HPLC quantification of biogenic amines in cheeses: correlation with PCR-detection of tyramine-producing microorganisms

The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.     

The effect of heat treatment and starter culture on colour intensity and sensory properties of Kulek cheese

The effect of the heat treatment on colour intensity and sensory properties of Kulek cheese made from raw milk with (RS) or without starter culture (R) and heated milk with starter culture (HS) was investigated during ripening. Colour L, a and b values of Kulek cheeses were determined for both interior and surface. The values for cheese R were 82.4 and 74.5 for L, ?5.2 and ?5.5 for a, 31.0 and 34.7 for b interior and surface respectively. The equivalents for RS cheese were 82.8 and 75.7 for L, ?4.8 and ?5.0 for a, 31.0 and 34.0 for b. The values for HS cheese were 88.9 and 88.4 for L and ?3.3 and ?3.3 for a, 22.8 and 24.2 for b. L values (lightness) were the highest in cheeses made from heated milk while b and negative a values were the highest in cheeses made from raw milk. Aroma scores of raw and heat‐treated milk cheeses made with starter were not significantly different. The panelists scored cheeses made from raw milk without starter as the best in body and texture.     

THE CONTRIBUTION OF HERBS TO THE ACCUMULATION OF HISTAMINE IN "OTLU" CHEESE

The aim of this study was to determine the effect of herbs and the use of raw milk on histamine accumulation in “Otlu” (Herby) cheese during ripening. Cow's milk was used for the cheese production. The milk was divided into two main groups: one was used raw and the other was pasteurized at 65C for 30 min. Each group was further divided into two subparts, one of which was used as control (without herbs), while 2% (w/v) of the herbs were added into the other to produce Herby cheese. All cheeses were ripened at 7C for 90 days. The cheese samples were analyzed in terms of histamine content, titratable acidity, dry matter, salt and degree of ripening on days 5, 30, 60 and 90. Total mesophilic aerobic bacteria (MAB) were also counted during ripening. The use of raw milk and the addition of herb both increased histamine formation in Otlu cheeses (P < 0.05). Moreover, higher water‐soluble nitrogen values, as degree of ripening, were obtained from both raw milk and herb‐added cheeses. The number of MAB was higher in raw cheeses (P < 0.05) and also herb‐added cheeses. The study suggests that the addition of herbs may facilitate histamine formation in Herby cheese.     

Influence of pasteurised milk, raw milk and different ripening cultures on biogenic amine concentrations in semi-soft cheeses during ripening

In semi-soft cheeses, produced with pasteurised milk, raw milk and different starter cultures, the concentrations of cadaverine, histamine, phenylethylamine, putrescine and tyramine were investigated. The cultures (pasteurised milk cultures, raw milk cultures and starter cultures) strongly influenced the biogenic amine concentrations in the cheeses ripened for 5 months. Two cheeses made with identical pasteurised milk, but different ripening cultures, differed greatly in their total biogenic amine concentrations (51 vs 371?mg/kg). In general, the biogenic amine concentrations increased markedly between month 2 and month 3 of cheese ripening. The high content of enterococci and Enterobacteriaceae yielded the biogenic amine concentrations. In contrast, Lactobacilli did not seem to be important. However, unspecified bacteria have to be considered, since cheeses with comparable microbiological profiles differed enormously in their biogenic amine concentrations. Semi-soft cheeses produced from pasteurised milk showed remarkably lower total biogenic amine concentrations compared to semi-soft cheeses produced from raw milk (51–1096?mg/kg vs 1011–3133?mg/kg, depending also on the ripening cultures). The highest total biogenic amine concentration (4817?mg/kg) was detected in a cheese produced from raw milk that had been stored for 36?h. In this cheese, the concentrations of cadaverine, phenylethylamine, putrescine and tyramine were higher than in all other cheeses. The highest histamine concentration was found to be in another raw milk cheese (573?mg/kg).     

Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese

The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp. cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese. Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media. Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6. Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses. Tyramine and histamine were detected in all batches of cheese from day 30. Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively). Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses. After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture.     

Proteolysis in goat cheese made from raw,pasteurized or pressure-treated milk

Primary and secondary proteolysis of goat cheese made from raw (RA), pasteurized (PA; 72 °C, 15 s) and pressure-treated milk (PR; 500 MPa, 15 min, 20 °C) were examined by capillary electrophoresis, nitrogen fractionation and HPLC peptide profiles. PA milk cheese showed a more important hydrolysis (P<0.05) of α_s1-casein than RA milk cheese at the first stages of ripening (15 days), while PR milk cheese had a level between those seen in PA and RA milk cheeses. Degradation of β-casein was more important (P<0.05) in PA and PR than in RA milk cheeses at 15 days of ripening. However, from thereon β-casein in PR and RA milk cheeses was hydrolyzed at essentially similar rates, but at lower rates (P<0.05) than in PA milk cheeses. Pressure treatment could induce proteolysis of β-casein in a way, which is different from that produced by heat treatment. There was an increase in 4.6-soluble nitrogen (WSN) and in trichloroacetic acid (TCASN) throughout ripening in cheeses, but higher contents (P<0.05) in PA and PR milk cheeses at the end of ripening were observed. PR milk cheeses contained considerably higher content (P<0.05) of free amino acids than RA or PA milk cheeses. In general, heat and pressure treatments had no significant effect on the levels of hydrophobic and hydrophilic peptides.     

Effect of post-ripening processing on the histamine and histamine-producing bacteria contents of different cheeses

Histamine is the most active biogenic amine and the one most commonly involved in food-borne intoxications. In this work, the effect of the post-ripening processing (cutting, slicing and grating) of different types of cheese on their histamine and histamine-producing bacteria contents was analysed. The average histamine content detected in Emmental cheeses was 220.8 mg kg⁻¹; the highest concentrations (up to 734.1 mg kg⁻¹) were recorded in grated samples. Significantly more histamine-producing bacteria were detected by real-time quantitative PCR (RT-qPCR) in the post-ripening-processed samples than in entire cheeses in all the cheese types analysed. A good association was obtained between the number of histamine-producing bacteria determined by RT-qPCR and histamine concentrations as determined by HPLC. Results for the analysis of entire Emmental cheeses and their post-ripening processed products suggested that processing had an important influence on the presence of histamine in cheese.     

Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model

Tyramine and histamine are the biogenic amines (BAs) most commonly found in cheese, in which they appear as a result of the microbial enzymatic decarboxylation of tyrosine and histidine respectively. Given their toxic effects, their presence in high concentrations in foods should be avoided. In this work, samples of three cheeses (Zamorano, Cabrales and Emmental) with long ripening periods, and that often have high BA concentrations, were screened for the presence of BA-degrading lactic acid bacteria (LAB). Seventeen isolates were found that were able to degrade tyramine and histamine in broth culture. All 17 isolates were identified by 16S rRNA sequencing as belonging to Lactobacillus casei. They were typed by plasmid S1-PFGE and genomic macrorestriction-PFGE analysis. Two strains (L. casei 4a and 5b) associated with high degradation rates for both BAs were selected to test how this ability might affect histamine and tyramine accumulation in a Cabrales-like mini-cheese manufacturing model. The quantification of BAs and the monitoring of the strains' growth over ripening were undertaken by RP-HPLC and qPCR respectively. Both strains were found to reduce histamine and tyramine accumulation. These two strains might be suitable for use as adjunct cultures for reducing the presence of BAs in cheese.     

Study of the chemical composition,proteolysis, volatile compounds,and textural properties of industrial and traditional Beaten (Bieno sirenje) ewe milk cheese

The objective of this study was to determine the gross composition, proteolysis, and volatile and texture profiles during ripening of industrial (IND) and traditional (TRD) Beaten (Bieno sirenje) cheeses made by using ewe milk. In the course of the analyses, statistical differences were determined in some physicochemical parameters, nitrogen fractions, and total free amino acid levels between TRD and IND types of cheese. Higher levels of proteolysis were observed in IND cheeses than in TRD cheeses during ripening. Levels of residual β- and α_s-caseins were 72.2 and 48.7%, respectively, in 180-d-old TRD cheeses. However, the residual levels were 52.8% for β-casein and 18% for α_s-casein in IND cheeses. Similar differences were noted for the reversed-phase HPLC peptide profiles of 2 types of cheeses. Also, higher concentrations of peptides were eluted in IND cheeses than in TRD cheeses during ripening. A total of 73 volatile compounds, including alcohols (16), esters (17), acids (14), terpenes (7), ketones (5), aldehydes (4), and miscellaneous (10) were identified. The IND cheeses contained higher levels of carboxylic acids, esters, alcohols, and terpenes than the TRD cheeses; however, the same levels of methyl ketones were determined in the 2 types of cheeses at the end of ripening. These may be due to some differences (e.g., pasteurization and scalding temperature, among other factors) in the manufacture of the 2 types of Beaten cheeses. The textural profile of Beaten cheeses showed that TRD production method resulted in firmer, less fracturable, and stiffer cheeses than the IND production method. In conclusion, the results suggest that the use of industrial production method (pasteurization of cheese milk and curd scalding at 70°C) in the manufacture of Beaten ewe milk cheese enriched the volatile profile of the cheese.     

Proteolysis during ripening of Manchego cheese made from raw or pasteurized ewes' milk. Seasonal variation

Changes in nitrogen compounds during ripening of 40 batches of Manchego cheese made from raw milk (24 batches) or pasteurized milk (16 batches) at five different dairies throughout the year were investigated. After ripening for six months, degradation of p-kappa- and beta-caseins was more intense in raw milk cheese and degradation of alpha(s2)-casein in pasteurized milk cheese. Milk pasteurization had no significant effect on breakdown of alpha(s1)-casein. Hydrophobic peptide content did not differ between raw and pasteurized milk cheese, whereas hydrophilic peptide content was higher in raw milk cheese. There were no significant differences between seasons for residual caseins, but hydrophobic peptides were at a higher level in cheese made in autumn and winter and hydrophilic peptides in cheese made in winter and spring. Raw milk cheese had a higher content of total free amino acids and of most individual free amino acids than pasteurized milk cheese. The relative percentages of the individual free amino acids were significantly different for raw milk and pasteurized milk cheeses. The relative percentages of Lys and lie increased, while those of Val, Leu and Phe decreased during ripening. There were also seasonal variations within the relative percentages of free amino acids. In raw milk cheeses, Asp and Cys were relatively more abundant in those made in autumn, Glu and Arg in cheeses made in winter, and Lys and Ile in cheeses made in spring and summer. Biogenic amines were detected only in raw milk cheese, with the highest levels of histamine, tryptamine and tyramine in cheeses made in spring, winter and spring, respectively.     

Effect of the addition of nitrate to milk on the formation of volatile N-nitrosamines and apparent total N-nitroso compounds

 Saint-Paulin, a French semihard cheese, was manufactured from milk with and without added NO₃ ^–. Reference and experimental cheeses were analysed at different stages of ripening for the presence of apparent total N-nitroso compounds (ATNC) by chemical denitrosation and chemiluminescence detection (thermal energy analyser). The fate of NO₃ ^– and NO₂ ^–was also scrutinized. The levels of ATNC and of volatile N-nitrosamines were followed during ripening. After 60 days of ripening, ATNC were detected at concentrations of 27 μg NO-N/kg and 64 μg NO-N/kg in the cheeses obtained from milk with and without added NO₃ ^–, respectively. The NO₃ ^– contents of both cheeses were nearly the same, and below 5 mg/kg. The occurrence of NO₂ ^–was not observed. No relationship was found between the NO₃ ^– contents and the contamination of the cheeses by volatile N-nitrosamines. At the beginning of ripening, the cheeses manufactured with added NO₃ ^- showed high levels of ATNC (mean value 5800 μg N-NO/kg with large variations of 570–15210 μg NO-N/kg). These ATNC contents rapidly decreased during the first days of ripening (96%), but decreased much more slowly during the following days. An hypothetical mechanism, attempting to explain the disappearance of ATNC, involving the formation of alkylated species in the cheese was proposed. Received: 30 March 1998 / Revised version: 7 October 1998     

SENSORY CHARACTERISTICS OF EWE MILK CHEESE MADE WITH THREE TYPES OF COAGULANT: CALF RENNET, POWDERED VEGETABLE COAGULANT AND CRUDE AQUEOUS EXTRACT FROM CYNARA CARDUNCULUS

This article reports on a study of the sensorial characteristics of ewe milk cheese (Los Pedroches) made with animal rennet and two types of coagulant obtained from the cardoon Cynara cardunculus. The cheeses made with rennet displayed a less odor intensity: pungent odor and acid odor; a less acid taste; and a slightly lighter color. They were harder and firmer, but less creamy than those made with the vegetable coagulant (powdered vegetable coagulant [PVC] and crude aqueous extract [CAE] from C. cardunculus). However, the cheeses made with PVC showed organoleptic characteristics very similar to the cheeses made with CAE from C. cardunculus. After 90 days' ripening, the cheeses made with vegetable coagulant displayed a slightly more bitter taste than those made with rennet. In general terms, increased ripening time prompted the increased scores for most of the sensory attributes studied.     

Behavior of Enteropathogenic Escherichia coli in Camembert Cheese Made From Ultrafiltered Milk

Skimmilk retentates from ultrafiltration (UF) were used in combination with concentrated cream (67% milk fat) to prepare a liquid precheese mixture that with the addition of rennet, lactic starter culture and a mold spore preparation of Penicillium candidum was transformed readily into Camembert cheese upon ripening. Yield increases from the retention of soluble milk protein, closer weight control tolerances for individual cheeses and reduction in rennet in comparison to the conventional Camembert process were observed. The behavior of enteropathogenic E. coli (ECC) serotypes was demonstrated in the Camembert cheese made from UF processes. Relationships were determined in UF Camembert as to microbial type and numbers used to inoculate the precheese, and as to site of survival and growth of different EEC serotypes. Differences which were observed in the physicochemical properties between conventional and UF cheesemilk mixtures predisposed the UF Camembert cheeses to greater E. coli survival and growth than in the Camembert cheeses made conventionally.     

FORMATIONS OF BIOGENIC AMINES IN TURKISH STYLE SAUSAGE DURING RIPENING AND STORAGE PERIODS

Changes of biogenic amine (histamine, β‐phenylethylamine, serotonin, spermine, spermidine, cadaverine, putresine, 1,7‐diamino heptane, tryptamine and tyramine) concentrations and pH values of Turkish style sausages were investigated during ripening and storage. Cadaverine and β‐phenylethylamine were not detected during the ripening and storage periods for all sausage types. Storage time had a significant (P<0.05) effect on the formation of all biogenic amines. Starter culture usage decreased (P<0.05) biogenic amine formation. Histamine levels were higher in sausages prepared without additives and lower in the sausages made with starter culture than the sausages made without starter culture. There was no effect of starter culture and additives on formation of tryptamine, 1,7‐diamino heptane, serotonin or spermidine. Starter culture and additives affected (P<0.05) the formation histamine, putrescine and spermine. pH values of sausages sharply decreased (P< 0.05) from 5.98 to about 4.53 during 3 days of ripening. After that, pH values increased slowly and were nearly constant during the storage period.     

The effect of varying casein/fat ratio on physicochemical and sensory qualities of Feta‐type cheese made using buffalo milk

The Feta‐type cheese was prepared with different casein/fat (C/F) ratios of buffalo milk using microbial rennet. The manufactured Feta cheeses were subjected to physicochemical and sensory quality at 15‐day interval up to 60 days of ripening. Sensory analysis discriminated the different level of C/F ratio of buffalo milk cheeses predominantly by age. There was no significant difference (P < 0.01) observed in cheese made from C/F ratio of 0.6–0.7 in terms of flavour. The titratable acidity (TA), soluble protein and free fatty acid appear to be age‐dependent and increased throughout the ripening in all experimental cheeses.     

Comparison of the compositional, microbiological, biochemical, and volatile profile characteristics of nine Italian ewes' milk cheeses

Nine Italian ewes’ milk cheeses were compared for compositional, microbiological, biochemical, and volatile profile characteristics. Mean values for the gross composition were rather similar among cheeses. The lowest pH values were found for cheeses that used primary starters. At the end of ripening, cheeses made from raw milk contained >6.0 log₁₀ cfu/g of nonstarter lactic acid bacteria. Several species of lactobacilli were identified, but Lactobacillus plantarum and Lactobacillus paracasei were dominant. Random amplified polymorphic DNA-PCR analysis showed the biodiversity among the strains, and in several cases a relationship with the cheese of provenance. Cheeses differed mainly for secondary proteolysis, as shown by the principal component analysis applied to reversed-phase fast protein liquid chromatography data of the pH 4.6-soluble fractions and by determination of the free AA. A total of 113 volatile components were identified in the Italian Pecorino cheeses by solid-phase microextraction coupled with gas chromatography-mass spectrometry analysis. The volatile profiles of the 9 cheeses differed significantly. Quantitatively, alcohols were the most abundant chemical class for some cheeses, whereas ketones were the most abundant for other cheeses. Esters and carboxylic acids were largely found. Specific volatile components seemed to distinguish specific cheeses.     

Compositional,biochemical and textural changes during ripening of Tulum cheese made with different coagulants

In the present study, biochemical, chemical and texture changes in Tulum cheeses made using calf rennet and microbial rennets (Aspergillus niger protease and Rhizomucor miehei protease) were compared during ripening for up to 90 days. A total of 15 free fatty acids (FFAs) were detected in the cheese samples. The peroxide values (PV) of the cheeses increased significantly (P < 0.05) during ripening and the cheese made with calf rennet had the highest PV. Proteolysis in the cheeses increased as the ripening time increased. α_s1‐casein and β‐casein degradation was higher in cheeses manufactured with R. miehei protease. Cheeses made with calf rennet were significantly (P < 0.05) harder, more adhesive, more cohesive and more resilient than those made with microbial rennet.     

Triacylglycerol composition of protected designation of origin cheeses during ripening. Authenticity of milk fat

Triacylglycerol (TAG) composition by carbon number in 2 protected designation of origin cheeses, Mahón (cheese from cow milk) and Manchego (cheese from ewe milk) that were manufactured by 3 different producers was analyzed during cheese ripening using gas chromatography with a short capillary column. The TAG composition at different times during cheese ripening was also analyzed in cheeses from different batches produced at the same plant. Lipolysis levels in the Mahón and Manchego cheeses during ripening were low; free fatty acid values ranged from 2,500 to 4,000 ppm at the end of ripening. The TAG composition did not change significantly during ripening. The TAG values obtained from each cheese sample were substituted into the multiple regression equations that have been proposed to detect foreign fats in milk fat. The values obtained using the equations for bovine (proposed by the European Union) and ovine milk (proposed by our laboratory) were within the normal range. Accordingly, these equations can be considered useful for detecting foreign fat in these cheeses during the ripening period contemplated during this study.     

Utilization of interesterified fat in the production of Turkish white cheese

The chemical, physicochemical, proteolysis, sensory, and texture characteristics of white cheeses made from interesterified fat were examined throughout ripening for 90 days. The water-soluble nitrogen based ripening indexes of cheeses increased throughout the ripening period. However, there were not large quantitative differences between the peptide profiles of the all cheese samples. Cheeses produced by using fully interesterified fat had higher values for hardness, chewiness, and gumminess than that of control cheese (p<0.05). The polyunsaturated to saturated fatty acid ratios of cheeses were increased due to the presence of interesterified fat. The cholesterol values of cheeses decreased at the rate of between 58.83–89.04% depending on interesterified fat addition. In the sensory analysis, similar scores were obtained for both the control cheese and the other cheeses. The results showed that interesterified fat in cheese production could be used to fully or partially replace the milk fat in cheese.     

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.