Reduction of Cadaverine and Tyramine Formation by Proteolytic Enzymes in Model System

Abstract

Cadaverine and tyramine are biogenic amines that are commenly found in fermented foods. This study focused on the reduction of cadaverine and tyramine formation by a new method using proteolytic enzymes (trypsin and chymotrypsin). Cadaverine and tyramine were synthesized from lysine and tyrosine in the presence of their decarboxylase enzymes, respectively. In this study, trypsin and chymotrypsin were used separately or in combination to reduce formation of cadaverine and tyramine in model system. Tyramine synthesis was reduced by trypsin and chymotrypsin as 57.2 and 52.1%, respectively. Moreover, the reduction of cadaverine was 80.4 and 83.0% by trypsin and chymotrypsin, respectively. Maximum reduction values were obtained when trypsin and chymotrypsin were used together for the reduction of tyramine (59.1%) and cadaverine formation (84.8%). In model system, cadaverine and tyramine formation were (p < 0.05) significantly reduced. It was concluded that trypsin and chymotrpsin could be used to reduce tyramine and cadaverine formation.

Keywords:

• Biogenic amine reduction
• Trypsin
• Chymotrypsin
• Tyramine
• Cadaverine

INTRODUCTION

Biogenic amines (BA) are low molecular weight, organic basic nitrogenous compounds. They are synthesized in the presence of decarboxylases by removal of α-carboxyl group from a precursor amino acid. Enzymes in raw material or produced by microorganisms lead to formation of BA during ripening and storage of food.^[1,2] The presence of BA has been used as an indicator of quality and/or acceptability in some foods. Intake of foods containing high concentrations of certain BA causes a health hazard through the direct toxic effect of these compounds and their interaction with some medicaments.^[3] In the human body there is a detoxification system which degrades BA to physiologically less active forms. Diamine oxidase and monoamine oxidase are two enzymes that are responsible for detoxification. However, intake of high concentrations of BA causes deactivation of this detoxification system.^[4] In addition, some drugs reduce the activity of monoamine oxidase. In that case, this enzyme cannot act, leading to the absorption of BA by the human body and occurrence of toxic effects. Also, BA are considered as precursors of carcinogenic amines such as N-nitrosamines that are produced from nitrites and secondary amines.^[4,5]

BA are the most commonly studied quality and safety parameters in fermented products.^[6,7] Tyramine is mainly related to the activity of fermentative lactic acid bacteria while cadaverine is usually resulted of the action of non-fermentative strains.^[8] The presence of high cadaverine concentrations has been associated with poor quality meat used in the manufacturing of meat products and mainly, with high levels of microbial contamination.^[9] Among the microbial groups producing BA, Enterobacteriaceae seem to have an important role in cadaverine production, as high Enterobacteriaceae counts are directly related to high cadaverine concentrations.^[10]

In fact, presence of cadaverine indicates low quality of raw materials in which high proliferation of microorganisms occurs.^[10] Cadaverine is not considered toxic individually; however, it can enhance the effect of histamine and tyramine by interacting with the amino oxidases and interfering with the detoxifying mechanism.^[11] Numerous efforts have been made to reduce or to prevent formation of BA in food. The prevention of BA formation can be achieved by using temperature control,^[12] high-quality raw material,^[13] good manufacturing practice,^[14] use of enzymes to oxidize amine,^[15] use of microbial modeling to assess favorable conditions,^[16,17] packaging techniques,^[18] high hydrostatic pressure (HHP),^[19] irradiation,^[20] and food additives.^[21] HHP and irradiation processes are used easily in laboratory conditions, however, could not be used extensively in the food industry due to the high cost of the processes.^[22] Additives could show inhibitory effect on BA formation. Further investigation is needed for reducing of BA formation in variety of foods. Controlled temperature is a way to control BA formation except for some bacteria produce BA at temperatures below 5^○C.^[23,24]

The amount of BA will be decreased by limiting the activity of enzymes which converts free amino acids to BA. It was reported that trypsin and chymotrypsin were effective on the reduction of decarboxylase activity in rats.^[25] They studied the effect of proteolytic enzymes (trypsin and chymotrypsin) on the histamine formation in rats. Use of these enzymes reduced the formation of histamine in rats. In the literature, there is no information about the reduction of BA formation in foods or model systems by use of proteolytic enzymes (trypsin and chymotrypsin). Therefore, the aim of this study was to develop a new method to reduce cadaverine and tyramine formation by proteolytic enzymes trypsin and chymotrypsin in model system.

MATERIALS AND METHODS

Chemicals

Cadaverine dihydrochloride, tyramine hydrochloride, trypsin (one Nα-Benzoyl-L-arginine ethyl ester (BAEE) unit will produce a A253 of 0.001 per min at pH 7.6 at 25°C using BAEE as a substrate), chymotrypsin (one unit will hydrolyze 1.0 μmole of N-Benzoyl-L-Tyrosine Ethyl Ester (BTEE) per min at pH 7.8 at 25°C), tyrosine, lysine decarboylase (LDC), tyrosine decarboxylase (TDC), and lysine were obtained from Sigma (St. Louis, MO). Cadaverine dihydrochloride and tyramine hydrochloride were used as BA standards, sodium hydroxide, 25% ammonium and sodium bicarbonate were obtained from Merck (Darmstadt, Germany), acetone from Reidel De Haen (Germany), dansyl chloride from Sigma Co. (St. Louis, MO), ammonium acetate from Merck (Darmstadt, Germany), and perchloric acid from JT Baker (Holland). All chemicals except acetonitrile were of analytical grade (extra pure) and acetonitrile was high-performance liquid chromatography (HPLC) grade.

Preparation of Standards

Standard solutions of BA (cadaverine and tyramine) and amino acids (lysine and tyrosine) were prepared and diluted to 1 mL with 0.4 M perchloric acid to give concentrations from 0.5 to 10 μg/mL.

Determination of BA and Amino Acids

Liquid chromatography (LC) mobile phases

The chromatographic method was used for the determination of the BA and amino acids.^[26] The HPLC consisted of a Shimadzu gradient pump (Shimadzu LC 20AB, Shimadzu Solvent Delivery Module, Kyoto, Japan), a Shimadzu auto injection unit (Shimadzu SIL20AHT, Kyoto, Japan), a Shimadzu ultra violet (UV) detector (Shimadzu SPD 20A, Kyoto, Japan) and a RP-18 guard column. The wavelength of UV detector was 254 nm. The HPLC column was Spherisorb ODS2, 200 μm and 4.6 × 200 mm (Inertsil, ODS-2). Ammonium formate solution (0.4 M) prepared by ultra-pure water (Millipore Elix 10UV and Milli-Q, Millipore S.A.S. 67120 Molsheim, France) and acetonitrile were filtered through a 0.45 μm Millipore filter (Billerica, MA). Ammonium formate and acetonitrile were used as the LC mobile phases. A gradient elution program was used with mobile phases of acetonitrile (solvent A) and 0.4 M ammonium formate (solvent B), starting with 50% solvent A and 50% solvent B and finishing with 90% solvent A and 10% solvent B after 35 min. The flow rate was 1.0 mL/min.

Derivatization method

One mililiter of the each sample (lysine, tyrosine, cadaverine, and tyramine) was made alkaline by adding 200 μL of 2 N NaOH solution; 300 μL of saturated sodium bicarbonate was also added as buffer. Two milliliters of dansyl chloride solution (10 mg/mL in acetone) was added to each sample and incubated for 45 min at 40°C. Residual dansyl chloride was removed by adding 100 μL of 25% ammonia. After 30 min, the solution was adjusted to 5 mL with acetonitrile, centrifuged for 5 min at 1790xg. The supernatant was filtered (0.45 μm), and 20 μL was then injected onto the HPLC. Lysine, tyrosine, cadaverine, and tyramine content was found from calibration curves.

Synthesis and determination of BA in model system

One mililiter of standard solutions of lysine were mixed with 1 mL of its decarboxylase enzyme (2 mg/mL lysine decarboxylase). Lysine concentration in solutions was adjusted to 25, 50, 100, 150, and 200 ppm. The solutions were incubated in orbital shaker (Innova 40) at 30°C with 70 rpm for 30 min. After 30 min, derivization procedure was applied and then analyzed by HPLC. Figure 1 shows the peaks of residual lysine and formed cadaverine. The same procedure was applied for tyrosine by using 2 mg/mL of TDC enzyme. Tyrosine concentration in solutions was adjusted to 25, 40, 75, 100, and 125 ppm.

FIGURE 1 Peaks of residual lysine and formed cadaverine.

Reduction of BA in Model System

Each model systems were prepared as two replicates and dublicate analyses were carried out in each replicate.

Application of trypsin

One mililiter of lysine solution, 1 mL of trypsin (2 mg/mL), and 1 mL of lysine decarboxylase enzyme (2 mg/mL) were mixed and incubated with incubation shaker at 30°C with 70 rpm for 30 min. Lysine concentration in solutions was adjusted to 25, 50, 100, 150, and 200 ppm. After 30 min, derivization procedure was applied to find formation and reduction in cadaverine and then analyzed by HPLC. The same procedure was applied for standard solution of tyrosine by using 2 mg/mL of TDC enzyme and 1 mg/mL of trypsin to detect tyramine formation. Tyrosine concentration in solutions was adjusted to 25, 40, 75, 100, and 125 ppm.

Application of chymotrypsin

One mililiter of lysine solution, 1 mL of chymotrypsin (2 mg/mL), and 1 mL of its decarboxylase enzyme (2 mg/mL) were mixed and incubated with incubation shaker at 30°C with 70 rpm for 30 min. Lysine concentration in solutions was adjusted to 25, 50, 100, 150, and 200 ppm. After 30 min, derivization procedure was applied to find cadaverine formation and then analyzed by HPLC. The same procedure was applied for standard solution of tyrosine by using 2 mg/mL of TDC enzyme and 1 mg/mL of chymotrypsin enzyme to detect tyramine formation. Tyrosine concentration in solutions was adjusted to 25, 40, 75, 100, and 125 ppm.

Application of trypsin and chymotrypsin

One mililiter of standard solution of lysine (25, 50, 100, 150, and 200 ppm), 1 mL of chymotrypsin (2 mg/mL), 1 mL of trypsin (2 mg/mL), and 1 mL of its decarboxylase enzyme (2 mg/mL) were mixed and incubated with incubation shaker at 30°C with 70 rpm for 30 min. Lysine concentration in solutions was adjusted to 25, 50, 100, 150, and 200 ppm. After 30 min, derivization procedure was applied to find cadaverine formation and then analyzed by HPLC. The same procedure was applied for standard solution of tyrosine by using 1 mg/mL of TDC enzyme, trypsin enzyme (1 mg/mL), and chymotrypsin enzyme (1 mg/mL) to detect tyramine formation. Tyrosine concentration in solutions was adjusted to 25, 40, 75, 100, and 125 ppm.

Determination of Percent Reduction of Cadaverine and Tyramine Formation

Percent reduction of cadaverine and tyramine was calculated as:

A is the amount of cadaverine or tyramine produced with their decarboxylase enzyme, B is the amount of cadaverine or tyramine produced with their decarboxylase enzyme in the presence of proteolytic enzymes (trypsin and chymotrypsin).

Statistical Analysis

The results were analyzed statistically using the statistical packages SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA). The one-way analysis of variance (ANOVA test) and Duncan’s multiple range test was performed. Values of p < 0.05 were used to indicate significant differences.

RESULTS AND DISCUSSION

Synthesis and Reduction of Tyramine

Tyramine is one of the BA that can cause some health disorders in sensitive individuals.^[27] Table 1 shows the tyramine synthesis in model system in the presence of TDC enzyme. Concentrations of synthesized tyramine increased (p < 0.05) when the concentrations of standard solution of tyrosine were increased (Table 1). Conversion of tyrosine to tyramine was achieved by a range from 78.4 to 96.7%. At 100 ppm, the percentage of tyrosine converted to tyramine is at its maximum value, and then that value decreases (Table 1). This may be due to the maximal reaction rate is obtained at 100 ppm tyrosine concentration and increasing tyrosine concentration did not increase the tyramine concentration afterall in which enzyme is saturated with substrate.

TABLE 1 Synthesis of tyramine and cadaverine

Concentration of tyrosine/lysine (ppm)	Residual tyrosine/lysine concentration (ppm)	Produced tyramine/cadaverine concentration (ppm)	% Synthesis of tyramine/cadaverine
Tyrosine + TDC
25	2.4 ± 0.5a	21.3 ± 1.1a	85.2
40	5.4 ± 0.9b	34.6 ± 2.0b	86.4
75	14.3 ± 0.4d	60.7 ± 1.5c	81.0
100	12.9 ± 0.9c	96.7 ± 1.8d	96.7
125	27.0 ± 0.5e	98.0 ± 0.7e	78.4
Lysine + LDC
25	7.3 ± 0.1a	17.7 ± 0.1a	70.8
50	15.2 ± 0.1b	34.8 ± 0.2b	69.6
100	25.3 ± 0.2c	74.4 ± 0.1c	74.4
150	68.0 ± 0.1d	82.0 ± 0.1d	54.7
200	84.9 ± 0.3e	115.0 ± 0.2e	57.5

Different letters indicate statistical difference at p < 0.05 level in each column.

Trypsin and chymotrypsin were applied to reduce tyramine formation in model system. Constant concentration of trypsin, chymotrypsin enzymes, and decarboxylase enzymes were used for different concentrations of tyrosine and it was observed that trypsin and chymotrypsin reduce efficiently (p < 0.05) tyramine synthesis (Table 2). Tyrosine (100 ppm) was converted to 96.75 ppm tyramine in the presence of TDC enzyme (Table 1). It was reduced to 82.2 ppm (15.0%) by chymotrypsin, to 69.8 ppm (27.8%) by trypsin, and to 63.4 ppm (34.5%) by a mixture of trypsin and chymotrypsin (Table 2). A maximum of 57.2 and 52.1% tyramine reduction was observed when trypsin and chymotrypsin enzymes were used, respectively. Also 59.1% reduction was observed by use of trypsin and chymotrypsin together (Table 2).

TABLE 2 Effect of trypsin and chymotrypsin on tyramine synthesis

Concentration of tyrosine (ppm)	Residual tyrosine (ppm) concentration	Produced tyramine concentration	% Reduction of tyramine synthesis
Tyrosine+Trypsin+TDC
25	15.9 ± 0.7a	9.1 ± 1.1a	57.2
40	17.6 ± 0.9a	22.4 ± 3.6b	35.2
75	41.1 ± 2.7b	33.9 ± 4.6c	44.1
100	30.2 ± 4.1c	69.8 ± 5.5c	27.8
125	51.5 ± 4.8d	73.5 ± 6.8d	25.0
Tyrosine+Chymotrypsin+TDC
25	14.8 ± 0.7a	10.2 ± 1.3a	52.1
40	15.8 ± 0.9a	24.2 ± 3.4b	30.0
75	30.6 ± 1.4b	44.4 ± 4.9c	26.8
100	17.8 ± 1.9c	82.2 ± 6.9d	15.0
125	34.3 ± 1.9c	90.6 ± 8.4e	7.5
Tyrosine+Trypsin+Chymotrypsin+TDC
25	16.3 ± 0.1a	8.7 ± 0.0a	59.1
40	20.2 ± 0.1b	19.8 ± 0.1b	42.8
75	37.0 ± 0.2c	38.0 ± 0.2c	36.4
100	38.6 ± 0.1d	63.4 ± 0.1d	34.5
125	44.4 ± 0.1e	85.6 ± 0.1e	12.6

Different letters indicate statistical difference at p < 0.05 level in each column.

As the concentration of tyrosine was increased, the percentage of the tyramine synthesis decreased. This could be explained by the insufficient concentrations of trypsin and chymotrypsin to reduce tyramine formation. Results show that 1 mg/mL of trypsin and chymotrypsin concentrations were not enough to reduce tyramine synthesis so that higher trypsin and chymotrypsin concentrations could be needed. It was concluded that use of a mixture of proteolytic enzymes results in highest reduction of tyramine. Trypsin shows better inhibitory effect on tyramine formation compared to chymotrypsin. Inhibition mechanism of trypsin and chymotrypsin did not obey any of reversible inhibition types. The inhibition mechanism was probably irreversible and proteolytic enzymes could alter the active site of the decarboxylase enzymes of amino acid residues. Proteolytic enzymes have been already used in the food industry to accelerate the fermentation but their actions on reduction of BA was first investigated by this study.

In the literature there is no study about reduction of formation of BA by proteolytic enzymes in model systems; however, there are different methods, such as controlled temperature, starter culture, HPP, irradiation, and using preservatives, that were studied to reduce formation of BA in foods (Table 3). Ruiz-Capillas et al.^[28] studied the reduction of formation of BA with high hyrostatic pressure at 350 MPa for 15 min. They observed a 17.0% reduction of tyramine formation compared to sausage not treated with HHP. Also, 17.7% tyramine reduction was observed by application of irradiation.^[29] Results found by other researches in foods are quite lower than that found in this study.

TABLE 3 Reduction of formation of biogenic amines by different methods

Method/technique	% Reduction	Reference
Additive	20.8% histamine reduction 31.2% tyramine reduction	[38]
Additive	Putrescine, cadaverine, histamine, spermidine reduced between 63% and 73% compared to control	[21]
HHP	84.4% tyramine reduction	[19]
HHP	8.7% putrescine reduction	[28]
HHP	88.0% putrescine reduction 98.0% cadaverine reduction	[36]
Irradiation	57.4% putrescine reduction 62.3% tyramine reduction 21.1% spermine reduction	[34]
Irradiation	100.0% putrescine reduction 17.7% tyramine reduction 28.8% spermidine reduction 56.2% spermine reduction	[29]
Packaging	16.7% histamine reduction 46.8% cadaverine reduction 32.5% spermidine reduction 36.0% putrescine reduction	[32]
Controlled temperaure	99.6% histamine reduction	[12]
Starter culture	16% reduction of total biogenic amine content	[41]

Novella-Rodriguez et al.^[19] studied the effect of HHP on BA reduction and they obtained higher (84%) reduction of tyramine during the ripening of goat cheese compared to the results obtained in this study. In addition, tyramine concentration dropped approximately from 200 to <100 ppm (about >50% reduction) in Indian mackerel (whole) by use of turmeric (curcumin), red pepper (capsaicin), and black pepper (piperine),^[30] and tyramine was reduced by 31.2% in Myeolhi-Jeot (fermented anchovies) by preservatives.^[21] Tyramine concentration dropped from 24.7 to 9.3 (62.3%) ppm in beef by the application of irradiation.^[31] Although many studies show the inhibitory effect of irradiation, there is an unfavorable point that is public acceptance.^[32–34] Results showed that using trypsin and chymotrypsin together had acceptable reduction of synthesis of tyramine compared to the other reduction methods.

Synthesis and Reduction of Cadaverine

Cadaverine has less pharmacological activity than the aromatic amines but it is probably potentiators of their toxicity.^[35] Table 1 shows the synthesis of cadaverine from lysine in the presence of lysine decarboxylase enzyme. According to the results, as the concentrations of lysine increased up to 100 ppm, concentrations of synthesized cadaverine increased (p < 0.05) as well (Table 1). Synthesized cadaverine changed between 54.7–74.4% (Table 1). A significant drop (p < 0.05) was observed in cadaverine formation when trypsin (maximum 80.4% reduction) and chymotrypsin (maximum 83% reduction) were used (Table 4). Also, the reduction of cadaverine formation was significant (p < 0.05), maximum 84.8%, when trypsin and chymotrypsin were used together (Table 4).

TABLE 4 Effect of trypsin and chymotrypsin on cadaverine synthesis

Concentration of lysine (ppm)	Residual lysine (ppm) concentration	Produced cadaverine concentration	% Reduction of cadaverine synthesis
Lysine+Trypsin+ +LDC
25	12.5 ± 2.5a	12.5 ± 1.5a	29.3
50	26.2 ± 8.7a	23.8 ± 1.8bc	31.6
100	69.0 ± 3.8b	31.0 ± 1.4d	58.3
150	124.4 ± 1.8c	25.6 ± 1.6c	68.7
200	177.5 ± 0.8d	22.5 ± 0.8bc	80.4
Lysine+Chymotrypsin+LDC
25	14.5 ± 0.3a	10.5 ± 1.3ab	40.6
50	29.9 ± 0.9a	20.1 ± 1.5c	42.2
100	84.6 ± .9c	15.4 ± 2.1a	79.3
150	127.8 ± 2.0d	22.2 ± 2.2b	73.0
200	180.4 ± 2.4e	19.6 ± 1.3ab	83.0
Lysine+Chymotrypsin +Trypsin+LDC
25	10.3 ± 0.7a	14.7 ± 1.0a	17.0
50	30.9 ± 3.7b	19.1 ± 1.3bc	45.1
100	79.8 ± 2.5d	20.2 ± 1.4bc	72.8
150	129.0 ± 1.2e	21.1 ± 1.5c	74.2
200	182.6 ± 1.5f	17.4 ± 1.2ab	84.8

Different letters indicate statistical difference at p < 0.05 level in each column.

According to the results of this study, when 100 ppm lysine was treated with lysine decarboxylase enzyme, 74.4 ppm cadaverine was detected. It was reduced to 31.0 ppm (58.3%) by trypsin, reduced to 15.4 ppm (79.3%) in the presence of chymotrypsin and to 20.2 ppm (72.8%) when chymotrypsin and trypsin were used together (Table 4). Synthesized cadaverine concentration increased as the initial concentration of lysine was increased. Cadaverine formation reduced significantly (p < 0.05) by use of trypsin, chymotrypsin, and the mixture of trypsin and chymotrypsin. The use of trypsin separately had less inhibitory effect than the other formulation of trypsin and chymotrypsin enzymes. Chymotrypsin showed better reduction of cadaverine formation compared to trypsin. However, the use of both trypsin and chymotrypsin enzymes together inhibited cadaverine formation more than the use of trypsin and chymotrypsin enzymes alone, as about 85%.

Ruiz-Capillas et al.^[28] were observed lower reduction of cadaverine (12.5%) by HHP compared to our results. In another study, 98% cadaverine reduction was observed by use of HHP.^[36] HHP provides significant reduction or control of BA; however, it has some limitations. Reduction of BA formation depends on the level of pressure applied.^[22] Low-pressure treatment of 50 MPa for 72 h increased BA content, while a high-pressure treatment of 400 MPa for 5 min plus 50 MPa for 72 h showed a slight decrease.^[19] Also, it was observed that cadaverine concentration was approximately dropped from 200 to 100 ppm (50.0%) in Indian mackerel (whole) by use of preservatives,^[30] and dropped from 18.9 to 10.1 ppm (53.4%) by Mbarki et al.^[32] Although there has been many studies that show inhibitory effects of food preservatives and additives on BA,^[21,37,38] some researches show promoting effects on BA formation.^[39,40] On the other hand, sufficient knowledge about effectiveness of additives on different BA is not present and needs to be tested in variety of food systems.

CONCLUSION

Researches have been trying to reduce BA formation due to their toxic risks, especially on people suffering from allergic reactions, and have a role as indicators of quality and/or acceptability in some foods. In this study a new method, the use of proteolytic enzymes (trypsin and chymotrypsin), was applied to reduce formation of BA in model system. Significant reduction of cadaverine and tyramine synthesis were detected (p < 0.05) in model system. Compared to the separate use of trypsin and chymotrypsin, in most cases, using trypsin and chymotrypsin mixture gave better results for cadaverine and tyramine reduction. Although use of trypsin separately showed better inhibitory effect on tyramine reduction compared to chymotrypsin, the use of trypsin had no similar effect on cadaverine inactivation. In cadaverine inactivation the use of chymotrypsin resulted in higher reduction compared to that of trypsin. It was found that the use of trypsin and chymotrypsin could be used to reduce selected BA formation in model system. However, further studies are required to determine the effect of trypsin and chymotrypsin in food systems on different BA.