Characteristics of ripened Tronchón cheese from raw goat milk containing legally admissible amounts of antibiotics

The aim of this study was to evaluate the transfer of the most widely used antibiotics in dairy goats from milk to cheese as well as their effect on the cheese-making process and cheese characteristics during ripening. Antibiotic-free milk was spiked individually with 7 veterinary drugs (amoxicillin, benzylpenicillin, cloxacillin, erythromycin, ciprofloxacin, enrofloxacin, and oxytetracycline) at an equivalent concentration of the European Union maximum residue limit. Spiked goat milk was used to make mature Tronchón cheeses, which were analyzed at 0, 30, and 60 d of maturation to determine pH, chemical composition, proteolytic and lipolytic activities, and color and textural properties. A sensory evaluation of 60-d ripened cheeses was carried out. Cheeses from raw antibiotic-free goat milk were made simultaneously to be used as reference. The cheese-making process was unaffected by the presence of most antibiotics evaluated. Only erythromycin and oxytetracycline significantly increased the time required for cheese production (122 ± 29 and 108 ± 25 min, respectively). However, variable amounts of antibiotics, ranging from 7.4 to 68%, were transferred from milk to cheese, with oxytetracycline and quinolones showing the highest retention rates. In general, antibiotic residues present in the cheeses at the beginning of maturation decrease significantly along time. Thus, β-lactams and erythromycin residues were not detectable after 30 d of ripening. However, relatively high concentrations of enrofloxacin (148 ± 12 µg/kg) and ciprofloxacin (253 ± 24 µg/kg) residues were found in the cheeses after 60 d of maturation. The quality characteristics of the Tronchón cheeses were only slightly affected by such substances, with few significant differences in the free fatty acid concentration and color and textural properties of the cheeses. Results herein indicate that the use of goat milk containing antibiotics, such as quinolones, at the European Union maximum residue limit for cheese production could adversely affect the safety of the final products because relatively high concentrations of these substances could be retained in soft and semi-mature cheeses, making it necessary to assess the risk for consumer health. Studies on the partition of the antibiotic substances during cheese-making, using specific technologies, would be convenient to guarantee the safety of cheese and related products.     

Distribution and stability of aflatoxin M1 during processing, ripening and storage of Telemes cheese

Telemes cheeses were produced using milk that was artificially-contaminated with aflatoxin M1 at the levels of 0.050 and 0.100 microg/l. The cheeses produced in the two cheese-making trials were allowed to ripen for 2 months and stored for an additional 4 months to simulate commercial production of Telemes cheese. Concentrations of aflatoxin M1 in whey, curd, brine, and the produced cheeses were determined at intervals by liquid chromatography and fluorometric detection coupled with immunoaffinity column extraction. Concentrations of aflatoxin M1 in the produced curds were found to be 3.9 and 4.4 times higher than those in milk, whereas concentrations in whey were lower than those in curd and milk. Aflatoxin M1 was present in cheese at higher concentrations at the beginning than at the end of the ripening/storage period, and it declined to concentrations 2.7 and 3.4 times higher than those initially present in milk by the end of the sixth month of storage. Concentrations of aflatoxin M1 in brine started low and increased by the end of the ripening/storage period but only a portion of the amounts of aflatoxin M1 lost from cheese was found in the brine. Results showed that Telemes cheeses produced from milk containing aflatoxin M1 at a concentration close to either the maximum acceptable level of 0.05 microg/l set by the European union (EU) or at double this value, will contain the toxin at a level that is much lower or slightly higher, respectively, than the maximum acceptable level of 0.250 microg of aflatoxin M1/kg cheese set by some countries.     

Production of cyclopiazonic acid by molds isolated from Taleggio cheese.

Twenty-seven strains of Penicillium were isolated from the rind of Taleggio, a typical Italian cheese, so that we could test their capacity to produce cyclopiazonic acid (CPA); all strains produced CPA. The production was strongly influenced by the strain variety and growth conditions. Strains incubated at 25 degrees C for 7 days always produced CPA in mannitol broth, with concentrations ranging from 0.02 to 1 microg/ml, whereas only 33% of strains grown in yeast-extract broth produced CPA, with a maximum value of 0.1 microg/ml. In milk, maximum production (1.6 microg/ml) was observed after 14 days of incubation at 25 degrees C. In order to evaluate the presence of the toxin and its capacity for migrating into the cheeses, the rind, the cheese near the rind, and the cores from six Taleggio cheeses were analyzed. CPA was present in five cheeses, with a maximum concentration of 0.25 mg/kg in one rind, and in one cheese, the toxin migrated to the core. A positive correlation between CPA production and surface mold was found.     

Zinc supplementation of Friesian cows: Effect on chemical-nutritional composition and aromatic profile of dairy products

Zinc represents an essential microelement for several biochemical mechanisms. The body's inability to store zinc necessarily requires a constant dietary supply to avoid alteration of physiological functions. The aim of the present study was to investigate the effect of dietary enrichment with zinc on chemical-nutritional and aromatic properties of milk and cheese. Thirty commercial dairy cows, balanced for parity, milk production, and days in milk, were randomly assigned to 2 groups. The control group was fed with a conventional complete diet (22 kg of dry matter/animal per day), whereas the experimental group received a daily zinc supplementation of 60 mg per kg of dry complete feed. During the experimental period, the milk yield was monitored and samples of milk and caciotta cheese were collected to obtain information about the chemical-nutritional composition and aromatic profile. Dietary zinc integration did not influence milk yield and composition, but induced a marked reduction of somatic cell count and improved the oxidative stability of ripened caciotta cheese. In both milk and cheese, the experimental group samples were characterized by a lower concentration of saturated fatty acids and an increase in oleic acid, vaccenic acid, and rumenic acid. The aromatic profile of dairy products was also positively affected by dietary zinc intake, with an increase in concentration of carboxylic acids, aldehydes, and esters. The present results suggest a positive role of zinc in improving animal health and nutraceutical properties of milk and corresponding cheese. Taking into account the analysis of volatile compounds, zinc dietary supplementation of dairy cows should contribute to the production of cheeses with interesting organoleptic properties, although more studies are necessary to confirm the consumer acceptability of these changes.     

Survival of Listeria monocytogenes during manufacture, ripening and storage of soft lactic cheese made from raw goat milk

Soft lactic cheeses were manufactured with raw goat milk inoculated with Listeria monocytogenes. The physico-chemical and microbiological characteristics of curds and cheeses were determined after each processing step as well as during ripening and refrigerated storage. The fate of Listeria monocytogenes was evaluated by enumeration on PALCAM agar and by a qualitative detection after a double selective enrichment procedure. The results showed that the physico-chemical and microbiological characteristics of lactic cheeses caused a decrease of Listeria monocytogenes counts. However, this decrease did not lead to the complete disappearance of the pathogen and Listeria monocytogenes was able to survive in soft lactic cheeses made with raw goat milk.     

60-day aging requirement does not ensure safety of surface-mold-ripened soft cheeses manufactured from raw or pasteurized milk when Listeria monocytogenes is introduced as a postprocessing contaminant

Because of renewed interest in specialty cheeses, artisan and farmstead producers are manufacturing surface-mold-ripened soft cheeses from raw milk, using the 60-day holding standard (21 CFR 133.182) to achieve safety. This study compared the growth potential of Listeria monocytogenes on cheeses manufactured from raw or pasteurized milk and held for > 60 days at 4 degrees C. Final cheeses were within federal standards of identity for soft ripened cheese, with low moisture targets to facilitate the holding period. Wheels were surface inoculated with a five-strain cocktail of L. monocytogenes at approximately 0.2 CFU/ cm2 (low level) or 2 CFU/cm2 (high level), ripened, wrapped, and held at 4 degrees C. Listeria populations began to increase by day 28 for all treatments after initial population declines. From the low initial inoculation level, populations in raw and pasteurized milk cheese reached maximums of 2.96 +/- 2.79 and 2.33 +/- 2.10 log CFU/g, respectively, after 60 days of holding. Similar growth was observed in cheese inoculated at high levels, where populations reached 4.55 +/- 4.33 and 5.29 +/- 5.11 log CFU/g for raw and pasteurized milk cheeses, respectively. No significant differences (P < 0.05) were observed in pH development, growth rate, or population levels between cheeses made from the different milk types. Independent of the milk type, cheeses held for 60 days supported growth from very low initial levels of L. monocytogenes introduced as a postprocess contaminant. The safety of cheeses of this type must be achieved through control strategies other than aging, and thus revision of current federal regulations is warranted.     

SDS-PAGE of Proteins in Goat Milk Cheeses Ripened under Different Conditions

Proteolytic characteristics of five varieties of commercial goat milk cheeses and a cow milk Cheddar aged under different conditions were evaluated by SDS-PAGE and an advanced densitometry system. All fresh goat cheeses had distinctively lower intensities of α_S1-casein (CN) bands than those of cow milk Cheddar, whereas intensities of β-CN were much greater in the goat cheeses. The PAGE patterns clearly displayed α_S2-CN in all goat cheese, but it was negligible in cow milk Cheddar. The greater protein degradation in hard goat cheeses than cow milk Cheddar at 4°C and 22°C strongly correlated with water-soluble nitrogen compound concentrations and densitometric values of corresponding cheeses.     

Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese

The Brazilian market for dairy products made from goat milk is increasing despite the seasonality of production and naturally small milk production per animal, factors that result in high-priced products and encourage fraud. In Brazil, no official analytical method exists for detecting adulteration of goat dairy products with cow milk. The aim of this study was to design a strategy to investigate the adulteration of frescal (fresh) goat cheeses available in the Rio de Janeiro retail market, combining analysis of cheese composition and the perception of adulteration by consumers. Commercial goat cheeses were tested by using a duplex PCR assay previously designed to authenticate cheeses, by targeting the mitochondrial 12S ribosomal RNA genes of both species simultaneously. The PCR test was able to detect 0.5% (vol/vol) cow milk added during goat cheese formulation. The analysis of 20 locally produced goat cheeses (20 lots of 4 brands) showed that all were adulterated with cow milk, even though the labels did not indicate the addition of cow milk. To estimate the ability of consumers to perceive the fraudulent addition of cow milk, a triangle test was performed, in which cheeses formulated with several different proportions of goat and cow milk were offered to 102 regular consumers of cheese. Detection threshold analysis indicated that almost half of the consumers were able to perceive adulteration at 10% (vol/vol) cow milk. Effective actions must be implemented to regulate the market for goat dairy products in Brazil, considering the rights and choices of consumers with respect to their particular requirements for diet and health, preference, and cost.     

Fate of ivermectin residues in ewes' milk and derived products

The fate of ivermectin (IVM) residues was studied throughout the processing of daily bulk milk from 30 ewes (taken up to 33 d following subcutaneous administration of 0.2 mg IVM/kg b.w.) in the following milk products: yoghurt made from raw and pasteurized milk; cheese after pressing; 30- and 60-day ripened cheese; and whey, secondary whey and whey proteins obtained after cheese-making (albumin cheese). The concentration of the H2B1a component of IVM was analysed in these dairy products using an HPLC method with fluorescence detection. The mean recovery of the method was, depending on the matrix, between 87 and 100%. Limits of detection in the order of only 0.1 microg H2B1a/kg of product were achieved. Maximum concentrations of IVM were detected mostly at 2 d after drug administration to the ewes. The highest concentration of IVM was found on day 2 in 60-day ripened cheese (96 microg H2B1a/kg cheese). Secondary whey was the matrix with the lowest concentration of IVM (<0.6 microg H2B1a/ kg). Residue levels fell below the limits of detection between day 5 (for secondary whey) and day 25 (for all cheese samples). In the matrices investigated, linear correlations between daily concentrations of IVM, milk fat and solid content were evident. During yoghurt production, fermentation and thermal stability of IVM was observed. During cheese production, approximately 35% of the IVM, present in the raw (bulk) milk samples, was lost. From the results it was concluded that the processing of ewes' milk did not eliminate the drug residues under investigation. The consequences of IVM in the human diet were discussed. Milk from treated animals should be excluded from production of fat products like cheese for longer after treatment with IVM than for lower fat products.     

Microbiological changes throughout ripening of goat cheese made from raw,pasteurized and high-pressure-treated milk

The bacteriological quality during ripening of raw (RA), pasteurized (PA; 72°C, 15 s) and pressure-treated (PR; 500 MPa, 20°C, 15 min) goat milk assessed by enumeration of total bacteria, psychrotrophic bacteria, Enterobacteriaceae, lactobacilli, enterococci, Micrococcaceae and lactococci was evaluated. The high pressure treatment applied was as efficient as pasteurization in reducing the bacterial population of milk. Experimental cheeses were made from RA, PA and PR milks to study the microbial population during ripening. Lactobacilli and lactococci were the predominant microbiota present during ripening in all the cheeses. There were no differences in numbers of starter bacteria during ripening. However, lactobacilli counts for RA milk cheese were significantly higher than for PA and PR cheeses in all the ripening stages studied. Micrococcaceae and enterococci remained at a secondary level, and no differences were observed between cheeses at the end of ripening. On the other hand, the number of Enterobacteriaceae decreased during ripening, but faster in PR milk cheese than in PA and RA milk cheeses. The results of this study suggest that goat cheese made from PR milk had similar microbiological characteristics to PA milk cheeses.     

High-Pressure Treatment and Freezing of Raw Goat Milk Curd for Cheese Manufacture: Effects on Cheese Characteristics

High-pressure treatment of raw goat milk curd was investigated as an alternative to thermal treatment of milk in cheese manufacture, and curd freezing as a procedure to surmount the seasonality of goat milk production. Experimental cheeses were made by mixing (70:30) fresh cow milk curd with frozen curd from pasteurized goat milk (PGC), frozen curd from raw goat milk (RGC), or frozen pressurized curd from raw goat milk (PRGC). Control cheese was made from a mixture (70:30) of pasteurized cow and goat milk. RGC cheese showed the highest counts of staphylococci, Gram-negative bacteria and coliforms, whereas PRGC cheese had maximum aminopeptidase activity, esterase activity, and overall proteolysis. Control cheese exhibited the highest dry matter content and peptide levels, the lowest concentration of free amino acids, the highest concentration of volatile compounds such as free fatty acids, alcohols and esters, and the firmest texture. Differences in sensory characteristics between experimental and control cheeses were of minor importance. High-pressure treatment of curd allowed the production of cheese of bacteriological quality similar to that of control cheese made using pasteurized milk, while curd freezing did not alter the sensory characteristics of experimental cheeses with respect to control cheese.     

Effect of artisanal kid rennet paste on lipolysis in semi-hard goat cheese

This study examined the use of hygienised kid rennet pastes in model cheese systems and also in goat milk semi-hard cheeses to promote lipolysis. The results obtained indicated that the use of rennet paste caused greater lipolysis and increased, mostly, the short-chain free fatty acid (FFA) content. The model systems made with whole goat’s milk using rennet paste and commercial rennet mixture exhibited a higher FFA content than did the rennet paste-free controls (31,600 vs. 25,600 μmol/kg cheese). For the pilot cheeses made with bovine rennet and rennet paste mixture, the increase in FFA level after 45 days of ripening compared with the cheeses prepared only with commercial rennet was as much as 6600 (μmol/kg cheese) and the increase in the butyric acid content was also 1650 (μmol/kg cheese). Moreover, after 15 days of ripening, industrially prepared cheeses made with rennet paste exhibited greater levels of FFA than did the cheeses made with commercial rennet (11,500 μmol/kg at 45 days of ripening). Their flavour was stronger and the organoleptic characteristics were better accepted, which implies less ripening time for commercial cheese manufacture.     

The effects of heat applications on macrocyclic lactone-structured antiparasitic drug residues in cows’ milk

ABSTRACT

The study aimed to evaluate the effects of heat on macrocyclic lactone residues in cows’ milk. Ivermectin, abamectin, doramectin, eprinomectin and moxidectin were added to raw milk in three concentrations. The milk was then pasteurised (40 seconds at 74°C or 1 minute at 80°C) and boiled (10 minutes at 100°C). The analyses were performed with a validated method: LC-MS/MS. Thermal treatment resulted in a statistically significant decrease in the abamectin, eprinomectin, and moxidectin concentrations in the milk; however, the residues did not completely degrade. Boiling resulted in a greater decrease in the moxidectin concentrations than was observed with pasteurisation. The high pasteurisation and boiling processes had a greater effect on the eprinomectin residues than did the low pasteurisation process. The pasteurisation and boiling processes did not have an effect on the doramectin and ivermectin. The study concluded that the macrocyclic lactones are generally resistant to such processes.     

Linear regression models for estimating the effect of technological factors on the sensory characteristics of goat cheeses

Linear regression models were applied to predict the effect of the breed and technological factors on the sensory characteristic of cheeses. Seventy goat cheeses from forty local producers were analysed. They represented the totality of the Andalusian cheese-making sector (different goat breeds, type of coagulant, ripening time and heat treatment of milk). Sensory attributes of cheeses were influenced by all the factors studied. Ripening affects all sensory attributes while the breed and the heat treatment are the most influential factors on the flavour and the type of coagulant on the texture attributes of these cheeses. Linear regression models were found to be suitable for studying which factors had an independent influence on the sensory attributes of these cheeses. This work could help researchers and producers to a better sensory characterisation of their products.     

Effects of atropine on plasma amino acid profiles and on the synthesis of individual milk proteins in dairy cows fed with pasture

Effects of atropine on blood plasma amino acid profile and on the yields and concentration of milk components were investigated in 12 Friesian cows in early lactation. Cows were housed indoors and fed with cut pasture ad libitum. Each cow received four treatments over 12 d during a replicated 4 x 4 Latin square experiment. Treatments were: control (saline); low dose (L; 30 microg atropine/kg body weight (BW)); medium dose (M; 40 microg atropine/kg BW); and 2 x L dose, 2 h apart (2 x L). On each of four treatment days, cows were milked at about 7.00, after which treatments were administered by subcutaneous injection. Cows were milked again at 2 h, 6 h and 10 h after injection. Milk samples were collected at each milking. Immediately after the 2 h milking, blood samples were drawn from each cow and the second injection was given for the 2 x L treatment. Atropine reduced hourly milk yield, and concentrations and hourly yields of total protein, casein, whey protein, alpha-casein, beta-casein, kappa-casein, beta-lactoglobulin and alpha-lactalbumin, but by differing amounts. Milk concentrations of bovine serum albumin and immunoglobulin G were increased by atropine, and overall yields of these proteins were mostly unchanged. Atropine lowered concentrations of most, but not all, amino acids in blood plasma, with essential amino acids reduced more than non-essential amino acids. Concentrations of alpha-amino N in whole blood, and glucose and insulin in blood plasma, fell after atropine injection. There was no difference between the L and M doses of atropine, but the 2 x L dose had greater effects on milk composition than the single doses. For yields of milk and milk components, the effect of the 2 x L dose was also more persistent. The results highlight the differential synthesis of individual milk proteins, and suggest that atropine might be useful for evaluating the mechanisms regulating milk protein composition.     

Microbial dynamics during the ripening of a mixed cow and goat milk cheese manufactured using frozen goat milk curd

To overcome the seasonal shortage of goat milk in mixed milk cheese manufacture, pasteurized goat milk curd and high-pressure-treated raw goat milk curd manufactured in the spring were held at −24°C for 4 mo, thawed, and mixed with fresh cow milk curd for the manufacture of experimental cheeses. Control cheeses were made from a mixture of pasteurized cow and goat milk. The microbiota of experimental and control cheeses was studied using culture-dependent and culture-independent techniques. Bacterial enumeration by classical methods showed lactic acid bacteria to be the dominant population in both control and experimental cheeses. In total, 681 isolates were grouped by partial amplified rDNA restriction analysis (ARDRA) into 4 groups and identified by 16S rRNA gene sequencing as Lactococcus lactis ssp. lactis (563 isolates), Leuconostoc pseudomesenteroides (72 isolates), Lactobacillus spp. (34 isolates), and Lc. lactis ssp. cremoris (12 isolates). Temporal temperature gradient gel electrophoresis (TTGE) analysis of cheese showed (1) the predominance of Lc. lactis in all cheeses; (2) the presence of Leu. pseudomesenteroides population in all cheeses from d 15 onward; (3) the presence of a Lactobacillus plantarum population in control cheese until d 15 and in experimental cheeses throughout the ripening period. Due to the most diverse and complete set of peptidases present in the genus Lactobacillus, the prevalence of this population in experimental cheeses could give rise to differences in cheese flavor between experimental and control cheeses.     

Nutritional and sensory characteristics of Minas fresh cheese made with goat milk,cow milk,or a mixture of both

This study aimed to assess and compare the nutritional, technological, and sensory characteristics of Minas fresh cheese made with goat milk, cow milk, or a mixture of the two stored in cold conditions for 21 d. The yield and centesimal composition of the cheeses were not affected by the type of milk used in their preparation. Reductions were observed in the moisture content, pH, proteolysis index, and instrumental hardness; moreover, increases were observed in the syneresis, acidity index, and depth of proteolysis index in all cheeses. The percentages of caprylic, capric, oleic, and linoleic fatty acids were higher in goat milk cheese and cheese made with a mixture of goat and cow milk compared with cow milk cheese, and a sensory evaluation revealed differences in color, flavor, and aroma between the cheeses. The preparation of Minas fresh cheese with a mixture of goat and cow milk can be a viable alternative for dairy products in the market that can be characterized as high-quality products that meet consumer demands.     

Proteolysis and Lipolysis of Goat Milk Cheese

Numerous varieties of goat milk cheeses are produced worldwide. Maturation or ripening of goat and other species milk cheeses is governed by interplay of many different factors. Proteolysis and lipolysis are two major biochemical processes in the multifaceted phenomenon of cheese aging, which involves a variety of chemical, physical, and microbiological changes under controlled environmental conditions. Proteolysis of cheeses in general is influenced by several factors including plasmin, chymosin, protease from starter and nonstarter bacteria, pH and moisture levels of the curds, storage temperature and time, salt content, salt-to-moisture ratio, and humidity. Primary factors affecting lipolysis in cheeses are fatty acid composition, lipolytic enzymes, lipolytic microorganisms, moisture, temperature, storage time, oxygen, and surface area, etc. Several analytical techniques have been used to measure proteolysis of goat and (or) cow milk cheeses during ripening, such as solubility of peptides and amino acids in various solvents or precipitants, liberation of reactive functional groups, various forms of chromatography, and different forms of electrophoresis. Lipolysis of goat milk cheeses has been estimated by acid degree value (ADV), acid value, and free fatty acid concentration, while lipid oxidation of dairy goat products can be determined by peroxide value, thiobarbuturic acid value (TBA). Recent reports have shown that goat cheeses had greater rates of protein degradation than cow counterparts, and that aging time and temperature synergistically elevated most of proteolytic and lipolytic indices in goat cheeses. This paper will further discuss proteolytic and lipolytic characteristics of goat milk cheeses.     

Efficacy of UV light for the reduction of Listeria monocytogenes in goat's milk

Certain types of goat's cheeses are produced using unpasteurized milk, which increases the food safety concerns for these types of products. Popularity and consumption of goat's milk products have increased, and the niche market includes gourmet goat's cheeses. The U.S. Code of Federal Regulations and the Pasteurized Milk Ordinance both address the possibility for processing alternatives to heat treatment, and the use of UV light treatment may be a viable alternative that still ensures the safety of the product. Fresh goat's milk was inoculated with Listeria monocytogenes (L-2289) at 10(7) CFU/ml and exposed to UV light using the CiderSure 3500 apparatus (FPE Inc., Macedon, NY). Inoculated milk was exposed to a UV dose range between 0 and 20 mJ/cm2 to determine the optimal UV dose. A greater than 5-log reduction was achieved (P < 0.0001) when the milk received a cumulative UV dose of 15.8 +/- 1.6 mJ/cm2. The results of this study indicate that UV irradiation could be used for the reduction of L. monocytogenes in goat's milk.     

Characterisation of Urda whey cheese: Evolution of main biochemical and microbiological parameters during ripening and vacuum packaged cold storage

In this study, the main biochemical and microbiological characteristics of Urda, a traditional Greek whey cheese, were determined during ripening at 19 ± 2 °C for 25 days followed by vacuum packaging and storage at 5 °C until day 360. Few differences in pH, water activity, acid degree value, moisture, salt, protein and fat contents were observed between Urda cheeses produced from sheep or goat milk whey at all sampling days (1, 25, 90, 180 and 360). Cheese microbiota was dominated by mesophilic lactic acid bacteria, but high numbers of enterococci, aerobic gram-negative bacteria and Enterobacteriaceae were also present. Increases in all the soluble nitrogen fractions of cheeses occurred, primarily during cold storage. Ketones and terpenes were the most abundant volatile compounds in fresh (1-day) sheep and goat cheeses, respectively, whereas free fatty acids were the most abundant compounds in mature (180-day) cheeses, followed by ketones.