Assessment of Cheddar Cheese Quality by Chromatographic Analysis of Free Amino Acids and Biogenic Amines

The free amino acids and biogenic amines extracted from normal and late-gassing Cheddar cheeses were derivatized with heptafluorobutyric anhydride and trifluoroacetic anhydride, respectively, before quantification by gas-liquid chromatography. On a microgram scale, twenty amino acids were positively identified in both types of cheese, but only high levels of γ-amino acid butyrate (0.3 to 19.4 mg/g) and small quantities of arginate were found to be associated with “poorly aged” Cheddar cheeses. Histamine (1.54 and 1.22 mg/g) and tyramine (0.32 and 0.43 mg/g) were the bioamines present in highest concentrations in both cheeses.     

BIOGENIC AMINE CONTENT OF SOME TURKISH CHEESES

Biogenic amines in ten samples of Kaşar (aged), five samples of Kaşar (fresh), four samples of Mihaliç, nine samples of Van Otlu (herbed), nine samples of Örgü, nine samples of Urfa and six samples of Civil cheeses were obtained from local supermarkets of Ankara. Tryptamine (TRY), phenylethylamine (PHA), putrescine (PUT), cadaverine (CAD), histamine (HIT), tyramine (TYA), spermine (SPM) and spermidine (SPD) contents of Turkish cheese samples were screened by high performance liquid chromatography (HPLC).
PUT, CAD, HIT, TYA and SPD were the predominant biogenic amines in maturated Kaşar (aged), vacuum-packed Kaşar (fresh), Mihaliç, Otlu (herbed) and Örgü cheeses. PHA, CAD, TYA and SPD were the predominant biogenic amines in Urfa cheese samples.
Civil cheeses were found to contain the highest HIT and TYA content with an average of 94.76 mg/100 g and 138.16 mg/100 g, respectively, much higher than the toxic dose. HIT level of Mihaliç cheeses were also higher than the accepted limit. The concentration of amines in all other cheeses was much lower than the toxic dose limits.     

牦牛乳硬质干酪成熟过程中生物胺与细菌群落结构分析

成熟干酪易产生生物胺,生物胺对人体健康具有影响,因此,为了探究牦牛乳硬质干酪的质量安全性,本实验利用高效液相色谱和高通量测序技术对干酪成熟过程中生物胺和细菌群落结构进行测定和分析。结果表明：牦牛乳硬质干酪中主要生物胺为腐胺、2-苯乙胺、酪胺、组胺和尸胺,在1～6 个月成熟过程中,干酪中各生物胺含量呈现增加趋势。干酪中生物胺含量最高阶段均出现在成熟期5～6 个月。干酪中组胺、酪胺和总生物胺含量分别低于推荐安全剂量50、100 mg/kg和1 000 mg/kg。干酪中游离氨基酸含量与除组胺之外的各生物胺含量、总生物胺含量、成熟时间之间具有显著正相关性（P＜0.01,P＜0.05）,各生物胺含量之间也存在正相关性。不同成熟期干酪包含相同属的微生物,其中链球菌属（Streptococcus）为优势菌属,平均相对丰度为84.63%,明串珠菌属（Leuconostoc）次之,平均相对丰度为6.91%,其他分别为乳杆菌属（Lactobacillus）、拉乌尔菌属（Raoultella）、不动杆菌属（Acinetobacter）、假单胞菌属（Pseudomonas）、肠杆菌属（Enterobacter）和无分类的肠杆菌科（unclassified_Eunnterobacteriaceae）。不同成熟期干酪中各菌属的相对丰度具有差异。本研究可为评估牦牛乳硬质干酪的质量安全性和生物胺形成机理提供理论依据。     

Factors controlling histamine production in Swiss cheese inoculated with Lactobacillus buchneri

Swiss cheese was made from raw milk inoculated with various concentrations of a histamine-producing strain of Lactobacillus buchneri. Histamine production in these cheeses was proportional to the initial number of L. buchneri present in the raw milk. The highest inoculum level tested was 10(5) L. buchneri/ml. This cheese contained 80 mg of histamine/100 g of cheese after 90 d of storage. Only 15 mg of histamine/100 g of cheese were detected after 90 d at the lowest inoculum level, 10(2) L. buchneri/ml. No histamine was detected in any of the Swiss cheese samples until after the brining stage. Perceptible growth of L. buchneri also did not occur until after the warm room treatment. Therefore, control of histamine formation in Swiss cheese requires control of the number of histamine-producing bacteria in the raw milk. A 5.5% NaCl concentration in DeMan, Rogosa, Sharpe (MRS) broth inhibited the production of histamine by L. buchneri, but the concentrations of NaCl typically found in Swiss cheese were not inhibitory. The histamine-producing isolate of L. buchneri survived heating at 49 to 80 degrees C for 10 min, suggesting that this organism would easily survive the normal heating process applied to raw milk used prior to making Swiss cheese.     

Public Health Significance of Amines in Cheese

Research designed to identify factors which may contribute to elevated biogenic amines (e.g., histamine, tryptamine, tyramine) in cheese is described. Free tyrosine and histidine in market cheese ranged from 6 to 29 and 1.5 to 12 mg/100 g. These concentrations of substrate would provide nontoxic quantities of corresponding amines, indicating that cheese proteolysis is important when toxic amounts occur. Pyridoxal phosphate concentration in 15 cheeses ranged from 42 to 215 μg/100 g, which appears to be sufficient to saturate amino acid decarboxylases required for amine production. Commercial preparations and cheese isolates of Propronibacterium species were tested for carbon dioxide production from histidine, tryptophan, and tyrosine in the presence and absence of pyriodoxal-5-phosphate. In the presence of cofactor, maximums were 10.9, 2.9, and 12.7 μl of carbon dioxide per h/mg cell dry weight. Over 150 isolates from 15 cheeses were tested for amine producing potential by measuring carbon dioxide production from histidine, tryptophan, and tyrosine; isolates were most active on tyrosine, producing as much as 26 μl of carbon dioxide per h/mg cell dry weight. Cheese slurries also were tested for carbon dioxide production from carboxyl carbon-14 labeled amino acids. Cheese isolates producing amines were tentatively identified as strains of Streptococcus faecium, Streptococcus mitis, Lactobacillus bulgaricus, Lactobacillus plantarum, and streptococci of the viridans group.     

Real time quantitative PCR detection of histamine-producing lactic acid bacteria in cheese: Relation with histamine content

Biogenic amines (BAs) are low molecular weight organic bases with biological activity, mainly formed by the microbial decarboxylation of amino acids. The consumption of food containing large amounts of some BAs can have toxicological consequences. Histamine is the most active BA and the most frequently involved in food-borne intoxications. This article reports the concentrations of histamine found in 80 cheeses made from different types of milk and subjected to different ripening periods. Histamine was detected in 41.25% of the samples, with values ranging from 20 to over 1000 mg per Kg of cheese. The highest histamine concentrations were recorded in long ripening cheeses made from raw milk. The presence and quantification of histamine-producing lactic acid bacteria was determined by RT-qPCR, and a good association was obtained between the C_t values and histamine concentrations determined by HPLC.     

Effect of forage type,season, and ripening time on selected quality properties of sheep milk cheese

The aim of this research was to study changes in the microbial populations, free AA profile, biogenic amine content, and sensory characteristics of ripened cheeses (100 and 180 d) produced in different seasons (summer, autumn, winter, and spring) from pasteurized sheep milk from 8 commercial flocks fed hay or silage diets. Twenty-one individual AA and 6 biogenic amines were determined by ultra-high performance liquid chromatography. Type of conserved forage for sheep feeding did not affect the variables studied, which is of great interest because hay and silage are low-cost ingredients for sheep feeding. Proteolysis led total free AA concentrations ranging between 35,179.26 and 138,063.71 mg/kg of cheese at 180 d of ripening. γ-Aminobutyric acid, which has been associated with beneficial effects on human health, was the second most abundant AA in all cheese samples, accounting for 15% of total free AA. Spring cheeses showed 2-fold higher concentrations of γ-aminobutyric acid than summer and autumn cheeses at the end of ripening. Overall, spring, winter, and autumn cheeses had lower average concentration of biogenic amines (431.99 mg/kg of cheese) than summer cheeses (825.70 mg/kg of cheese) as well as better sensory characteristics. Therefore, this study could provide the dairy industry with useful information for producing cheeses with valuable nutritional and sensory quality for consumers.     

Application of Fourier transform infrared spectroscopy for monitoring short-chain free fatty acids in Swiss cheese

Short-chain free fatty acids (FFA) are important sources of cheese flavor and have been reported to be indicators for assessing quality. The objective of this research was to develop a simple and rapid screening tool for monitoring the short-chain FFA contents in Swiss cheese by using Fourier transform infrared spectroscopy (FTIR). Forty-four Swiss cheese samples were evaluated by using a MIRacle three-reflection diamond attenuated total reflectance (ATR) accessory. Two different sampling techniques were used for FTIR/ATR measurement: direct measurement of Swiss cheese slices (∼0.5 g) and measurement of a water-soluble fraction of cheese. The amounts of FFA (propionic, acetic, and butyric acids) in the water-soluble fraction of samples were analyzed by gas chromatography-flame ion-ization detection as a reference method. Calibration models for both direct measurement and the water-soluble fraction of cheese were developed based on a cross-validated (leave-one-out approach) partial least squares regression by using the regions of 3,000 to 2,800, 1,775 to 1,680, and 1,500 to 900 cm⁻¹ for short-chain FFA in cheese. Promising performance statistics were obtained for the calibration models of both direct measurement and the water-soluble fraction, with improved performance statistics obtained from the water-soluble extract, particularly for propionic acid. Partial least squares models generated from FTIR/ATR spectra by direct measurement of cheeses gave standard errors of cross-validation of 9.7 mg/100 g of cheese for propionic acid, 9.3 mg/100 g of cheese for acetic acid, and 5.5 mg/100 g of cheese for butyric acid, and correlation coefficients >0.9. Standard error of cross-validation values for the water-soluble fraction were 4.4 mg/100 g of cheese for propionic acid, 9.2 mg/100 g of cheese for acetic acid, and 5.2 mg/100 g of cheese for butyric acid with correlation coefficients of 0.98, 0.95, and 0.92, respectively. Infrared spectroscopy and chemometrics accurately and precisely predicted the short-chain FFA content in Swiss cheeses and in the water-soluble fraction of the cheese.     

Biogenic amine content in Chilean Gauda cheese: physico‐chemical and microbiological factors that may influence this content

The presence of four biogenic amines in Chilean Gauda cheese was studied, and the effect of certain factors may influence the production of those amines. Histamine, tyramine, putrescine and cadaverine were quantified with high‐performance liquid chromatography (HPLC), in cheeses from the three major Chilean cheese manufacturers. Multiple regression analysis revealed that water activity, free amino acid content and the Enterobacteriaceae count all favoured production of biogenic amines. The total amount of biogenic amines in the samples studied was between 47.9 and 150.5 mg/kg of cheese, much lower than that associated with a possible consumer health risk (1000 mg/kg).     

Biogenic amines in Brazilian cheeses

The levels of biogenic amines in Brazilian cheeses were investigated for the first time. Ninety-two samples of 10 types of cheese were purchased at retail stores. The amines were extracted using hydrochloric acid, partitioned into diethyl ether, separated through reversed-phase ion-pair chromatography and quantified fluorimetrically after postcolumn derivatization with o-phthaldehyde. Overall, spermine was the most prevalent amine (93%), followed by histamine (65%), 2-phenylethylamine (62%), spermidine (61%), putrescine (60%), cadaverine (59%), serotonin (44%), agmatine (38%), tyramine (37%) and tryptamine (29%). Spermine, agmatine, 2-phenylethylamine, serotonin, spermidine and tryptamine were detected at low levels (< 4.10 mg/100g). Cadaverine, tyramine, histamine and putrescine were present at levels up to 111.00, 21.25, 19.65 and 17.37 mg/100g, respectively. There was variability on the type and levels of amine in each kind of cheese suggesting that amine formation and accumulation in cheese could be prevented. Efforts should be made to understand amine formation in cheese in order to optimize technology and secure low amine levels. Susceptible individuals should be advised to consume cheeses with low biogenic amines contents.     

Detection of biogenic amine producer bacteria in a typical Italian goat cheese

The aim of this study was to research decarboxylating bacterial strains and biogenic amine content in a typical Italian goat cheese (Robiola di Roccaverano). The study was performed on fresh and ripened samples of goat cheese manufactured from industrial and artisanal producers. Sixty-seven bacterial strains isolated showed decarboxylating activity, and Enterococcus faecalis was the most widespread decarboxylating species in all artisanal and industrial products. Pediococcus acidilactici and Enterococcus malodoratus were also identified as biogenic amine producers in Robiola di Roccaverano cheese. All the E. faecalis strains isolated in this study were able to decarboxylate tyrosine. Tyramine was the most abundant biogenic amine in cheese samples, while histamine was the most widespread. High amounts of these two biogenic amines were found in ripened samples (up to 2,067 mg/kg for tyramine and 1,786 mg/kg for histamine), whereas 2-phenylethylamine and tryptamine were present in almost all ripened cheeses at low concentrations. The detection of strains producing biogenic amines and the high concentrations of tyramine and histamine found in ripened Robiola di Roccaverano could represent a potential risk to the consumer.     

Apulian Cacioricotta goat's cheese: technical interventions for improving yield and organoleptic characteristics

The production process for making Cacioricotta, the most common goat cheese of Apulia, was modified to improve both its yield and organoleptic characteristics. The changes made to the traditional cheese-making process consisted in reducing the amounts of rennet to be added from 100 to 40 ml/q (q=quintal=100 kg) and in introducing a mesophilic starter mixed with the conventional thermophilic starter. The traditional cheese was compared to the modified product by means of routine analyses and measurements of the cheese yields and determination of free aminoacids and free fatty acids. The analytical techniques utilized were high pressure liquid chromatography (HPLC) and gas chromatography (GC). Both the cheeses had the same level of proteolysis while lipolysis was significantly lower (p<0.001) in the modified product with about 20% fewer free fatty acids. The amounts of short and medium chain free fatty acids (C₄-C₁₄), responsible for the marked pungent flavor of goat dairy products, differed significantly (p<0.001) in the two products: 203.41 mg/100 g cheese for the traditional product and 166.22 mg/100 g for the modified product. Finally, cheese yields with the modified procedure were 12% greater than the yield obtained with the traditional process.     

Interrelationships among microbiological, physicochemical, and biochemical properties of Terrincho cheese, with emphasis on biogenic amines

Changes in the microbiological, physicochemical, and biochemical characteristics of Terrincho cheese as represented by native microflora, pH, water activity, soluble nitrogen fractions, free amino acids, and biogenic amines (e.g., ethylamine, dimethylamine, tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, cystamine, and spermine) during ripening were monitored. Terrincho is a traditional Portuguese cheese manufactured from raw ewe's milk. The main groups of microorganisms (lactococci, lactobacilli, enterococci, pseudomonads, staphylococci, coliforms, yeasts, and molds) were determined following conventional microbiological procedures. Free amino acids and biogenic amines were determined by reverse-phase high-performance liquid chromatography, following extraction from the cheese matrix and derivatization with dabsyl chloride. The total content of free amino acids ranged from 1,730 mg/kg of dry matter at the beginning of the ripening stage to 5,180 mg/kg of dry matter by day 60 of ripening; such an increase was highly correlated with the increase of water-soluble nitrogen in total nitrogen, 12% trichloroacetic acid-soluble nitrogen in total nitrogen, and 5% phosphotungstic acid-soluble nitrogen in total nitrogen throughout ripening. Histamine was consistently present at very low levels, whereas putrescine, cadaverine, and tryptamine were the dominant biogenic amines and increased in concentration during ripening. Ethylamine, tryptamine, phenylethylamine, and cystamine reached maxima by 30 days of ripening and decreased thereafter. Significant correlations between amino acid precursors and corresponding biogenic amines, as well as between biogenic amines and microbial viable numbers, were observed.     

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.     

Monitoring of biogenic amines in cheeses manufactured at small-scale farms and in fermented dairy products in the Czech Republic

The aim of the study was the monitoring of six biogenic amines (histamine, tyramine, phenylethylamine, tryptamine, putrescine, and cadaverine) and two polyamines (spermidine and spermine) in 112 samples of dairy products purchased in the Czech Republic, namely in 55 cheeses made in small-scale farms and in 57 fermented dairy products. The products were tested at the end of their shelf-life period. Neither tryptamine nor phenylethylamine was detected in the monitored samples; histamine was found only in four cheese samples containing up to 25 mg/kg. The contents of spermine and spermidine were low and did not exceed the values of 35 mg/kg. Significant amounts of tyramine, putrescine, and cadaverine occurred especially in cheeses produced from ewe’s milk or in long-term ripened cheeses. In about 10% of the tested cheeses, the total concentration of all the monitored biogenic amines and polyamines exceeded the level of 200 mg/kg, which can be considered toxicologically significant. In fermented dairy products, the tested biogenic amines occurred in relatively low amounts (generally up to 30 mg/kg) that are regarded safe for the consumer’s health.     

Profiles of Organic Acid and Volatile Compounds in Acid-Type Cheeses Containing Herbs and Spices (Surk Cheese)

A study was conducted to evaluate the basic chemical composition, organic acids, volatile compound profiles, and overall acceptability of Surk cheese (acid cheese). The organic acids were determined by reverse phase high performance liqued chromatography method, and volatile compounds were analyzed by static headspace/gas chromatography/mass spectrometry technique. A total of 134 volatile compounds, including 42 esters, 40 terpenes, 15 alcohos, 11 free fatty acids, 6 ketones, 5 aldehydes, 4 alkenes, 4 phenyl propanoids, 3 phenolics, and 4 other compounds, were identified in the Surk cheeses. The main compounds were found to be carvacrol, γ-terpinene, p-cymene, hexanoic acid, octanoic acid, decanoic acid, butanoic acid, and eugenol. The mean total organic acid content of the Surk cheese was 1.71 g/100 g. The main organic acid in the Surk cheese was lactic acid (1067 mg/100 g), followed by acetic, propionic, oxalic, formic, citric, pyruvic, orotic, hippuric, and uric acids.     

Effect of temperature on evolution of free amino acid and biogenic amine contents during storage of Azeitão cheese

A study on the evolution of free amino acids and biogenic amines in Azeitão cheese during 4 weeks at different temperatures of storage (4 and 25°C) was performed. Free amino acids and biogenic amines were determined by RP-HPLC with visible detection, following extraction from the cheese and derivatization with dabsyl chloride. The method presented a linear relation between peak area and concentration from 2–200 mg/l. The detection limit value was less than 1.5 mg/l. The average repeatability was less than 4%. The major free amino acids were proline, valine, isoleucine and leucine and the major amines were tyramine, cadaverine and histamine. Room temperature (25°C) promoted a significant increase of the contents of valine, leucine, tyramine and putrescine, expressed as g/kg of dry matter. These two free amino acids and two biogenic amines may serve as indicators of temperatures changes in ripened cheese.     

Determination of Biogenic Amines in Herby Cheese

Herby cheese (Otlu peynir) is widely produced and consumed in eastern parts of Turkey, and is generally made from sheep milk. The biogenic amines and organic acids content, microbiological, and chemical properties of Herby cheese were determined on 30 samples obtained from retail markets in East Anatolia Region, Turkey. The most important biogenic amines were tyramine (range 18.0–1125.5 mg kg^?1), followed by cadaverine varied from not detected to 1844.5 mg kg^?1. Histamine content generally was found higher than 100 mg kg^?1. The concentration of amines in some cheeses was much higher than the toxic dose limits. Generally, total amine content was found high in samples that had high nitrogen fraction and organic acids value. Levels of biogenic amines were significantly correlated with the organic acids of Herby cheese. The basic organic acid was found the lactic acid ranging from 585.4 to 26480.3 mg kg^?1, followed by butyric acid varying between 314.8 and 7329.6 mg kg^?1 values. Lipolysis and nitrogen fraction were determined high in ripened Herby cheese samples. Coliform counts of samples showed lower than 100 cfu g^?1 in the samples. Significant (p < 0.01) positive correlation was observed between acetic acid and phenylethylamine, putrescine, cadaverin, histamine, and tyramine.     

The effect of different packaging methods on the formation of biogenic amines and organic acids in Kashar cheese

The effects of packaging methods (nonvacuum and vacuum) on biogenic amines (cadaverine, putrescine, tyramine, tryptamine, phenylethylamine, and histamine) and organic acids (citric, lactic, formic, acetic, propionic, and butyric) during storage for 180 d at 4°C were investigated in Kashar cheese. Dry matter, titratable acidity, total nitrogen, water-soluble nitrogen, trichloroacetic acid-soluble nitrogen, phosphotungstic acid-soluble nitrogen, free amino group (proteolysis), pH, fat, and acid degree value were also determined. Storage period had a significant effect on all of the biogenic amines. When compared with vacuum packaging, nonvacuum packaging resulted in no large differences among the amounts of biogenic amines. Vacuum-packaged cheeses had more lactic, formic, acetic, and butyric acids than did cheeses packaged without vacuum. Water-soluble nitrogen, trichloroacetic acid-soluble nitrogen, phosphotungstic acid-soluble nitrogen, proteolysis, pH, and acid degree values of the cheese samples increased continuously until the end of the ripening in all the samples. No significant change was observed in total nitrogen, dry matter, or fat content within the ripening period, whereas titratable acidity values changed significantly in vacuum-packaged cheese and decreased slightly in the non-vacuum-packaged cheeses. The results of this study showed that storage period and packaging method had significant effects on the quality of Kashar 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).