MATERIALS AND METHODS

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1 Biogenic Amines in Some Natural and Processed Cheeses Sold in Laguna Province, Philippines Ma. Jannine M. Vallejos 1, Laura J. Pham 2, and Virginia L. Barraquio 3* 1,3* Animal and Dairy Sciences Cluster (Dairy Training and Research Institute), College of Agriculture, University of the Philippines Los Baños, College, Laguna 2 National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños, Laguna Natural and processed cheese samples from the different supermarkets around Laguna Province in the Philippines were analyzed for the presence and quantity of biogenic amines using thin layer chromatography with Biosoft Quantiscan program. The histamine concentrations were ± 1.8 ppm, ± 1.0 ppm, and 49.9 ± 3.6 ppm in Blue, Cheddar, and Edam cheese, respectively. The histamine limit in cheese is 100 mg/kg, hence the level found in Cheddar cheese needs to be looked into. The tyramine contents were ± 3.6 ppm, ± 2.0 ppm and ± 1.9 ppm in Blue, Cheddar, and Edam cheese, respectively. Because the limit for tyramine in cheese is 200 mg/kg, the levels found in Blue and Cheddar cheese are of serious concern. Brie, processed cheese and white soft cheese (kesong puti) were negative for both histamine and tyramine. All cheeses were negative for cadaverine. High levels of histamine and tyramine found in the cheese samples analyzed indicate the need to expand sampling to other natural cheese varieties sold in the province. Then, actual or anticipated human exposure risk to BAs in cheese can be determined to ascertain the necessity for the Food and Drug Administration and other government agencies concerned with food safety to take action. Key Words: biogenic amines, cheese, histamine, tyramine INTRODUCTION Biogenic amines (BAs) have been involved in many cases of food poisoning which were attributed to fermented foods that contain them in high amounts. In fermented foods, the presence of BAs are due to the activity of the lactic acid bacteria (LAB) which are used as starter cultures while for non-fermented foods BA s are produced by contaminating microorganisms (Muñoz 2008). This means that BAs can be an indicator of the quality of the raw materials or the hygienic condition during processing (Rodriguez et al. 2002). Biogenic amines are formed through the decarboxylation of amino acids or through amination and transamination of aldehydes and ketones. Particular bacterial genera such as Bacillus, *Corresponding author: vlbarraquio@gmail.com Citrobacter, Clostridium, Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella, Photobacterium, and LAB Lactococcus, Pediococcus, and Streptococcus are capable of decarboxylating amino acids. Aside from being genera and strain specific, there are also conditions which favor the decarboxylation of amino acids. One of them is the availability of free amino acids produced by microbial strains with high proteolytic enzyme activity. The conditions which favor bacterial growth, decarboxylase synthesis, and decarboxylase activity are also factors that affect the formation of BA s. Acidic environment, such as ph between 4.0 and 5.5, favors the decarboxylation of amino acids. The bacteria produce decarboxylase enzyme as a defense mechanism against Reproduced from Philippine Journal of Science 141: (2012). Virginia L. Barraquio: Participant of the 3rd UM,

2 There are several biogenic amines identified in fermented foods and each one can cause serious illness to humans. Histamine, one of the most common BA, is a vasoactive substance which causes dilatation of blood vessels, capillaries and arteries that can lead to headache, hypertension, gastrointestinal distress and edemas. Tyramine can act as a vasoconstrictor which increases the noradrenaline concentration in the blood (Muñoz 2008) that leads to hypertension, migraine, brain hemorrhage, and heart failure (Elsanhoty et al. 2009). Putrescine and cadaverine are also given much attention as these amines can be precursors of carcinogenic nitrosamines which cause cancer in humans (Muñoz 2008). Different research institutes around the world have set legal limits for biogenic amine content of food products to ensure safety of consumers. The Nutritional Codex of the Slovak Republic has set the legal limit for histamine and tyramine in fish and fish products at 200 mg/kg. The same agency set the legal limit for tyramine in cheese at 200 mg/kg. The histamine limit in fish set by the Codex Alimentarius Commission is 200 mg/kg. For meat products, the Netherlands Institute for Dairy Research recommended an upper limit of mg/kg for Next to fish, cheese is the most implicated item associated with biogenic amine food poisonings (Maijala and Eerola 2003). Therefore, the dairy industry must be vigilant with regards to BA in cheese products, as its presence is an indicator of microbial spoilage which could cause serious food poisoning. BAs are formed from free amino acids produced during cheese ripening. Ripening involves storing the curd or freshly made cheese under controlled temperature and relative humidity for a period of several months to over a year depending on cheese variety. Such cheeses are called natural cheeses. The unique taste of natural cheese is usually attained after ripening. Ripening develops the flavor, body, texture and color of the cheese (Lampert 1975). During ripening, the amino acids and small peptides from casein are catabolized by different enzymes into sapid and aromatic compounds such as carbonyls, Processed cheese on the other hand, is cheese made from blends of old (>5 months) and young (<5 months) natural cheeses that were ground/shredded, added with emulsifying salts and flavors, moisture content is adjusted (to produce either spreadable or sliceable type processed cheese), then pasteurized. The age and flavor characteristics of natural cheeses to be used in processed cheese manufacture should be properly selected to ensure the quality of the processed cheese product. The use of very young cheese, ripened for less than 10 days, yields a rubbery and firm product. On the other hand, the use of very old cheese yields a weak and grainy product (Lampert 1975). To our knowledge, BAs are not among those substances in foods monitored by our local food and drug regulatory agency and no limits have been set to date. This study was conducted to evaluate some natural and processed cheeses for the presence and levels of three (3) commonly encountered biogenic amines: specifically, histamine, cadaverine, and tyramine. MATERIALS AND METHODS This study was conducted from May to October 2010 at the Fats and Oils Laboratory, National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños, Laguna. A total of ten (10) samples: five (5) each of natural and processed cheeses were purchased from different supermarkets in Laguna. All chemicals used were of analytical grade and obtained commercially. BA standards tyramine hydrochloride, histamine dihydrochloride and cadaverine hydrochloride were obtained from Sigma Aldrich (St. Louis, MO). Extraction of BA Extraction of BA s was done using the method of Elsanhoty et al. (2009). Twenty five grams (25g) of grated cheese was added with 125 ml of 5% trichloroacetic acid (TCA) to precipitate the protein. The sample was blended for 3 minutes and then filtered using Whatman No. 1.10m filter paper. Twenty (20) ml filtrate was transferred to a 20 ml test tube then added with 4g NaCl to prevent turbidity. One (1) ml of 50% NaOH was added to calibrate acidity. Thin Layer Chromatography Evaluation of the BAs was carried out using modified methods developed at the Fats and Oils Laboratory of BIOTECH, UPLB (Pham and Gregorio 2008). Samples were spotted on thin layer plates coated with silica gel G60 and developed with the solvent system consisting of ethanol:nh 4 OH (80:20 v/v). Spots were visualized in a chamber with iodine vapor. Biogenic amines were identified by comparing the retention factor (Rf) of the samples with those of authentic standards. The Rf value was calculated as the distance traveled by the compound divided by the distance traveled by the solvent. Biogenic Amine Quantification BAs were quantified using the Biosoft Quantiscan for Windows (Biosoft 2004) program. The program has the same functionality to sophisticated densitometers but at a fraction of the cost. The image of the chromatogram was loaded to the Quantiscan program which was then 198

3 processed and analyzed. Reports can be generated from peaks detected by the position, height, width, and net area. Based on the net area, the concentration of BA was calculated from the standard curve. For the standard curve, different concentrations of standards were also spotted and developed. The concentrations used were: 1.00, 0.75, 0.50, 0.25, 0.10 and 0.05 mg ml -1. This standard curve was used to estimate the concentrations of BAs present in the extracts. The BA concentration of the samples was estimated using a simple linear regression formula: y = mx + b (Skoog et al. 2004), where y is the net area of the chromatogram of the BA spot and x is the concentration of the BA in the sample. The m (slope) and b (y intercept) values were obtained from the net area (y) and the concentration (x) of the BA standard. Substituting them in the given equation y = mx + b, the value of x (the concentration of the BA in the sample) was computed. RESULTS AND DISCUSSION Biogenic Amine Identification The Rf values of the cheese samples and the biogenic amine standards are presented in Table 1. Tyramine and histamine were found in the natural cheeses: Blue, Cheddar and Edam. Other spots were not identified due to the limited number of standards used (only three, histamine, tyramine, and cadaverine). The unknowns could probably be other BAs. Biogenic Amine Concentration The mean concentrations of BAs in the cheese samples are shown in Table 2. Three (3) out of the ten (10) cheese samples were found to contain the BAs, histamine and tyramine. Cheddar cheese has the highest histamine concentration of ± 1.0 ppm which exceeded the limit of 100 ppm (Ten Brink et al. 1990; Durlu-Özkaya 2002). The histamine concentration in Blue cheese was ± 1.8 ppm. Edam cheese contained the lowest histamine concentration of 49.9 ± 3.6 ppm. Blue cheese was found to have the highest concentration of tyramine, ± 3.6 ppm which was way above the acceptable limit of 200 ppm set by the Nutritional Codex Rabie et al. (2011) reported 2010 mg/kg tyramine in blue cheese. According to another study, tyramine concentration was higher at the edge part than in the core of Niva blue cheese because of the higher number of enterococci and Enterobacteriaceae (Standarová et al. 2009). Cheddar and Edam cheese were found to contain ppm ± 2.0 and ± 1.9 ppm of tyramine, respectively. No BAs were detected in Brie, kesong puti, and processed cheeses. Formation of BA was affected by ripening period of cheese as longer storage period increases the BA content (Elsanhoty et al. 2009; Aliakbarlu et al. 2009; Standarová et al. 2009; Komprda et al. 2005; Rodríguez et al. 2002). Brie cheeses are usually ripened for three to four weeks only which is shorter than the ripening period for other natural cheeses. Shorter ripening period means less time for BA formation. Cheese is a favorable environment for the formation of BA due to the free amino acids produced from milk proteins during ripening (Elsanhoty et al. 2009). The kesong puti (white soft cheese) was used as the negative control in this study. Since it is unripened there was no chance for the formation of BA. Another factor for BA formation is the quality of raw milk used for cheese making. Pasteurization of milk eliminates the bacteria responsible for BA formation. The standardization of milk quality, selection of appropriate starter culture, and hygienic cheese manufacturing will reduce the BA content in cheese (Elsanhoty et al. 2009). Although the processed cheese sample extracts were concentrated by evaporation of the extracting solution to increase the concentration of BAs in the processed cheese samples to detectable level, no BAs were found. BAs could come from the natural cheeses used as ingredient of processed cheese (Stratton et al. 1991). Pasteurization or cooking during processed cheese manufacturing cannot eliminate BAs. Thus, the quality of raw materials such as natural cheese should be good and hygiene should be observed during manufacturing to avoid the growth of microorganisms causing spoilage (Silla-Santos 1996). The absence of BAs in the processed cheese samples could probably be due to the fact that the processed cheese processors in the Philippines use curd rather than old stock cheese as main ingredient since it is cheaper. Curd is also known as green cheese or unripened cheese. The total curd importation of the Philippines was 7.99 thousand MT in early 2010 (Philippine Dairy and Products Annual 2010). Cadaverine which is usually produced during the early stage of decomposition was not detected since all samples analyzed were still in good condition or has not reached their expiration or best before dates. Filipinos in general are not cheese-eaters but they include cheese in some recipes and snacks. The histamine and tyramine contents of the natural cheese samples analyzed were mostly beyond the safe levels, indicating the need to expand sampling to cover other natural cheese varieties sold in the province. Then, actual or anticipated human exposure risk to BAs in cheese can be determined to ascertain the necessity for the Food and Drug Administration and other government agencies concerned with food safety to take action. 199

4 Table 1. Rf values of the standards and cheese samples sold in Laguna Province, Philippines (n=2). Standards and Cheese Samples Peaks Distance Traveled by Rf value Identity of Compound Compound (cm) Cadaverine Histamine Tyramine Blue Cheese Tyramine Brie Cheese 0 Cheddar Cheese Tyramine Adam Cheese Tyramine White Soft Cheese (kesong puti) 0 Process Cheese A 0 Process Cheese B 0 Process Cheese C 0 Process Cheese D 0 Process Cheese E 0 Distance traveled by solvent = 8.0 cm. Table 2. Biogenic amine content of the cheese samples sold in Laguna Province, Philippines (n=2). Cheese samples Biogenic amine content (ppm) Histamine Tyramine Blue Cheese ± ± 3.6 Cheddar Cheese ± ± 2.0 Edam Cheese 49.9 ± ± 1.9 Brie Cheese 0 0 White Soft Cheese (kesong puti) 0 0 Processes Cheese A 0 0 Processes Cheese B 0 0 Processes Cheese C 0 0 Processes Cheese D 0 0 Processes Cheese E 0 0 REFERENCES ALIAKBARLU A, ALIZADEH M, ROHANI SM, VAHABZADEH Z, SAEI SS, AGH N Effects of processing factors on biogenic amines production in Iranian white brined cheese. Res J Biol Sci 4: TEN BRINK BT, DAMINK C, JOOSTEN HMLJ, HUIS IN T VELD JHJ Occurrence and formation of biologically active amines in foods. In: Silla-Santos, M.H. Biogenic amines: their importance in foods. Int J Food Microbiol 29: DURLU-ÖZKAYA F Biogenic amine content of some Turkish cheeses. J Food Process Preserv 26: ELSANHOTY R M, MAHROUS H, GHANAIMY G Chemical, microbial counts and evaluation of biogenic amines during ripening of Egyptian soft domiati cheese made from raw and pasteurized buffaloes milk. Int J Dairy Sci 4 (2): FOX PF Biochemistry of Cheese Ripening. In: Encyclopedia of Dairy Sciences. Roginski H, Fuquay JW & FOX PF (eds.) New York: Academic Press. p

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