, by the late vegetative stage, and reached a maximum of 20.6 and 25.6 mg N plant^-1, respectively, by the late pod-filling stage. These values accounted for 30.5% and 34.9% of the total plant N for the peat and granular inoculant, respectively, by late pod-filling. For the liquid inoculant treatment, the amount of N₂ fixed increased from 2.3 mg N plant^-1 by the late vegetative stage to a maximum of 9.6 mg N plant^-1 which was 22.2% of total plant N by the mid pod-filling stage. The highest daily N₂ fixation rate for the peat and granular inoculant was 0.9 mg N plant^-1 and occurred between flowering and early pod-filling, whereas that for the liquid occurred between early and mid pod-filling stages (0.23 mg N plant^-1). After the mid pod-filling stage, little or no N₂ was fixed in all treatments. Plant dry matter increased from the late vegetative stage to physiological maturity but the greatest dry matter accumulation occurred between the late vegetative and early pod-filling stages in all treatments.     

Evaluation of APHA and AOAC methods for phosphatase in cheese

Varieties of market cheese were analyzed for alkaline phosphatase by the modified rapid colorimetric method of the American Public Health Association (APHA) and the official AOAC method, 16.304-16.306. In the APHA method, 5 g cheese (pH less than 7.0) is macerated with 2 mL 1:1 carbonate buffer, or 2 mL water (for cheese with pH greater than 7.0). Addition of 0.1 mL magnesium acetate (1 mg magnesium) to test portions of cheese extracts yielded reproducible and quantitative recovery of added phosphatase. In the AOAC method, macerating 0.5 g cheese with 1 mL borate buffer before adding milk phosphatase improved recovery among cheeses. Addition of magnesium ion increased phosphatase activity in some cheeses. Phosphatases in blue mold-ripened and Swiss cheeses were inactivated by heat faster than was milk phosphatase, yet milk phosphatase added to various soft cheeses was completely inactivated at 60 degrees C for 10 min. The lability of phosphatase was due to the heat-denaturing effect of NaCl present in finished cheeses. Some Mexican style soft cheeses contained both heat-labile and heat-stable phosphatases. These data suggest that the phosphatase test to differentiate milk and microbial phosphatases on the basis of repasteurization and analysis of cheese is no longer valid.     

Enzyme immunoassay for mycophenolic acid in milk and cheese

Mycophenolic acid (MPA) was reacted with N-hydroxysuccinimide and conjugated to keyhole limpet hemocyanin (KLH), and to horseradish peroxidase (HRP), respectively. The MPA-KLH was used to produce anti-MPA antiserum in rabbits. A competitive direct enzyme immunoassay (EIA) for MPA was established with anti-MPA antiserum and MPA-HRP conjugate. The mean 50% inhibition and detection limit of MPA standard curves (n = 103) were 197 +/- 67 and 81 +/- 48 pg/mL, respectively. The EIA was specific for MPA and its synthetic 2-morpholinoethyl ester, mycophenolate mofetil (91% relative cross-reactivity). Raw bulk milk and pasteurized milk, with and without beta-glucuronidase pretreatment, were analyzed by EIA. No MPA was found in milk, at a detection limit of 100 pg/mL (recovery 58-66% at 0.125-2 ng/mL). Blue-veined cheese from the German market (n = 53) was analyzed by EIA, and the detection limit was at 0.5 ng/g (recovery 68-79% at 5-100 ng/g). All but two cheeses contained MPA, although mostly (66%) at levels of <10 ng/g. MPA at 400-1200 ng/g was found in Roquefort cheeses. Highest levels (4-11 microg/g) were found in a German soft cheese preparation. MPA levels in mycelium-rich parts of cheese were 3 times higher than in mycelium-free parts.     

Swiss concept of soil protection

The maintenance of soils as a basis for sustaining life is an important subject of Swiss environmental policies. A prominent instrument to achieve this goal is the Ordinance of 1998Relating to Impacts on the Soil (OIS). In 13 articles a guiding framework for the protection of the soil is given. For a better understanding of the legal background, SAEFL has published specific commentaries. They explain the intention of the legislator and thus contribute to a consistent and mutual implementation of the soil regulations in practice. Comments are made on respective articles, on the Swiss concept of soil protection and also on the guide, trigger and cleanup values for hazardous substances in the soil. The commentaries are available in German, French and English languages. The extent of the Commentaries (including OIS and excerpts of the Swiss Federal Law of 1983 Relating to the Protection of the Environment) is 44 pages. Mail-address: sol@huwal.admin.ch     

Instability of PR toxin in blue cheese.

PR toxin was unstable in solvent extracts of blue cheese and in strongly acidic solutions. However, it was appreciably stable over a 2.5 hr period in moderately acidic methanol-water extracts (pH 2--3) of blue cheese. Using this extraction mixture, it was determined that PR toxin was not stable in blue cheese itself. PR toxin reacted with model neutral and basic amino acids and the formation of PR imine from PR toxin in the presence of blue cheese was demonstrated. While PR imine added to blue cheese could be recovered on analysis after 5 min, over a 2--5 day period it too was unstable in the cheese.     

Time course study of aluminum-induced callose formation in barley roots as observed by digital microscopy and low-vacuum scanning electron microscopy

To clarify the mechanism(s) involved in the short-term inhibition of root elongation by AI, we monitored the morphological changes of barley roots by digital microscopy. Within 30 min after exposure to 37 µM AI, the surface of the root epidermis in the region of a distance of 1.5 mm from the root tip became rough and began to show signs of damage. After 38 min, callose was rapidly excreted from the junction between the root cap and the root epidermis, and formed a spherical lump approximately 60 µm in diameter. The fine structure of the callose deposits on the root surface was analyzed by low-vacuum scanning electron microscopy. After 50 min, there was a significant increase in the callose contents in the distal 0.6 mm part. At the same time, root elongation stopped completely. Fluorescence staining indicated that callose was localized on the surface of the cell elongation area (the elongation zone of primary roots and root hairs), but not on the surface of the meristem. The root growth reduction associated with AI treatment may be due to the use of sugar substrates for callose formation instead of cellulose formation.     

Thin layer chromatographic determination of sterigmatocystin in cheese

A one-dimensional thin layer chromatographic method has been developed for determining sterigmatocystin in cheese. Cheese is extracted with acetonitrile-4% KCl (85 + 15). A simplified liquid-liquid partition cleanup is used, and the sample extract is passed through a cupric carbonate column for final purification. Sterigmatocystin is visualized by spraying the plate with aluminum chloride. The fluorescence of the spot is enhanced 10-fold by additional plate spraying with a silicone-ether mixture, enabling sterigmatocystin detection and quantitation at 2 and 5 micrograms/kg, respectively. Average recoveries were 88.3 and 86.4% at the 10 and 25 micrograms/kg levels, respectively.     

Fluorimetric determination of aflatoxin M1 in cheese

A simple and sensitive method is proposed for the determination of aflatoxin M1 in cheese. The ground cheese sample is extracted with acetone-water (3 + 1). Acetone is evaporated under vacuum, and the aqueous phase is passed through a C18 disposable cartridge. After the cartridge is washed with acetonitrile-water (1 + 9), the toxin is eluted with acetonitrile. The extract is then cleaned up on a silica cartridge. Final analysis is performed by 2-dimensional thin layer chromatography (TLC) combined with fluorodensitometry or by liquid chromatography on a reverse phase C18 column with fluorescence detection. Recovery is greater than 90%, and the coefficient of variation is 6% or less. The detection limit is in the range of 10 ng/kg. The identity of aflatoxin M1 is confirmed by formation of the M2a or acetyl-M1 derivative and rechromatography.     

Identification of bitterness-masking compounds from cheese

Bitterness-masking compounds were identified in a natural white mold cheese. The oily fraction of the cheese was extracted and further fractionated by using silica gel column chromatography. The four fractions obtained were characterized by thin-layer chromatography and nuclear magnetic resonance spectroscopy. The fatty acid-containing fraction was found to have the highest bitterness-masking activity against quinine hydrochloride. Bitterness-masking activity was quantitated using a method based on subjective equivalents. At 0.5 mM, the fatty acid mixture, which had a composition similar to that of cheese, suppressed the bitterness of 0.008% quinine hydrochloride to be equivalent to that of 0.0049-0.0060% and 0.5 mM oleic acid to that of 0.0032-0.0038% solution. The binding potential between oleic acid and the bitter compounds was estimated by isothermal titration calorimetry. These results suggest that oleic acid masked bitterness by forming a complex with the bitter compounds.     

Chemometric analysis of Ragusano cheese flavor

Ragusano cheeses were produced in duplicate from milk collected from pasture-fed and total mixed ration (TMR)-fed cattle at four time intervals. The cheeses were subjected to chemical analysis, conventional sensory testing, and gas chromatography-olfactometry (GCO). Data from each type of analysis were examined by principal component and factor analysis and by pattern recognition (SIMCA) to see if sufficient information for classification into pasture-fed and TMR-fed groups was contained therein. The results clearly indicate that there are significant differences in sensory panel and chemical analysis results between the two cheeses. The data were also examined to see if models of sensory responses as a function of analytical or GCO results or both could be constructed with the modeling technique partial least-squares regression (PLS). Strong PLS models of some sensory responses (green and toasted odor; salt, pungent, bitter, and butyric sensations; and smooth consistency) were obtained.     

Study of light-induced volatile compounds in goat's milk cheese

Light-induced volatile compounds in goat cheese were studied by a combination of solid phase microextraction (SPME)-gas chromatography (GC)-mass spectrometry (MS), headspace oxygen depletion, and sensory evaluation. Samples stored under fluorescent light for 2 days at 30 degrees C had 90% more volatile compounds and 4 times more headspace oxygen depletion than samples stored in the dark at 30 degrees C. The volatiles 1-heptanol, heptanal, nonanal, and 2-decenal were formed and increased only in the light-stored samples, which may be formed from singlet oxygen oxidation of unsaturated fatty acids. Sensory evaluation showed that samples stored under light had significantly more off-flavor than samples stored in the dark at 30 degrees C (P < 0.05), and 1-heptanol, heptanal, nonanal, and 2-decenal increased the goat cheese off-flavor significantly (P < 0.05).     

Factors affecting the retention of rennet in cheese curd

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify the effects of several milk-related factors and parameters during cheese manufacture on the retention of coagulant in cheese curd. The amount of coagulant retained in curd was determined by its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) using reversed-phase HPLC. The retention of chymosin in cheese curd increased significantly when the pH of milk was reduced at rennet addition below pH 6.1, the pH at whey drainage below pH 5.7, or the average casein micelle size in milk and when the ionic strength of milk was increased. The casein content of milk and the quantity of chymosin added to milk had no significant effect on the retention of chymosin in curd; the quantity of coagulant bound per gram of casein remained unchanged.     

Determination of phosphorus in processed cheese: collaborative study

A second interlaboratory collaborative study of the determination of phosphorus in processed cheese products by the molybdenum blue method verifies that this method is prone to producing a laboratory-induced systematic error. It would be useless to continue to make minor modifications in the details of the method, which will improve only the within-laboratory precision, until an accuracy control of the final measurement step is incorporated into the method.     

Time course study of substrate utilization by Aspergillus flavus in medium simulating corn (Zea mays) kernels.

Utilization of the three major corn reserve materials, starch, triglycerides (refined corn oil), and zein (storage protein), by Aspergillus flavus was monitored in vitro over a 7-day fermentation. Medium composition in which proportions of reserve materials initially approximated proportions in mature corn kernels changed little over the first 18 h. Subsequently, hydrolysis of both starch and triglycerides occurred simultaneously, with peak concentrations of glucose and free fatty acids on day 2 of the fermentation period. Fatty acid concentrations dropped relatively rapidly after day 2 but increased again after day 6. Aflatoxin B(1) production increased after 36 h, with a peak at day 4. Aflatoxin B(1) production paralleled fungal biomass production during the exponential growth phase. A. flavus did not appear to preferentially utilize any of the released fatty acids. A number of fungus-specific metabolites were detected, including arabitol, erythritol, mannitol, trehalose, and kojic acid. Mannitol exceeded the other metabolites in concentration, and the timing of mannitol production closely paralleled that of aflatoxin B(1). Kojic acid concentrations peaked at day 6. In contrast to previously described selective use of simple carbohydrates by A. flavus, less discrimination was displayed when faced with utilization of complex substrates such as starch or triglycerides.