Reducing Biogenic-Amine-Producing Bacteria, Decarboxylase Activity, and Biogenic Amines in Raw Milk Cheese by High-Pressure Treatments

Javier Calzada; Ana del Olmo; Antonia Picón; Pilar Gaya; Manuel Nuñez

doi:10.1128/AEM.03368-12

. 2013 Feb;79(4):1277–1283. doi: 10.1128/AEM.03368-12

Reducing Biogenic-Amine-Producing Bacteria, Decarboxylase Activity, and Biogenic Amines in Raw Milk Cheese by High-Pressure Treatments

Javier Calzada ¹, Ana del Olmo ¹, Antonia Picón ¹, Pilar Gaya ¹, Manuel Nuñez ^1,^✉

PMCID: PMC3568586 PMID: 23241980

Abstract

Biogenic amines may reach concentrations of public health concern in some cheeses. To minimize biogenic amine buildup in raw milk cheese, high-pressure treatments of 400 or 600 MPa for 5 min were applied on days 21 and 35 of ripening. On day 60, counts of lactic acid bacteria, enterococci, and lactobacilli were 1 to 2 log units lower in cheeses treated at 400 MPa and 4 to 6 log units lower in cheeses treated at 600 MPa than in control cheese. At that time, aminopeptidase activity was 16 to 75% lower in cheeses treated at 400 MPa and 56 to 81% lower in cheeses treated at 600 MPa than in control cheese, while the total free amino acid concentration was 35 to 53% higher in cheeses treated at 400 MPa and 3 to 15% higher in cheeses treated at 600 MPa, and decarboxylase activity was 86 to 96% lower in cheeses treated at 400 MPa and 93 to 100% lower in cheeses treated at 600 MPa. Tyramine, putrescine, and cadaverine were the most abundant amines in control cheese. The total biogenic amine concentration on day 60, which reached a maximum of 1.089 mg/g dry matter in control cheese, was 27 to 33% lower in cheeses treated at 400 MPa and 40 to 65% lower in cheeses treated at 600 MPa. On day 240, total biogenic amines attained a concentration of 3.690 mg/g dry matter in control cheese and contents 11 to 45% lower in cheeses treated at 400 MPa and 73 to 76% lower in cheeses treated at 600 MPa. Over 80% of the histidine and 95% of the tyrosine had been converted into histamine and tyramine in control cheese by day 60. Substrate depletion played an important role in the rate of biogenic amine buildup, becoming a limiting factor in the case of some amino acids.

INTRODUCTION

Biogenic amines (BA) are low-molecular-weight organic bases showing biological activity (1). Monoamines, diamines, and polyamines consist of an aliphatic, aromatic, or heterocyclic structure with one, two, or more attached reactive amino groups, respectively. The main monoamines are tyramine, a potent vasoconstrictor with an effect on healthy individuals usually limited to headache or migraine (2), and histamine, also vasoactive, which may cause urticaria, hypotension, headache, flushing, and abdominal cramps (3). The diamines putrescine and cadaverine can react with nitrite to form carcinogenic nitrosamines (4). Polyamines and diamines may be converted into stable carcinogenic N-nitroso compounds and enhance the growth of chemically induced aberrant crypt foci in the intestine (5). Accumulation of BA in cheese and other foods is therefore a matter of public health concern.

The sensitivity of individuals to BA varies considerably. Concentrations of histamine above 500 to 1,000 mg/kg of food are regarded as potentially dangerous for human health (6), but cheeses with lower histamine contents have been involved in outbreaks (7). Alcohol and other potentiating factors may increase the effect of biogenic amines. For this reason, acceptable BA levels for foods have not been established as a general rule. Only an upper limit for histamine, 100 mg/kg, has been set for certain fish and fish products. Threshold values of 100 to 800 mg/kg for tyramine and 30 mg/kg for phenylethylamine have been suggested as the toxic dose (6).

During cheese manufacture and ripening, lactic starter cultures, together with other microorganisms, milk enzymes, and coagulant enzymes, degrade milk proteins into peptides, which are further hydrolyzed to free amino acids (FAA). Bacterial decarboxylases are responsible for the conversion of precursor amino acids into monoamines and diamines (8), while polyamines can also be formed by “deureation,” an alternative metabolic pathway (9). Tyramine, phenylethylamine, histamine, tryptamine, cadaverine, and putrescine are formed through the decarboxylation of tyrosine, phenylalanine, histidine, tryptophan, lysine, and ornithine or arginine (via agmatine), respectively. Enterococci and heterofermentative lactobacilli have been considered the main tyramine and histamine formers, respectively, but other lactic acid bacteria (LAB) and some Gram-negative bacteria may also be involved in BA formation in cheese (10, 11).

The main source of decarboxylase-positive bacteria in cheese is raw milk. Bacterial reduction procedures for milk, such as bactofugation, pasteurization, pressurization, or high-pressure homogenization (12–15), may diminish the levels of decarboxylase-positive bacteria and BA in cheese. Also, bacteriocinogenic strains of LAB have been shown to inhibit decarboxylase-positive bacteria, thus hindering BA formation (16). Even irradiation has been investigated to control BA buildup in cheese, decreasing BA contents with respect to a nonirradiated control but showing a deleterious effect on sensory characteristics (17). High-pressure (HP) treatments have been successfully applied to inactivate microbial contaminants in raw milk cheese (18, 19). To impede BA formation, HP treatments have been assayed only on pasteurized goat milk cheese, but the low BA concentration in control cheese did not permit an evaluation of the efficacy of the procedure (20).

In spite of these and other works, there are aspects of BA formation in cheese by decarboxylase-positive bacteria and its control by HP treatments which need to be elucidated. To our knowledge, the fate of bacterial decarboxylases during ripening and the limiting conditions of substrate depletion or availability for decarboxylation reactions have not been investigated. Also, although the inactivation of glycolytic and proteolytic enzymes of milk- and cheese-borne bacteria by HP treatments has been studied, there is no information on the effect of high pressure on amino acid decarboxylases of bacterial origin in cheese. In the present work, the buildup of BA in raw milk cheeses, pressurized or untreated, throughout their ripening and refrigerated storage periods was investigated with regard to the populations of bacterial groups potentially able to form BA, the activity of decarboxylating enzymes present in cheese, and the concentration of amino acids as the substrates required for BA formation.

MATERIALS AND METHODS

Cheeses and high-pressure treatments.

Raw ewe milk (mean total viable counts, 4.78 log₁₀ CFU/ml) was used for the manufacture of Casar cheese in duplicate trials, carried out on consecutive days. Milk (400 liters) with no lactic cultures added was coagulated at 30°C for 60 min with cardoon extract. Curd was cut into 1-cm cubes, held at 30°C for 15 min, and distributed into cylindrical molds. Cheeses, 13 cm in diameter and 6 cm high, were pressed for 3 h and salted by rubbing dry salt onto all the surfaces. They were ripened at 8°C and in 92% relative humidity (RH) until day 60 and afterwards held at 4°C until day 240.

Cheeses from each trial were pressurized for 5 min at 400 or 600 MPa, after 3 or 5 weeks of ripening, and coded as cheeses 400W3, 600W3, 400W5, and 600W5. Before high-pressure (HP) treatment, cheeses were vacuum packaged in CN300 bags (Cryovac Grace S.A., Barcelona, Spain). A 120-liter-capacity isostatic press (NC Hyperbaric, Burgos, Spain) was used for HP treatments. Come-up times to reach 400 and 600 MPa were 1.85 and 2.83 min, respectively, and depressurization times were 7 and 8 s. The temperature of the water used as transmitting fluid remained below 14°C during the process. After HP treatments, cheeses were unpackaged, and ripening proceeded under the same conditions as the control cheese.

Microbiological methods.

Representative cheese samples (10 g) were homogenized with 90 ml of a sterile 2% (wt/vol) sodium citrate solution at 45°C in a Colworth Stomacher 400 (A. J. Seward Ltd., London, United Kingdom). Decimal dilutions of samples were prepared in a sterile 0.1% peptone solution. Total viable counts and counts of LAB, enterococci, and lactobacilli were determined in duplicate on plates of plate count agar (Biolife, Milano, Italy) incubated for 48 h at 30°C, De Man-Rogosa-Sharpe (MRS) agar (acidified at pH 5.7 with acetic acid; Biolife) incubated for 48 h at 30°C, Kenner Fecal (KF) Streptococcus agar (Oxoid, Basingstoke, United Kingdom) incubated for 48 h at 37°C, and Rogosa agar (acidified at pH 5.4 with acetic acid; Biolife) incubated anaerobically for 48 h at 37°C, respectively. Counts of Micrococcaceae were determined in duplicate on plates of mannitol salt agar (MSA; Oxoid) incubated for 72 h at 30°C, counts of coagulase-positive staphylococci were determined on Baird-Parker agar (Oxoid) with rabbit plasma fibrinogen (RPF) supplement II (Biolife) incubated at 37°C for 48 h, counts of Gram-negative bacteria were determined on MacConkey agar (Biolife) incubated for 24 h at 30°C, and counts of coliforms were determined on violet red bile agar (VRBA; Oxoid) incubated for 24 h at 37°C.

Chemical analyses.

BA and FAA were simultaneously extracted from duplicate samples according to procedures described previously by Krause et al. (12). Quantitative analysis of BA, after derivatization with dabsyl chloride, was carried out by reverse-phase high-pressure liquid chromatography (RP-HPLC), using a System Gold HPLC apparatus (Beckman Coulter, Palo Alto, CA) equipped with a Nova-pack C₁₈ column (Waters, Milford, MA). A standard mixture of BA (Sigma-Aldrich, Alcobendas, Spain) was used for their identification and quantification. Analysis of FAA was carried out by RP-HPLC after derivatization with Waters AccQ Fluor reagent, using a Waters AccQ Tag column. Concentrations of BA and FAA were expressed as mg per g of dry matter (DM). Cheese DM was determined in triplicate, after cheese grinding with sand, by drying to a constant weight in an oven at 102°C. Cheese pH was measured in triplicate directly by means of a Crison penetration electrode (model 52-3,2; Crison Instruments S.A., Barcelona, Spain) coupled to a Crison GPL 22 pH meter.

Enzymatic determinations.

Aminopeptidase activity was determined using an extract obtained by homogenizing 10 g of cheese with 20 ml of 100 mM sodium phosphate buffer (pH 7) for 1 min in an Ultra-Turrax T-10 blender (IKA) at high speed on ice, followed by centrifugation (10,000 × g for 30 min at 4°C) and filtering through Whatman no. 2 paper. Activity on duplicate samples was measured with lysine p-nitroanilide (Lys p-NA) and leucine p-nitroanilide (Leu p-NA) as substrates and expressed as nmol of p-nitroaniline produced per min per g of cheese DM.

For the determination of tyrosine decarboxylase (TDC) activity in cheese, a standard curve was obtained by adding up to 32 mAU/ml of the l-TDC apoenzyme (Sigma, Alcobendas, Spain). (One arbitrary unit [AU] of tyrosine decarboxylase liberates 1.0 μmol of CO₂ from tyrosine per min at pH 5.5 and 37°C.) Methods described previously by Børresen et al. (21), with some modifications, were followed. Briefly, each reaction was initiated by adding different volumes of l-TDC solution (0.625 mg/ml apoenzyme stock solution in 0.5 M acetate buffer [pH 5.5]) in a total volume of 2.2 ml acetate buffer (0.5 M, pH 5.5) in the presence of 1.5 mM l-tyrosine (Sigma) and 0.15 mM pyridoxal-5′-phosphate (Sigma). The reaction mixture was incubated at 37°C for 24 h and stopped by adding 0.55 ml of 1 M perchloric acid to the mixture. After centrifugation (10,000 × g for 15 min at 25°C), tyramine was quantified by HPLC, as described above, and tyramine concentrations were plotted against concentrations of the added TDC. To determine TDC concentrations in cheese, 10 g of cheese was mixed with 20 ml of a 2% sodium citrate solution, homogenized for 1 min in an Ultra-Turrax T-10 blender (IKA) at high speed on ice, and centrifuged at 10,000 × g for 30 min at 4°C. The supernatant was recovered and dialyzed against sterile double-distilled water for 24 h at 5°C in a dialysis tube with a nominal molecular mass cutoff of 10 kDa (Medicell International Ltd., London, United Kingdom). The dialysate was centrifuged at 10,000 × g for 20 min at 5°C, and the supernatant was stored at −20°C until analysis. TDC in cheese was assayed on 0.3 ml of this supernatant as described above, without the addition of the apoenzyme. Reaction mixtures were incubated at 37°C, and the tyramine concentration was determined by HPLC after 0 h and 24 h of incubation. TDC activity in cheeses was calculated from the increase in tyramine concentrations after 24 h by means of the TDC standard curve.

Statistical treatment.

Analysis of variance with HP treatment (four treatments and control) and cheese age as the main effects was performed on the analytical variables by means of the SPSS Win 14.0 program. Calculation of correlations and comparison of means by Tukey's test, with the significance assigned at a P value of <0.05, were carried out by using the same program.

RESULTS

Cheese microbiota.

Levels of total viable counts and LAB were over 7.5 log₁₀ CFU/g on day 1 (Fig. 1) and had increased to almost 9.5 log₁₀ CFU/g by day 21 (data not shown). HP treatments brought about significant (P < 0.05) decreases in counts of all microbial groups, which were particularly pronounced at 600 MPa. Immediately after pressurization, total viable counts were 0.88 to 1.33 log units lower and LAB counts were 1.32 to 1.64 log units lower in cheeses treated at 400 MPa than in control cheese, while the decreases in cheeses treated at 600 MPa were 3.99 to 4.43 log units for total viable counts and 6.03 to 6.51 log units for LAB counts. Afterwards, a slight recovery of LAB was recorded for cheeses treated with 600 MPa, with counts 4.14 to 5.17 log units lower in cheeses treated at 600 MPa than in control cheese on day 60. Similar patterns were found for counts of enterococci and lactobacilli. Micrococcaceae were more baroresistant than LAB, with decreases immediately after treatment of 1.17 to 1.44 log units in cheeses treated at 400 MPa and 2.78 to 3.89 log units in cheeses treated at 600 MPa. However, on day 60, coagulase-positive staphylococci were below the detection level in all pressurized cheeses. At that time, counts of Gram-negative bacteria and coliforms were more than 3 log units lower in 400W3 cheese than in control cheese and had declined below the detection level in the rest of the HP-treated cheeses. Differences in microbial counts between pressurized and control cheeses persisted throughout refrigerated storage, until day 240. Contrarily, minor differences were recorded for pH values, which ranged from pH 5.33 to 5.42 on day 60 and from pH 5.56 to 5.73 on day 240, and in dry matter content, which ranged from 50.41% to 53.40% on day 60 and from 54.51% to 57.81% on day 240 (data not shown).

Inline graphic — Bacterial counts (log CFU/g) during ripening and storage in control (■) cheeses and cheeses submitted to treatment at 400 MPa (□) or 600 MPa (▧) at 3 weeks and at 400 MPa (▤) or 600 MPa () at 5 weeks. Bars indicate standard errors of the means.

Aminopeptidase activity.

Cheese pressurization on day 21 reduced significantly (P < 0.05) the aminopeptidase activity recorded with Leu p-NA as the substrate, by 35% in 400W3 cheese and by 48% in 600W3 cheese, while the respective decreases were 26% and 37% with Lys p-NA as the substrate (data not shown). On day 35, the decreases in activity caused by pressurization were 21% in 400W5 cheese and 30% in 600W5 cheese with Leu p-NA as the substrate and 24% in 400W5 cheese and 28% in 600W5 cheese with Lys p-NA as the substrate (data not shown). Aminopeptidase activity increased in all cheeses during ripening, attaining its maximum values on day 60 (Table 1). Thereafter, a gradual decline of aminopeptidase activity was recorded, with few significant differences between cheeses at the last stages of refrigerated storage.

Table 1.

Aminopeptidase activity in control and pressurized cheeses during ripening and storage^a

Substrate	Cheese	Mean aminopeptidase activity (nmol p-NA/min per g cheese DM) ± SEM
Substrate	Cheese	Day 60	Day 120	Day 180	Day 240
Leu p-NA	Control	80.67 ± 11.11^D	30.95 ± 4.64^B	9.41 ± 0.28^AB	6.42 ± 0.60^A
	400W3	67.45 ± 4.96^CD	25.25 ± 0.83^B	9.74 ± 1.34^B	5.47 ± 0.31^A
	600W3	19.05 ± 1.32^A	9.70 ± 0.93^A	5.88 ± 0.87^A	4.45 ± 1.01^A
	400W5	41.27 ± 3.26^BC	27.58 ± 3.81^B	8.07 ± 0.40^AB	4.43 ± 0.43^A
	600W5	35.17 ± 3.15^AB	21.81 ± 1.59^B	7.75 ± 0.12^AB	5.78 ± 0.90^A
Lys p-NA	Control	38.10 ± 8.32^C	11.34 ± 1.74^B	7.07 ± 0.09^A	4.67 ± 0.13^AB
	400W3	25.69 ± 1.65^B	9.04 ± 0.46^B	6.76 ± 0.50^A	5.04 ± 0.83^B
	600W3	7.22 ± 0.70^A	4.15 ± 0.50^A	5.57 ± 1.19^A	2.81 ± 0.37^A
	400W5	9.42 ± 2.64^A	8.91 ± 1.02^B	6.73 ± 0.38^A	4.28 ± 0.49^AB
	600W5	14.15 ± 0.21^AB	8.24 ± 0.69^AB	6.64 ± 0.95^A	3.82 ± 0.14^AB

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On day 1, aminopeptidase activities in control cheese with Leu p-anilide and Lys p-nitroanilide as substrates were 60.93 ± 1.45 and 44.67 ± 0.83 nmol p-nitroaniline/min per g cheese DM, respectively. Mean values in the same column for the same substrate bearing the same superscript did not differ significantly (P > 0.05).

Free amino acids.

Accumulation of FAA during ripening proceeded at different rates in HP-treated and control cheeses from the 0.24 mg/g DM found in control cheese on day 1 (data not shown). Pressurization of cheese at 400 MPa, but not at 600 MPa, enhanced significantly (P < 0.05) the formation of FAA during ripening (Fig. 2). Leucine, valine, glutamic acid, and lysine were the most abundant FAA in control cheese on day 60. At the end of refrigerated storage, on day 240, concentrations of FAA totaled 23.31 mg/g DM in control cheese, 25.40 to 26.05 in cheeses treated at 400 MPa, and only 16.47 to 17.47 mg/g DM in cheeses treated at 600 MPa (Fig. 2). At that time, leucine, valine, lysine, and phenylalanine were the most abundant FAA in control cheese.

Fig 2 — Selected free amino acids (mg/g DM) during ripening and storage in control (■) cheeses and cheeses submitted to treatment at 400 MPa (□) or 600 MPa (▧) at 3 weeks and at 400 MPa (▤) or 600 MPa () at 5 weeks. Bars indicate standard errors of the means.

Biogenic amines.

None of the BA was detected in control cheese on day 1. However, by day 21, the concentration of total BA in control cheese had reached 0.371 mg/g DM, and by day 35, it reached 0.740 mg/g DM (data not shown). On day 60, the most abundant BA in control cheese were tyramine, putrescine, cadaverine, and histamine (Fig. 3). The concentration of total BA increased up to 1.089 mg/g DM in control cheese on day 60, while it remained at 0.728 to 0.794 mg/g DM in cheeses treated at 400 MPa and 0.377 to 0.656 mg/g DM in cheeses treated at 600 MPa. Formation of BA progressed during refrigerated storage, and on day 240, the concentration of total BA reached 3.690 mg/g DM in control cheese, 2.022 to 3.276 mg/g DM in cheeses treated with 400 MPa, and only 0.896 to 1.011 mg/g DM in cheeses treated with 600 MPa. The most abundant BA in control cheese on day 240 were tyramine, putrescine, cadaverine, and tryptamine (Fig. 3). Spermidine and spermine were not found in any of the cheeses at any stage of ripening or refrigerated storage. Concentrations of all individual BA in control cheese correlated significantly (P < 0.05) with ripening time. Values of r² ranged from 0.891 for histamine to 0.984 for tyramine when concentrations of individual BA in control cheese from day 1 to day 240 were plotted against time.

Fig 3 — Biogenic amines (mg/g DM) during ripening and storage in control (■) cheeses and cheeses submitted to 400 MPa (□) or 600 MPa (▧) at 3 weeks and at 400 MPa (▤) or 600 MPa () at 5 weeks. Bars indicate standard errors of the means.

Tyramine concentrations in control and experimental cheeses on days 60, 120, 180, and 240 correlated significantly (P < 0.05) with log counts of enterococci in the same cheeses. Some significant correlations were also found between tyramine concentrations and log counts of LAB or lactobacilli although with lower r² values than for enterococci. Correlations of histamine concentrations on days 120, 180, and 240 with log counts of lactobacilli in the same cheeses were significant (P < 0.05). Significant correlations between histamine concentrations and log counts of LAB or enterococci were occasionally found, with lower r² values than for lactobacilli. Log counts of Micrococcaceae did not correlate significantly with tyramine or histamine concentrations at any of the sampling times. Coagulase-positive staphylococci, Gram-negative bacteria, and coliforms were below detection levels in pressurized cheeses, and their correlations with BA could not be calculated. The correlations of histamine contents to histidine contents (r² = 0.128) and of tyramine concentrations to tyrosine concentrations (r² = 0.290) in all cheeses, at all times of ripening and storage, were nonsignificant.

Substrate depletion.

The proportion of FAA decarboxylated to the respective BA depended on the amino acid and on the HP treatment. Decarboxylated phenylalanine in control cheese up to day 60 represented 27.82% of the FAA formed during the first 60 days of ripening, while in pressurized cheeses, it ranged from 1.86% to 12.53% of the total free phenylalanine formed (Table 2). Similar proportions were recorded for other FAA such as lysine and tryptophan (data not shown). In the case of histidine, the decarboxylated FAA in control cheese up to day 60 was 82.82% of the free histidine formed until that time, and in pressurized cheeses, it accounted for 14.84% to 62.83% of the free histidine formed. Decarboxylated tyrosine in control cheese up to day 60 reached 96.01% of the free tyrosine formed during the first 2 months, while in pressurized cheeses, it ranged from 68.98% to 81.20%. On day 180, the differences in the availability of FAA between control and pressurized cheeses persisted (Table 2).

Table 2.

Decarboxylation^a of phenylalanine, histidine, and tyrosine to the respective biogenic amine in cheese during ripening and storage

Cheese	Decarboxylation (%)
	Phenylalanine		Histidine		Tyrosine
	Day 60	Day 180	Day 60	Day 180	Day 60	Day 180
Control	27.82	15.41	82.82	53.44	96.01	72.99
400W3	4.16	13.20	62.83	78.05	81.20	73.75
600W3	1.86	16.13	14.84	5.76	68.98	61.59
400W5	10.85	7.26	61.04	70.81	80.15	63.86
600W5	12.53	7.04	36.21	21.25	69.39	57.69

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Expressed as the percentage of decarboxylated FAA of the total amount of FAA formed, which was calculated as the sum of the present amount of FAA plus the maximum (present or past) amount of the respective biogenic amine plus the equimolecular CO₂ resulting from decarboxylation.

Decarboxylase activity.

The standard curve for the determination of tyrosine decarboxylase activity fit a linear model within the concentration range of 0 to 0.2 mAU of TDC/ml. It followed the equation y = 72.185x + 0.22219, in which y was μg tyramine formed/ml and x was mAU TDC/ml, with an r² value of 0.999. By means of this standard curve, TDC activity in control and pressurized cheeses was determined. On day 60, TDC activity was significantly (P < 0.05) higher in control cheese than in pressurized cheeses (Table 3). Activity increased during refrigerated storage, by approximately 4-fold in control cheese and 6-fold in cheeses treated at 400 MPa from day 60 to day 180, while a minor increase was observed for cheeses treated at 600 MPa. Differences between the TDC activity of pressurized and control cheeses persisted on day 180 (Table 3).

Table 3.

Tyrosine decarboxylase activity in control and pressurized cheeses during ripening and storage

Cheese	Mean tyrosine decarboxylase activity (mAU/g cheese DM) ± SEM^a
Cheese	Day 60	Day 180
Control	0.738 ± 0.027^B	3.008 ± 0.162^C
400W3	0.031 ± 0.023^A	0.183 ± 0.071^AB
600W3	0.052 ± 0.031^A	0.080 ± 0.053^A
400W5	0.103 ± 0.028^A	0.689 ± 0.221^B
600W5	ND	0.061 ± 0.044^A

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Means in the same column bearing the same superscript letter did not differ significantly (P > 0.05). On day 1, TDC activity of control cheese was below the detection level. ND, not determined (below the detection level).

DISCUSSION

Prerequisites for BA formation are availability of FAA, presence of decarboxylase-positive microorganisms, and environmental conditions allowing decarboxylase activity (8, 22). In the present work, HP treatments influenced the first two prerequisites, while the environmental conditions (pH value and dry matter content) were not substantially affected. pH values for control and pressurized cheeses appeared to be favorable for tyramine formation by LAB, while they might slightly hinder its formation by Enterobacteriaceae, according to results reported previously for the decarboxylase activity of various bacterial sources at different pH values (23).

According to our results, the overall availability of FAA did not appear to be a limiting factor for BA formation. On day 60, the concentration of total FAA in control cheese was 4.82 mg/g DM versus 1.09 mg/g DM of total BA, while on day 240, the respective concentrations were 23.31 and 3.69 mg/g DM. These concentrations of FAA and BA exceed considerably the respective levels previously reported for other cheese varieties (13, 24, 25). On day 60, total FAA attained higher concentrations in cheeses treated at 400 MPa than in control cheese, a result in apparent contradiction with the lower aminopeptidase activity recorded for the former cheeses. The enhanced proteolysis of HP-treated cheeses observed in the present work can be explained by the conformational changes in the caseins of pressurized cheeses, revealed by confocal laser scanning microscopy (26), which facilitate the access of enzymes to their substrates. Regarding some particular FAA, the phenylalanine concentration did not seem to restrain phenylethylamine formation, since less than 30% of the free phenylalanine formed in control cheese and less than 15% of that formed in pressurized cheeses had been decarboxylated on day 60, while on day 180, this proportion was below 20% in all cheeses (Table 2). Histidine content might limit histamine formation to a certain extent, particularly in control cheese, in which more than 80% of the free histidine had been decarboxylated by day 60, and in cheeses treated with 400 MPa, with over 60% decarboxylated histidine, while in cheeses treated with 600 MPa, the proportion of decarboxylated histidine remained below 40%. The availability of free tyrosine appeared to be strongly limiting for tyramine formation, with more than 95% of the free tyrosine being decarboxylated by day 60 in control cheese, while in cheeses pressurized at 400 and 600 MPa, more than 80% and almost 70% of tyrosine, respectively, had been decarboxylated. The remarkably higher tyrosine decarboxylase activity recorded for control cheese (Table 3), in which the enzyme had not been inactivated since high pressure had not been applied, than in pressurized cheeses, in particular when treated at 600 MPa, appears to be the main cause for tyrosine becoming practically exhausted in the former cheese.

Decarboxylase-positive microorganisms in cheese were negatively influenced by HP treatments, as expected. Pressurization reduced the populations of all the analyzed microbial groups, with a more marked effect on Gram-negative bacteria. Counts of Enterococcus, a genus known to harbor abundant decarboxylase-positive strains (11), suffered important declines in pressurized cheeses. A similar result was obtained for Lactobacillus, also rich in strains with decarboxylating activity (27). Decreases in the levels of all microbial groups were significantly more pronounced after treatments at 600 MPa than at 400 MPa, in agreement with results from previous studies regarding the effect of pressurization on the microbiota of raw milk cheeses (18, 19, 28).

Tyramine concentrations in control and experimental cheeses correlated significantly with the respective log counts of enterococci from day 60 onwards, and histamine concentrations correlated significantly with the respective log counts of lactobacilli from day 120 onwards. In spite of these correlations, the possible contribution of microorganisms from other genera to the formation of tyramine and histamine in the present work cannot be excluded. Strains of Lactobacillus, Lactococcus, Leuconostoc, Carnobacterium, and Enterobacteriaceae have been found to produce tyramine (23, 29), and strains of Enterococcus, Lactococcus, Oenococcus, Pediococcus, Streptococcus, and Enterobacteriaceae have been found to form histamine (30–32). On the other hand, bacterial strains belonging to genera frequently found in cheese, such as Lactobacillus, Micrococcus, and Pediococcus, have been reported to degrade tyramine and histamine, generally by means of monoamine oxidases and preferably under aerobic conditions (33). Also, the concentration of BA in a minicheese model (34) ripened for 4 months was approximately 95% lower when selected Lactobacillus casei strains capable of degrading tyramine and histamine in MRS broth were added to milk as adjunct cultures. This phenomenon was probably enhanced by the small size of the minicheeses, favorable for aeration. In the case of real-size cheeses, the low oxygen concentration present in their interior would probably preclude a significant contribution of oxidases to BA degradation. In another work (16), the use of a nisin-producing Lactococcus lactis strain as the main starter or two enterocin-producing Enterococcus faecalis strains as adjunct cultures inhibited a histamine-forming Lactobacillus buchneri strain and reduced histamine contents in 4-month-old cheese from 177 to 214 mg/kg to less than 10 mg/kg.

In the present work, pressurization had a negative effect on all microbial groups and on TDC activity. Levels of microbial groups decreased further during ripening and storage of all cheeses. On the contrary, TDC activity increased by approximately 4-fold from day 60 to day 180 in control cheese and, unexpectedly, even more in cheeses treated at 400 MPa, by approximately 6-fold. The increase of TDC activity in control cheese can be explained by the production of the enzyme by intact bacterial cells, followed by its release into the surrounding medium, favored by spontaneous cell lysis. In cheeses treated at 400 MPa, counts of bacterial groups after pressurization were lower than in control cheese, but the mild-pressure treatment would most probably enhance the lysis of LAB cells, as shown previously for lactococci (35), without inactivating the enzyme. The minor increase in TDC activity recorded for cheeses treated at 600 MPa might be due to a double effect of high pressure at this level, on the one hand promoting cell lysis, as in the case of cheeses treated at 400 MPa, and on the other hand inactivating the enzyme released into the medium.

In spite of the well-known need for sufficient amounts of FAA for BA formation, weak correlations between the concentrations of BA and the concentrations of their precursor FAA in control and pressurized cheeses of different ages were obtained. Pools of FAA and BA in cheese are subject to considerable variations during ripening. The FAA generated in cheese by microbial peptidases, besides being decarboxylated to BA, may be converted into a wide range of compounds through the catabolism processes initiated by aminotransferases, amino acid lyases, and deaminases (36). The BA formed in cheese through FAA decarboxylation may be degraded by oxidases and possibly by other enzymes of microbial origin. From a public health point of view, it must be taken into account that the increase in the concentration of FAA brought about by some of the manufacturing procedures designed to accelerate cheese ripening may enhance BA formation (37, 38), in particular in the presence of decarboxylase-positive microbiota.

According to the results obtained in the present work, it may be concluded that HP treatments were capable of reducing not only the population of potentially decarboxylating microbiota but also the level of decarboxylating enzymes and the concentrations of BA. Contrary to the results found for some FAA in control cheese, no substrate depletion occurred in pressurized cheeses. In spite of this fact, cheeses treated at 400 and 600 MPa showed total BA concentrations up to 45% and 76% lower, respectively, than those in control cheese.

ACKNOWLEDGMENTS

This work was supported by project AGL2009-07801 of the Spanish Ministry of Science and Innovation (MICINN). Javier Calzada was the recipient of a MICINN fellowship.

Footnotes

Published ahead of print 14 December 2012

REFERENCES

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24. Gaya P, Sánchez C, Fernández-García E, Nuñez M. 2005. Proteolysis during ripening of Manchego cheese made from raw or pasteurized ewes' milk. Seasonal variation. J. Dairy Res. 72:287–295 [DOI] [PubMed] [Google Scholar]
25. Mayer HK, Fiechter G, Fischer E. 2010. A new ultra-pressure liquid chromatography method for the determination of biogenic amines in cheese. J. Chromatogr. A 1217:3251–3257 [DOI] [PubMed] [Google Scholar]
26. O'Reilly CE, Kelly AL, Oliveira JC, Murphy PM, Auty MAE, Beresford TP. 2003. Effect of varying high-pressure treatment conditions on acceleration of ripening of cheddar cheese. Innov. Food Sci. Emerg. Technol. 4:277–284 [Google Scholar]
27. Joosten HMLJ, Northolt MD. 1989. Detection, growth and amine producing capacity of lactobacilli in cheese. Appl. Environ. Microbiol. 55:2356–2359 [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Rodríguez E, Arqués JL, Nuñez M, Gaya P, Medina M. 2005. Combined effect of high-pressure treatments and bacteriocin-producing lactic acid bacteria on the inactivation of Escherichia coli O157:H7 in raw milk cheese. Appl. Environ. Microbiol. 71:3399–3404 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. González de Llano D, Cuesta P, Rodríguez A. 1998. Biogenic amine production by wild lactococcal and leuconostoc strains. Lett. Appl. Microbiol. 26:270–274 [DOI] [PubMed] [Google Scholar]
30. Chaves-López C, De Angelis M, Martuscelli M, Serio A, Paparella A, Suzzi G. 2006. Characterization of the Enterobacteriaceae isolated from an artisanal Italian ewe's cheese (Pecorino Abruzzese). J. Appl. Microbiol. 101:353–360 [DOI] [PubMed] [Google Scholar]
31. Connil N, Le Breton Y, Dousset X, Auffray Y, Rincé A, Prévost H. 2002. Identification of the Enterococcus faecalis tyrosine decarboxylase operon involved in tyramine production. Appl. Environ. Microbiol. 68:3537–3544 [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Rossi F, Gardini F, Rizzotti L, La Gioia F, Tabanelli G, Torriani S. 2011. Quantitative analysis of histidine decarboxylase gene (hdcA) transcription and histamine production by Streptococcus thermophilus PRI60 under conditions relevant to cheese making. Appl. Environ. Microbiol. 77:2817–2822 [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Leuschner RG, Heidel M, Hammes WP. 1998. Histamine and tyramine degradation by food fermenting microorganisms. Int. J. Food Microbiol. 39:1–10 [DOI] [PubMed] [Google Scholar]
34. Herrero-Fresno A, Martínez N, Sánchez-Llana E, Díaz M, Fernández M, Martin MC, Ladero V, Alvarez MA. 2012. Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model. Int. J. Food Microbiol. 157:297–304 [DOI] [PubMed] [Google Scholar]
35. Malone AS, Shellhammer TH, Courtney PD. 2002. Effects of high pressure on the viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris. Appl. Environ. Microbiol. 68:4357–4363 [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Yvon M, Rijnen L. 2001. Cheese flavor formation by amino acid catabolism. Int. Dairy J. 11:185–201 [Google Scholar]
37. Fernández-García E, Tomillo J, Nuñez M. 1999. Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese. Int. J. Food Microbiol. 52:189–196 [DOI] [PubMed] [Google Scholar]
38. Leuschner RGK, Kurihara R, Hammes WP. 1998. Effect of enhanced proteolysis on formation of biogenic amines by lactobacilli during Gouda cheese ripening. Int. J. Food Microbiol. 44:15–20 [DOI] [PubMed] [Google Scholar]

[B1] 1. Silla-Santos MH. 1996. Biogenic amines: their importance in foods. Int. J. Food Microbiol. 29:213–231 [DOI] [PubMed] [Google Scholar]

[B2] 2. Til HP, Falke HE, Prinsen MK, Willems MI. 1997. Acute and subacute toxicity of tyramine, spermidine, spermine, putrescine and cadaverine in rats. Food Chem. Toxicol. 35:337–348 [DOI] [PubMed] [Google Scholar]

[B3] 3. Stratton JE, Hutkins RW, Taylor SL. 1991. Biogenic amines in cheese and other fermented foods. J. Food Prot. 54:460–470 [DOI] [PubMed] [Google Scholar]

[B4] 4. Hotchkiss JH, Scanlan RA, Libbey LM. 1977. Formation of bis(hydroxyalkyl)-n-nitrosamines as products of nitrosation of spermidine. J. Agric. Food Chem. 25:1183–1189 [DOI] [PubMed] [Google Scholar]

[B5] 5. Eliassen KA, Reistad R, Risøen U, Rønning HF. 2002. Dietary polyamines. Food Chem. 78:273–280 [Google Scholar]

[B6] 6. ten Brink B, Damink C, Joosten HMLJ, Huis in't Veld JHJ. 1990. Occurrence and formation of biologically-active amines in foods. Int. J. Food Microbiol. 11:73–84 [DOI] [PubMed] [Google Scholar]

[B7] 7. Taylor SL. 1986. Histamine food poisoning: toxicology and clinical aspects. Crit. Rev. Toxicol. 17:91–128 [DOI] [PubMed] [Google Scholar]

[B8] 8. Halász A, Baráth á, Simon-Sarkadi L, Holzapfel W. 1994. Biogenic amines and their production by microorganisms in food. Trends Food Sci. Technol. 5:42–49 [Google Scholar]

[B9] 9. Komprda P, Burdychová R, Dohnal V, Cwiková O, Sládková P. 2008. Some factors influencing biogenic amines and polyamines content in Dutch-type semi-hard cheese. Eur. Food Res. Technol. 227:29–36 [Google Scholar]

[B10] 10. Joosten HMLJ, Northolt MD. 1987. Conditions allowing the formation of biogenic amines in cheese. 2. Decarboxylase properties of some non-starter bacteria. Neth. Milk Dairy J. 41:259–280 [Google Scholar]

[B11] 11. Sarantinopoulos P, Andrighetto C, Georgalaki MD, Rea MC, Lombardi A, Cogan TM, Kalantzopoulos G, Tsakalidou E. 2001. Biochemical properties of enterococci relevant to their technological performance. Int. Dairy J. 11:621–647 [Google Scholar]

[B12] 12. Krause I, Bockhardt A, Klostenmeyer H. 1997. Characterization of cheese ripening by free amino acids and biogenic amines and influence of bactofugation and heat-treatment of milk. Lait 77:101–108 [Google Scholar]

[B13] 13. Lanciotti R, Patrignani F, Iucci L, Guerzoni ME, Suzzi G, Belletti N, Gardini F. 2007. Effects of milk pressure homogenization on biogenic amine accumulation during ripening of ovine and bovine Italian cheeses. Food Chem. 104:693–701 [Google Scholar]

[B14] 14. Novella-Rodríguez S, Veciana-Nogués MT, Trujillo-Mesa AJ, Vidal-Carou MC. 2002. Profile of biogenic amines in goat cheese made from pasteurized and pressurized milks. J. Food Sci. 67:2940–2944 [Google Scholar]

[B15] 15. Ordóñez AI, Ibañez FC, Torre P, Barcina Y. 1997. Formation of biogenic amines in Idiazábal ewe's-milk cheese: effect of ripening, pasteurization and starter. J. Food Prot. 60:1371–1375 [DOI] [PubMed] [Google Scholar]

[B16] 16. Joosten HMLJ, Nuñez M. 1996. Prevention of histamine formation in cheese by bacteriocin-producing lactic acid bacteria. Appl. Environ. Microbiol. 62:1178–1181 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. Rabie MA, Siliha HI, El-Saidy SM, El-Badawy AA, Malcata FX. 2011. Effect of γ-irradiation upon biogenic amine formation in blue cheese during storage. Int. Dairy J. 21:373–376 [Google Scholar]

[B18] 18. Arqués JL, Garde S, Gaya P, Medina M, Nuñez M. 2006. Inactivation of microbial contaminants in raw milk La Serena cheese by high pressure treatments. J. Dairy Sci. 89:888–891 [DOI] [PubMed] [Google Scholar]

[B19] 19. O'Reilly CE, O'Connor PM, Kelly AL, Beresford TP, Murphy PM. 2000. Use of hydrostatic pressure for inactivation of microbial contaminants in cheese. Appl. Environ. Microbiol. 66:4890–4896 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. Novella-Rodríguez S, Veciana-Nogués MT, Saldo J, Vidal-Carou MC. 2002. Effects of high hydrostatic pressure treatments on biogenic amine contents in goat cheeses during ripening. J. Agric. Food Chem. 50:7288–7292 [DOI] [PubMed] [Google Scholar]

[B21] 21. Børresen T, Klausen NK, Larsen LM, Sørensen H. 1989. Purification and characterization of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. Biochim. Biophys. Acta 993:108–115 [DOI] [PubMed] [Google Scholar]

[B22] 22. Linares D, Del Rio B, Ladero V, Martinez N, Fernandez M, Martin MC, Alvarez MA. 2012. Factors influencing biogenic amines accumulation in dairy products. Front. Microbiol. 3:180 doi:10.3389/fmicb.2012.00180 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23. Pircher A, Bauer F, Paulsen P. 2007. Formation of cadaverine, histamine, putrescine and tyramine by bacteria isolated from meat, fermented sausages and cheeses. Eur. Food Res. Technol. 226:225–231 [Google Scholar]

[B24] 24. Gaya P, Sánchez C, Fernández-García E, Nuñez M. 2005. Proteolysis during ripening of Manchego cheese made from raw or pasteurized ewes' milk. Seasonal variation. J. Dairy Res. 72:287–295 [DOI] [PubMed] [Google Scholar]

[B25] 25. Mayer HK, Fiechter G, Fischer E. 2010. A new ultra-pressure liquid chromatography method for the determination of biogenic amines in cheese. J. Chromatogr. A 1217:3251–3257 [DOI] [PubMed] [Google Scholar]

[B26] 26. O'Reilly CE, Kelly AL, Oliveira JC, Murphy PM, Auty MAE, Beresford TP. 2003. Effect of varying high-pressure treatment conditions on acceleration of ripening of cheddar cheese. Innov. Food Sci. Emerg. Technol. 4:277–284 [Google Scholar]

[B27] 27. Joosten HMLJ, Northolt MD. 1989. Detection, growth and amine producing capacity of lactobacilli in cheese. Appl. Environ. Microbiol. 55:2356–2359 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Rodríguez E, Arqués JL, Nuñez M, Gaya P, Medina M. 2005. Combined effect of high-pressure treatments and bacteriocin-producing lactic acid bacteria on the inactivation of Escherichia coli O157:H7 in raw milk cheese. Appl. Environ. Microbiol. 71:3399–3404 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. González de Llano D, Cuesta P, Rodríguez A. 1998. Biogenic amine production by wild lactococcal and leuconostoc strains. Lett. Appl. Microbiol. 26:270–274 [DOI] [PubMed] [Google Scholar]

[B30] 30. Chaves-López C, De Angelis M, Martuscelli M, Serio A, Paparella A, Suzzi G. 2006. Characterization of the Enterobacteriaceae isolated from an artisanal Italian ewe's cheese (Pecorino Abruzzese). J. Appl. Microbiol. 101:353–360 [DOI] [PubMed] [Google Scholar]

[B31] 31. Connil N, Le Breton Y, Dousset X, Auffray Y, Rincé A, Prévost H. 2002. Identification of the Enterococcus faecalis tyrosine decarboxylase operon involved in tyramine production. Appl. Environ. Microbiol. 68:3537–3544 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Rossi F, Gardini F, Rizzotti L, La Gioia F, Tabanelli G, Torriani S. 2011. Quantitative analysis of histidine decarboxylase gene (hdcA) transcription and histamine production by Streptococcus thermophilus PRI60 under conditions relevant to cheese making. Appl. Environ. Microbiol. 77:2817–2822 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Leuschner RG, Heidel M, Hammes WP. 1998. Histamine and tyramine degradation by food fermenting microorganisms. Int. J. Food Microbiol. 39:1–10 [DOI] [PubMed] [Google Scholar]

[B34] 34. Herrero-Fresno A, Martínez N, Sánchez-Llana E, Díaz M, Fernández M, Martin MC, Ladero V, Alvarez MA. 2012. Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model. Int. J. Food Microbiol. 157:297–304 [DOI] [PubMed] [Google Scholar]

[B35] 35. Malone AS, Shellhammer TH, Courtney PD. 2002. Effects of high pressure on the viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris. Appl. Environ. Microbiol. 68:4357–4363 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Yvon M, Rijnen L. 2001. Cheese flavor formation by amino acid catabolism. Int. Dairy J. 11:185–201 [Google Scholar]

[B37] 37. Fernández-García E, Tomillo J, Nuñez M. 1999. Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese. Int. J. Food Microbiol. 52:189–196 [DOI] [PubMed] [Google Scholar]

[B38] 38. Leuschner RGK, Kurihara R, Hammes WP. 1998. Effect of enhanced proteolysis on formation of biogenic amines by lactobacilli during Gouda cheese ripening. Int. J. Food Microbiol. 44:15–20 [DOI] [PubMed] [Google Scholar]

PERMALINK

Reducing Biogenic-Amine-Producing Bacteria, Decarboxylase Activity, and Biogenic Amines in Raw Milk Cheese by High-Pressure Treatments

Javier Calzada

Ana del Olmo

Antonia Picón

Pilar Gaya

Manuel Nuñez

Abstract

INTRODUCTION