Italian Journal of Food Safety 2015; volume 4:4700 Comparison of -aminobutyric present useful nutraceutical properties, such γ as γ-aminobutyric acid (GABA), while others, Correspondence: Gavina Manca, Laboratory of acid and biogenic amine such as biogenic amines (BA), may have neg- Commodity Science Technology and Quality, content of different types of ative effects on human health. Department of Economic and Business Sciences, ewe’s milk cheese produced in Over the last decade attention has been paid University of Sassari, via Muroni 25, 07100 to the presence of GABA in cheese and dairy Sassari, Italy. Sardinia, Italy Tel: +39.079.229579 - Fax +39.079.229579. product, as a result of its health-related benefits E-mail: [email protected] (Mills et al., 2009; Wang et al., 2010; Nejati et al., Gavina Manca,1 Arianna Porcu,1 2013). GABA is a non-protein amino acid acting Key words: GABA; Biogenic amines; Ewe’s milk 1 1 Antonio Ru, Margherita Salaris, as one of the main inhibitory neurotransmitters cheese; Pecorino; Casu Marzu. Mario A. Franco,1 Enrico P.L. De Santis2 in the sympathetic mammalian nervous system 1Laboratory of Commodity Science and exerts positive effects in the treatment of Conflict of interest: the authors declare no poten- tial conflict of interest. Technology and Quality, Department of sleeplessness, depression, chronic alcohol- Economic and Business Sciences, related symptoms (Oh et al., 2003) and Received for publication: 8 September 2014. University of Sassari; 2Department of Parkinson’s disease (De Jong et al., 1984). In Revision received: 12 March 2015. Veterinary Medicine, University of addition, it has shown antitumorigenic activity Accepted for publication: 17 March 2015. (Thaker et al., 2005) and, since it is a strong Sassari, Italy secregatogue of insulin from the pancreas, it This work is licensed under a Creative Commons can prevent diabetes (Hagiwara et al., 2004). Attribution 3.0 License (by-nc 3.0). GABA is produced by the decarboxylation of ©Copyright G. Manca et al., 2015 glutamate catalysed by the enzyme L-glutamic Abstract Licensee PAGEPress, Italy acid decarboxylase (GAD). This enzyme is pres- Italian Journal of Food Safety 2015; 4:4700 ent in different species of microorganism that doi:10.4081/ijfs.2015.4700 The bioactive compounds γ-aminobutyric could be used for the development of probiotic acid (GABA) and biogenic amines (BA), cheese (Wang et al., 2010). The most interest- together with protein-free amino acids, were ing are the lactic acid bacteria (LAB), in partic- measured by high-performance liquid chro- ular several strains of Lactobacillus brevis simultaneous determination of GABA and BA matography in ewe’s milk cheeses produced in (Mills et al., 2009), Lactobacillus plantarum, in Pecorino Sardo produced in cheese facto- Sardinia with different technological traits. Lactobacillus delbrueckii subsp. bulgaricus, ries, Pecorino produced in farmhouses, and The study included three types of cheese: Lactobacillus casei and Lactococcus lactis subsp. Casu Marzu, made from insect larvae (Piophila Pecorino Sardo PDO, Pecorino and Casu Lactis (Siragusa et al., 2007; Mills et al., 2009; casei), in order to assess the variability of Marzu. Farmhouse Casu Marzu and Pecorino Wang et al., 2010). The Enterococcus spp. and these nitrogenous compounds important to showed GABA content (maximum levels: Streptococcus spp. also include strains that have define cheese quality, in products obtained 1001.3 and 378.1 mg 100 g–1 respectively) that shown GAD activity (Hayakawa et al., 1997). with different technological traits. had never been found so high in cheese Other research has shown that these before, suggesting that these types of cheese species of bacteria are also involved in the pro- present ideal conditions to produce GABA. duction of BA (Galgano et al., 2001; Linares et These two types of cheese also showed high al., 2012; Lorencová et al., 2012), considered as Materials and Methods levels of BA (their total maximum levels were a serious health problem when present at sig- 1035.7 and 288.0 mg 100 g–1 respectively). nificant levels (Stratton et al., 1991). The study was carried out on traditional Pearson correlation analysis detected signifi- Tyramine, β-phenylethylamine, histamine, cheeses made in Sardinia (Italy) from whole cant correlation between GABA and the main tryptamine, cadaverine, putrescine, spermine ewe’s milk. Samples were collected at different BA present in the cheeses (tyramine, cadaver- and spermidine are considered as the most dairies and at different stages of ripening. The ine and putrescine), suggesting that the fac- important amines occurring in cheese. types of cheese considered were Pecorino tors affecting the production of GABA are the In cheese, BA are considered as an indicator Sardo protected designation of origin (PDO), same as those influencing BA formation. of poor hygienic conditions of raw material produced at cheese factories (13 samples with and/or manufacturing practices since their ripening time ranging from 30-360 days), farm- production and accumulation is often associat- house Pecorino (12 samples with ripening time ed with the activity of contaminant bacteria ranging from 60-360 days), and farmhouse Introduction (Loizzo et al., 2013). In general, artisanal Casu Marzu (9 samples with ripening time sheep cheeses are rich in GABA (Siragusa et ranging from 60-90 days). Pecorino Sardo PDO Consumers’ interest in the relationship al., 2007) but also in BA (Loizzo et al., 2013; produced in cheese factories is a semi-cooked between diet and health has increased the Mascaro et al., 2010). In fact, microbial decar- cheese made from thermised milk inoculated demand for information about the specific boxylase activity requires the availability of with a starter culture and coagulated with calf health characteristics of food. It is well known free amino acid precursors, produced as an rennet. Ripening takes place in ripening that food can contain ingredients that may outcome of proteolysis, and favourable pH and rooms, the temperature (between 6-12°C) and have physiological benefits and/or reduce the temperature conditions that can be achieved relative humidity (between 80-95%) of which risk of chronic disease, or even represent sig- during ripening (Siragusa et al., 2007; Linares are measured and controlled automatically. nificant risk factors influencing ill health. In et al., 2012; Dhakal et al., 2012). Some Pecorino cheese obtained from farmhouses cheese, compounds like amino acids are freed research has reported that GABA is also pres- is a semi-cooked cheese made from raw milk during proteolysis; they are substrates for sec- ent in Pecorino Sardo (Siragusa et al., 2007), without a starter culture and coagulated with ondary catabolic reactions by means of bacte- as are BA (Manca et al., 2000), even if these calf rennet. Ripening takes place in rooms ria with aminoacyl decarboxylase activity that studies were limited to a very few samples. For with no control over humidity or temperature, may yield bioactive compounds. Some of them these reasons this study has concerned the which may even reach room values. [Italian Journal of Food Safety 2015; 4:4700] [page 123] Article Casu Marzu (also called casu modde, casu Free amino acids and BA were determined Pearson correlation analysis was conducted to cundídu, or casu fràzigu in Sardinian lan- by the liquid chromatographic method determine the relationship between the differ- guage, which translates rotten cheese) is a described by Minocha and Long (2004). The ent compounds considered. Statistical signifi- Sardinian cheese produced with the use of lar- solvents used for separation were eluent A: cance was declared at P=0.01. vae of the cheese fly Piophila casei. It is pre- 100% acetonitrile (ACN) and eluent B: 20 mM pared in summer, when higher temperatures ammonium acetate buffer (pH 5.9) containing favour the life-cycle of the fly, and derived from 3% v/v 2-propanol. A gradient programme was Pecorino cheese that producers place in the implemented as follows: time=0, A:B (10:90), warmer rooms of the plant, uncovered, so that flow=0.5 mL min–1; time=1 min, A:B (10:90), Results and Discussion flies have better access to lay their eggs in it. flow=0.5 mL min–1; time=50 min, A:B (65:35), After 60-90 days of ripening the cheese is flow=0.5 mL min–1; time=65 min, A:B (100:0), On the basis of the original method ready to eat. flow=0.5 mL min–1; time=66 min, A:B (100:0), designed for the analysis of FAA and To extract the nitrogenous compounds con- flow=2 mL min–1; time=70 min, A:B (10:90), polyamines (Minocha and Long, 2004), we sidered, an amount of 1 g of ground cheese flow=0.5 mL min–1. An equilibration time of 10 developed a method that allowed extending the was weighed directly in a centrifuge tube and min was applied to achieve mobile phase sta- analysis to other seven BA. To do this, it was 20 mL of HCl 0.1 M added. The mixture was bilisation. necessary to extend the chromatographic run- then homogenised in an ULTRA TURRAX The identification of the nitrogenous com- ning time from 56 to 70 min. In Figure 1 the homogeniser (Zipperer, Staufen, Germany) for pounds was performed by comparison of the chromatograms of a cheese sample and of a 5 min. The cheese slurry obtained was cen- retention times of peaks in the samples to standard solution of amino acids and amines trifuged (ALC PK131R; ALC International S.r.l., those of standard solutions and by addition of are shown. Dansyl amino acids and dansyl BA Milan, Italy) at 7100 x g for 20 min at 4°C. The the suspected compound to the samples. A cal- were eluited in the following order: aspartic supernatant was recovered and the residue re- ibration curve was obtained by analysing stan- acid, glutamic acid, serine, threonine, glycine, extracted using the same procedure.