Screening of indoles in cheese

 Indole and skatole (3-methylindole) are formed from tryptophan by microbial activity. Depending on their concentration, they may either contribute to an unpleasant odour, as described for pig meat, or alternatively to a positive aroma profile, as during ripening of cheese. In a screening study, the two indoles in various types of cheese were determined, by both HPLC and GC. It was found that the two compounds mainly occur in mould cheese, such as Camembert and are even more pronounced in blue-veined cheese. The concentrations in unripened cheese were up to 700 ng/g fat for indole and 50 ng/g fat for skatole. The formation of the two indoles increases when proteolysis during ripening provides more tryptophan. Skatole formation appears to be specifically favoured by a low pH and anaerobic conditions. Received: 14 May 1997     

Ripening Changes in a Blue-Mold Surface Ripened Cheese from Goat's Milk

Samples of blue-mold surface ripened goat milk cheese were analyzed for fat and protein breakdown during ripening. After 2 wk, cheese had a pH of 4.96 and 9.13 micromoles of free fatty acids (F.F.A.)/g fat. After 6 wk, the pH increased to 6.43 and F.F.A. were 160.85 micromoles/g fat. At this time, the cheese rind pH was 7.18 with 253.81 micromoles FFA/g fat. Water soluble protein in the cheese increased from 20.47% at 2 wk of ripening to 26.62% after 4 wk of ripening.     

Electrophoretic patterns and thin-layer chromatography of common cheeses in Egypt: Comparison and quantification

The extent of cheese ripening and the type of proteolysis and lipolysis of common cheeses in Egypt were measured by concentration of each of soluble tyrosine; soluble trypophan; amino N; soluble N/total N; total volatile fatty acids and free fatty acids, and by quantitative gel electrophoresis and thin-layer chromatography. The effects of concentration-related factors (e.g. moisture, salt and pH) on cheese protein and fat hydrolysis were also studied.

The results showed that, as a heterogeneous group of cheese, differences were marked in gross chemical composition and both the extent of cheese ripening and the relative proportions of protein, fat and their degradation products. Among the selected cheeses, ras cheese has higher values of ripening indices, while kariesh cheese has lower values. Increasing the salt content of mish cheese caused an inhibition in degradation of its protein and fat.

The principal protein regions in electrophoretic patterns and fractions of fat in TLC patterns were similar in number and relative mobility. In most of the cheeses, _s-casein was degraded more extensively than β-casein, while the whole of the γ-caseins were resistant to further hydrolysis. Also, there was close correlation between _s-casein and its degradation products. In spite of the absence of significant relationships between the soluble nitrogen and the relative amounts of unattached _s-, β-, and γ-caseins, the amino nitrogen and soluble tyrosine and tryptophan were in close correlation with _s- and β-caseins and their degradation products.

A positive relationship was noted between pH (from 4·40 to 5·85) and both protein and fat hydrolysis. The fat of roquefort cheese was more hydrolysed than other cheeses; however, the fat of the soft cheeses was less hydrolysed. Moreover, negative and highly significant correlations between triglycerides and their degradation by both TLC and chemical analysis were obtained.     

Study of macromolecular interactions in low‐fat brined cheese modified with Zedu gum

The functionality of Zedu gum as a fat mimetic in low‐fat brined cheese was studied. The physicochemical, textural, rheological, microstructural and sensory properties of cheese samples modified with 0.1% and 0.25% of Zedu gum were compared to those of control cheeses (low‐fat and full‐fat cheeses with no fat mimetic) during ripening. To obtain further information about the cheeses' structure and interactions between macromolecules (casein protein and Zedu gum), other parameters were analysed by differential scanning calorimetry and Fourier transform infrared (FTIR) spectroscopy. Incorporation of Zedu gum into low‐fat cheese caused an open microstructure and softer texture in comparison with the control low‐fat cheese. The thermal properties and FTIR spectra of the cheeses were influenced by both fat mimetic and ripening time. On days 1 and 60 of ripening time, the lower value of enthalpy of the low‐fat cheese with 0.25 g of Zedu gum/kg of milk (AS 0.25) in comparison with control low‐fat cheese could have been due to the electrostatic nature of the interactions between Zedu gum and casein protein. On both days, the FTIR spectrum of AS 0.25 showed a well separated absorption at 1746 cm^?1 possibly due to the formation of ester groups as a result of the interaction of the carbonyl groups in Zedu gum with the hydroxyl groups of some amino acids in casein.     

Milk fat thermal properties and solid fat content in emmental cheese: a differential scanning calorimetry study

The experiments reported in this study give deeper insight into the crystallization of milk fat in Emmental cheese, which is the most widely consumed hard cheese in France. Differential scanning calorimetry (DSC) was used to monitor the thermal properties of milk fat after the main stages involved during manufacture of Emmental cheese. By heating the samples to 60°C to eliminate their thermal history and cooling them at 2°C/min, the liquid → solid phase transition of fat was investigated. Confocal laser scanning microscopy was used to characterize in situ the supramolecular organization of milk fat dispersed in the casein matrix. The destabilization of fat globules by aggregation or coalescence and the formation of free fat during the manufacture altered the thermal properties of milk fat by increasing the initial temperature of crystallization and by the formation of 2 overlapping exotherms. The melting properties of the crystalline structures formed by fat at the temperatures used for ripening (12, 21, and 4°C) were examined. Differential scanning calorimetry was used to determine the ratio of solid to liquid fat; that is, the amount of fat that is crystallized, by dividing the partial enthalpy of melting of the fat for ripening temperature by the total enthalpy of melting of the same fat extracted from cheese. This study shows, for the first time, that milk fat is partially crystallized in Emmental cheese: about 55.7 ± 3.5% of fat is solid at 4°C at the end of ripening. Polymorphic phase transitions of milk fat are also suggested during ripening of Emmental cheese.     

Proteolytic and lipolytic changes during the ripening of León raw cow's milk cheese, a Spanish traditional variety

Proteolytic and lipolytic changes were studied throughout ripening of five batches of León cow's milk cheese, a traditional variety made in the north of Spain. Total soluble nitrogen, non-protein nitrogen, oligopeptides nitrogen, amino nitrogen and ammonia nitrogen fractions increased slightly during the ripening process. The final values of these nitrogen fractions indicate that this cheese undergoes a very slight proteolysis as much in extent as in depth. This weak protein degradation is corroborated when the caseins and their degradation products were quantified by electrophoresis. β-Casein stayed practically intact throughout the ripening process and only 10% of α_s-casein became degraded. The content of total free amino acids increased progressively but in a slightly increased way during ripening, reaching final average values of 592 mg (100 g)⁻¹ of total solids. The most abundant free amino acid at the end of ripening was lysine, followed by leucine, glutamic acid, tryptophan, valine and phenylalanine. The acidity index of the fat values increased during ripening by a factor of 4.39. The final values of this parameter are in the range of those observed in other cow's milk cheeses ripened by bacteria. The content in total free fatty acids underwent an increase throughout ripening reaching final average values of 6669 ppm. The most abundant free fatty acid at the end of ripening was oleic acid followed by butyric and palmitic acids. The high content of short-chain fatty acids is outstanding, specially that of butyric acid.     

Lipolysis and proteolysis profiles of fresh artisanal goat cheese made with raw milk with 3 different fat contents

The objective of this study was to describe the proteolysis and lipolysis profiles in goat cheese made in the Canary Islands (Spain) using raw milk with 3 different fat contents (0.5, 1.5, and 5%) and ripened for 1, 7, 14, and 28 d. β-Casein was the most abundant protein in all cheeses and at all ripening times. Quantitative analysis showed a general decrease in caseins as ripening progressed, and degradation rates were higher for α_S1-casein than for β-casein and α_S2-casein. Furthermore, the degradation rate during the experimental time decreased with lower fat contents. The α_S2-casein and α_S1-casein levels that remained in full-fat and reduced-fat cheeses were less than those in low-fat cheese. In contrast, β-casein also showed degradation along with ripening, but differences in degradation among the 3 cheese types were not significant at 28 d. The degradation products increased with the ripening time in all cheeses, but they were higher in full-fat cheese than in reduced-fat and low-fat cheeses. The free fatty acid concentration per 100 g of cheese was higher in full-fat cheese than in reduced- and low-fat cheese; however, when the results were expressed as milligrams of free fatty acids per gram of fat in cheese, then lipolysis occurred more rapidly in low-fat cheese than in reduced- and full-fat cheeses. These results may explain the atypical texture and off-flavors found in low-fat goat cheeses, likely the main causes of non-acceptance.     

Quantitative determination of the boar taint compounds androstenone,skatole, indole, 3α-androstenol and 3β-androstenol in wild boars (Sus scrofa) reveals extremely low levels of the tryptophan-related degradation products

The major boar taint compounds androstenone and skatole as well as the minor compounds indole, 3α-androstenol and 3β-androstenol were determined in back fat samples of 23 male wild boars by applying a recently published SIDA–HS-SPME–GC/MS method. The boar pheromones androstenone, 3α-androstenol and 3β-androstenol were found in extraordinary high concentrations, resulting in mean values of 3329 ng/g androstenone, 1273 ng/g 3α-androstenol and 545 ng/g 3β-androstenol. Interestingly, skatole was not detectable in about 50% of the boars and negligibly low in all other samples as expressed by a mean skatole value of only 14 ng/g. Indole was also found in every sample, but again in low concentrations with a mean value of 40 ng/g. Possible factors explaining this remarkably low skatole deposition in wild boars such as intestinal flora and anatomy, dietary composition, housing or genetic predisposition are discussed in this paper.     

The effect of fat content on the microbiology and proteolysis in cheddar cheese during ripening dairy foods

We investigated the effect of incremental reduction in fat content, in the range 33 to 6% (wt/wt), on changes in the microbiology and proteolysis of Cheddar cheese, over a 225-d ripening period at 7 degrees C. A reduction of fat content resulted in significant increases in contents of moisture and protein and a decrease in the concentration of moisture in nonfat substance. Reduced fat had little effect on the age-related changes in the population of starter cells. The populations of nonstarter lactic acid bacteria decreased with fat content, and counts in the low fat cheese (6% wt/wt) were significantly lower than those in the full fat cheese (33% wt/wt) at ripening times >1 and <180 d. Proteolysis as measured by the percentage of total N soluble at pH 4.6 or in 70% ethanol decreased significantly as the fat content decreased. However, the content of pH 4.6 soluble N per 100 g of cheese was not significantly influenced by fat content. At ripening times >60 d, the content of 70% ethanol soluble N per 100 g of full fat (33% wt/wt) cheese was significantly lower than that in either the half fat (17% wt/wt) or low fat (6% wt/wt) cheeses. The concentration of AA N, as a percentage of total N, was not significantly affected by fat content. However, when expressed as a percentage of total cheese, amino acid N increased significantly with decreasing fat content. Analysis of pH 4.6 soluble N extracts by reverse phase- and gel permeation HPLC revealed that fat content affected the pattern of proteolysis, as reflected by the differences in peptide profiles.     

Effect of an Exopolysaccharide-Producing Strain of Streptococcus Thermophilus on the Yield and Texture of Mexican Manchego-Type Cheese

An exopolysaccharide-producing strain of Streptococcus thermophilus was evaluated in the production of Mexican manchego-type cheese. This ropy strain improved water and fat retention, and significantly increased cheese yield. Furthermore, the ropy strain cheese retained more moisture than control cheese during ripening, suggesting that exopolysaccharide strongly bound water within the protein matrix of the cheese. Scanning electron microscopy confirmed that exopolysaccharide bound to the protein matrix of the cheese, producing a dense network that helped to increase water and fat retention and leading to a more open structure of the cheese that gave a softer product, as confirmed by instrumental texture profile analysis and sensory evaluation. Comparison of scanning electron microscopy micrographs of the different sections of the cheese showed higher concentration of exopolysaccharide in the centre than in the outer sections, indicating that exopolysaccharide production continued during ripening and that the environment at the centre of the cheese (moisture and/or oxygen concentration) favoured exopolysaccharide production. Instrumental texture profile analysis also demonstrated that the ropy strain cheese was more cohesive and less elastic than the control; in contrast, exopolysaccharide did not affect chewiness. The changes in texture could be correlated to composition: hardness increased as water and fat decreased, while springiness decreased with increasing fat. The interactions of exopolysaccharide with the cheese protein matrix had an affect on the increase in cohesiveness of the ropy strain cheese.     

Rheological Properties of Cheddar Cheese as Influenced by Fat Reduction and Ripening Time

Fat reduction in Cheddar cheese resulted in an increase in viscoelasticity as evidenced by increases in G’and G”. Proteolysis during ripening led to softening of all cheeses and thus decreases in G’and G” for cheeses containing 34, 27, and 20% fat. Cheese with 13% fat showed a decrease in G’upon ripening, but no change in G”. This lack of change in viscous behavior may be important to the texture of reduced-fat Cheddar cheese and overall acceptability. Dynamic rheological testing was helpful in understanding rheological behavior associated with fat reduction in cheese.     

Different scalding techniques do not affect boar taint

The prevention of unpleasant boar taint is the main reason for castration of male piglets. For animal welfare reasons, castration is announced to be banned in the European Community. This study aimed to investigate whether androstenone, skatole and indole in backfat of boars may be reduced by different scalding technologies. To discriminate ante and post mortem effects, carcasses were sampled before and after scalding in two abattoirs using either horizontal (TANK) or vertical (TUNNEL) scalding. Backfat samples were analysed using gas chromatography (androstenone) and liquid chromatography (skatole, indole). Neither TANK nor TUNNEL scalding did significantly reduce malodorous compounds. Skatole and androstenone in backfat obtained after scalding averaged 112 ± 123 ng/g and 1196 ± 885 ng/g melted fat, respectively; significant differences between abattoirs were observed for skatole. Increased skatole levels were tentatively assigned to longer transport duration. Concluding from recent consumer research and subsequent application of suggested sensory rejection thresholds for androstenone (2000 ng/g) and skatole (150 ng/g), nearly 30% of the carcasses may be unacceptably tainted.     

A single nucleotide polymorphism in the CYP2E1 gene promoter affects skatole content in backfat of boars of two commercial Duroc-sired crossbred populations

The prevention of unpleasant boar taint is the main reason for castration of male piglets. This study aimed to investigate how the malodorous compound skatole is affected by a single nucleotide polymorphism (g.2412 C>T at -586 ATG) in the porcine cytochrome p450 II E1 (CYP2E1) gene. 119 boars of two commercial Duroc-sired crossbred populations raised at different farms were investigated. Skatole and androstenone in backfat averaged 114±125ng/g and 1206±895ng/g melted fat, respectively. The frequency of the genotypes CC, CT, and TT was 25, 52, and 23%, respectively. CC boars had the highest average skatole levels (175ng/g) compared to CT (92ng/g) and TT (93ng/g). Applying suggested sensory threshold levels for skatole (>150ng/g) and androstenone (>2000ng/g), 30% of the carcasses may be unacceptably tainted while the proportion of tainted carcasses is significantly higher within genotype CC (56.7%) compared to genotypes CT (24.3%) and TT (14.8%). Effective reduction of tainted carcasses appears feasible applying marker assisted selection.     

Viscoelastic Properties of Cheddar Cheese: Effect of Calcium and Phosphorus,Residual Lactose,Salt-to-Moisture Ratio and Ripening Time

The viscoelastic properties of eight different types of Cheddar cheeses prepared with two levels of calcium (Ca) and Phosphorus (P) content, two levels of residual lactose content and two levels of salt to moisture ratio (S/M) ratio were studied in a STRESSTECH viscoanalyzer. The elastic (G′) and viscous (G″) modulus were measured at 0, 1, 2, 4, 6, and 8 months of ripening during heating the cheese samples from 30 to 70°C. Low levels of Ca and P content (0.53 g Ca and 0.39 g P /100 g cheese) in the Cheddar cheese resulted up to 20.9% and 15.9% lower elastic and viscous modulus respectively, compared to Cheddar cheese prepared with high levels of Ca and P content (0.67 g Ca and 0.53 g P/100g cheese) during ripening up to 8 months. Low levels of residual lactose (0.78 g/100g) in the Cheddar cheese resulted in 39.1 and 78.1% lower elastic and viscous modulus, respectively, compared to Cheddar cheese with high levels of residual lactose (1.4 g/100g) during ripening up to 8 months. In the same way, low levels of S/M ratio (4.8) in the Cheddar cheese resulted in 40.7 and 40.5% lower elastic and viscous modulus, respectively, compared to high levels of S/M ratio (6.4) during ripening up to 8 months. Upon heating from 30 to 70°C, the elastic and viscous modulus of the eight different types of Cheddar cheeses reduced up to 91.7 and 95.1%, respectively, during ripening. Cheddar cheese recorded maximum elastic modulus at the end of 8 months of ripening, and maximum viscous modulus at the end of 4 months of ripening.     

Changes in the microbiological and chemical characteristics of an artisanal, low-fat cheese made from raw ovine milk during ripening

Changes in the microbial flora of batzos cheese made from raw ovine milk were studied during ripening. Lactic acid bacteria and Enterobacteriaceae were the predominant groups of micro-organisms. Cheeses manufactured in summer had higher microbial counts than those made in spring, with the exception of staphylococci. Nevertheless, Enterobacteriaceae and coliforms decreased more rapidly in cheese made in summer and counts at the end of storage were lower than those in spring cheese.
Enterococci predominated in the ripened curd of cheese made in spring, whereas lactobacilli were the most abundant lactic acid bacteria in cheese made in summer. Enterococcus faecium was the predominant species in spring, and Lactobacillus paracasei ssp. paracasei predominated in cheese made in summer. The pH of the cheeses was > 5.0 throughout ripening, and NaCl-in-moisture content (> 8.0%) permitted the growth and survival of salt-tolerant micro-organisms. α_s1-Casein degraded at a faster rate than β-casein; both caseins were hydrolysed more rapidly in spring than in summer. The free amino acid content became higher in summer cheese (566.24–3460.25 µg/g of glycine equivalent) than in spring cheese because of the progress of ripening. Moreover, the milk fat of the cheese was degraded more in the summer than in the spring. The results suggest that there could be advantages to using starter cultures and improving the level of hygiene during milk and cheese production in order to eliminate undesirable micro-organisms and standardize cheese quality.     

EFFECT OF LIPASE ENZYME ON THE RIPENING OF WHITE PICKLED CHEESE

The effect of addition of pregastric lipase enzyme on the accelerated ripening of white pickled cheese was investigated. Commercial pregastric lipase was added to milk before rennet addition at a level of 0,5, 8, 11 g per 100 L of milk and cheeses were made from this milk. Total solids, fat, total nitrogen, salt, titratable acidity, pH and free fatty acids (C₂-C_18:1) were analysed in the samples during 1–90 days of ripening period at 15 days intervals. Total solids, fat, total nitrogen, salt, titratable acidity, and pH of cheeses slightly increased during the ripening period. Free fatty acids and volatile free fatty acid contents in cheeses made from pregastric lipase added milk were affected by pregastric lipase and their contents were increased significantly (P<0.01) during the ripening period. Particularly, when cheese had a high level (11 g per 100 L milk) pregastric lipase, the amounts of butyric, caproic and caprylic acids in white pickled cheese were quite high. The relative amounts of volatile free fatty acids varied with storage time and pregastric lipase levels.     

Comparative analysis of an intestinal strain of Bifidobacterium longum and a strain of Bifidobacterium animalis subspecies lactis in Cheddar cheese

Bifidobacteria cultures were incorporated into Cheddar cheeses to conduct a comparative analysis between the commercially available strain Bifidobacterium animalis ssp. lactis Bb-12 and the wild-type intestinal isolate, Bifidobacterium longum DJO10A. They were incorporated as starter adjuncts in separate vats and as a mixed culture, and survival through manufacturing and cheese ripening was assessed. For cheese using only Bb-12, the cells may have grown during cheese manufacture as 133% of the inoculum was incorporated into the cheese, resulting in 8.00 log cfu/g. Counts remained high during ripening showing less than 1 log decrease over a 12-mo period. For cheese using a mixed culture of Bb-12 and DJO10A, both strains were incorporated at much lower levels: 3.02 and 1.11%, respectively. This resulted in cheese with 6.00 and 5.04 log cfu/g for Bb-12 and DJO10A, respectively. Bifidobacteria survival rates were low, most likely due to the moisture of the cheese being below 38%. The Bb-12 demonstrated almost 100% viability during ripening. Numbers of DJO10A started to decline after 2 mo of ripening and dropped below the level of detection (2 log cfu/g) after 4.5 mo of ripening. Neither DJO10A nor Bb-12 fortified cheeses produced detectable amounts of organic acids during ripening other than lactic acid, indicating the lack of detectable metabolic contribution from bifidobacteria during cheese production and ripening such as production of acetic acid. To determine if sublethal stresses could improve the viability of DJO10A, 2 more vats were made, 1 with DJO10A exposed to sublethal acid, cold, and centrifugation stresses, and 1 exposed to none of these stresses. Although stress-primed DJO10A survived cheese manufacture better, as 72.8% were incorporated into the cheese compared with 41.1% of the unprimed, the statistical significance of this difference is unknown. In addition, the difference in moisture levels in the cheese cannot be excluded as influencing this difference. However, the rate of decline during ripening was similar for both. After 6 mo of ripening, cell counts in cheese were 4.68 and 4.24 log cfu/g for primed and unprimed cultures, respectively. These results suggest that whereas priming bifidobacteria with sublethal stresses before incorporation in a cheese fermentation may improve the number of viable cells that get incorporated into the cheese, it does not affect viability during cheese ripening.     

Development and application of confocal scanning laser microscopy methods for studying the distribution of fat and protein in selected dairy products.

Confocal scanning laser microscopy (CSLM) methods were developed to identify fat and protein in cheeses milk chocolate and milk powders. Various fluorescent probes were assessed for their ability to label fat or protein in selected food products in situ. Dual labelling of fat and protein was made possible by using mixtures of probes. Selected probes and probe mixtures were then used to study (a) structure development of Mozzarella cheese during manufacture and ripening, and (b)) the distribution of fat and protein in milk chocolate made with milk powders containing varying levels of free fat. Microstructural changes in the protein and fat phases of Mozzarella cheese were observed at each major step in processing. Aggregation of renneted micelles occurred during curd formation; this was followed by amalgamation of the para-casein into linear fibres during plasticization. Following storage, the protein phase of the Mozzarella became more continuous; entrapping and isolating fat globules. Chocolate made with a high free-fat spray-dried powder blend showed a homogeneous fat distribution, similar to that of chocolate made with roller-dried milk. Chocolate made with whole milk powder containing 10 g free fat/100 fat showed a non-homogeneous fat distribution with some fat occluded within milk protein particles. These differences in fat distribution were related to Casson yield value and Casson viscosity of the chocolates.