The monitoring of biogenic amines in the raw food

The aim of this work was to evaluate microbial quality and the presence of biogenic amines in raw bars. This study was focused on microbiological research in order to determine the presence of selected indicator groups of microorganisms depending on the composition of raw food. Identification of microorganisms was carried out by MALDI-TOF MS. In the second part of the experiment, biogenic amines and polyamines were analyzed using high performance liquid chromatography with UV/VIS detection. An increased incidence of mold has been reported in the samples, which is associated with a risk of mycotoxin production. After identifying microorganisms, it was found out that genera Micrococcus, Bacillus and Staphylococcus were the most represented. The highest concentration of biogenic amines (tyramine 42.2 ±4.8 mg.kg-1; putrescine 54.0 ±2.9 mg.kg-1) was found in a sample containing the vegetable component. The average concentration of biogenic amines in the tested raw bars was <30 mg.kg-1 and therefore they do not pose a serious health hazard to a consumer. 



INTRODUCTION
Raw foods consist mainly or entirely of raw uncooked food. In the Czech Republic, we can also see the name "live diet", and supporters of this trend are called "vitarians" (Cunningham, 2004;Červenka, Brožková and Fišerová, 2016). Food is considered raw unless it has undergone a heat treatment of more than 48 °C. Generally, there is no single specific temperature in the literature, but the range is between 38 -48 °C (Cunningham, 2004;Červenka, Brožková and Fišerová, 2016). Food meets raw food parameters unless it is refined, pasteurized, treated with pesticides or otherwise processed industrially. Instead, this diet approves a variety of food modifications like mixing, lyophilization, soaking and germination. Raw diet is based on eating a plant-based diet, especially fruits, vegetables, nuts and seeds. Cereals and legumes are also allowed, but usually they are first soaked (Cunningham, 2004). Among the positive effects of eating raw food is the high intake of fiber, minerals and water-soluble vitamins (Craig, 2009). The controversial effects of eating raw food are reducing the risk of cancer, especially women-specific types of cancer (Lanou and Svenson, 2011; Tantamango-Bartley et al., 2013) when mortality decreases compared to people, who consumed animalbased food (Orlich et al., 2014). Raw food has a positive effect on the composition of the intestinal microflora, which exhibits a protective effect (Glick-Bauer and Yeh, 2014). Ling and Hänninen (1992) describe a significant decrease in the activity of some precarcinogenic enzymes formed by the intestinal microflora when the raw foods were eaten for a week. Consuming raw food also seems useful in terms of intake of protective nutrients and photochemicals and also of minimizing intake of substances that are involved in many chronic diseases (Dewell et al., 2008).
Limiting the animal fat in the diet decreases the intake of saturated fatty acids and cholesterol. These aspects lead to a lower incidence of cardiovascular disease. At the same time, HDL cholesterol is also reduced, as well as insufficient intake of vitamin B12 (Dewell et al., 2008;Koebnick et al., 2005). In the long term, insufficient intake of vitamin B12, iron, zinc, essential fatty acids and essential amino acids is considered to be the major drawback of this nutritional trend. The largest natural sources of vitamin B12 are meat, offal, seafood, eggs, milk and dairy products (Watanabe, 2016).
Deficiency of B12 may result in megaloblastic anemia, which causes interruption of the cell division process. Clinical manifestations are consequently fatigue, weakness, paleness and decreased muscle activity (Aslinia, Mazza and Yale, 2006). Another type of anemia associated with the raw diet is sideropenic anemia, which is associated with reduction of iron in the blood (Sahovic, Vukobrat-Bijedic and Sahovic, 2012). The best sources of iron in the raw diet are mainly nuts (cashews, almonds, hazelnuts) and legumes (lentils, beans, peas), which are first recommended to be soaked or germinated. In the long term, intake of raw foods leads to weight loss, but also to underweight in many cases. Increased consumption of raw No. 1/2019 food is therefore usually associated with low values of BMI (Koebnick et al., 1999;Craig, 2009). Eating raw food results in low intake of protein, calcium and vitamin D. Low density of bone tissue and increased risk of osteoporosis (Fontana et al., 2005) are often manifested in people following this diet. Ganss, Schlechtriemen and Klimek (1999) reported an increased incidence of tooth enamel erosion. Its decay is associated with excessive consumption of fruit, which contains easily fermentable sugars. Eating raw foods leads to insufficient intake of polyunsaturated fatty acids necessary for normal function and further development of the brain, especially in children and adolescents who are still growing (Fonseca-Azevedo and Herculano-Houzel, 2012).
Eating raw food is also associated with worse digestibility of plant proteins, correlated with reduced nutrient utilization due to the presence of antinutrients. Antinutrients act on the activity of some enzymes, vitamins and minerals. In legumes and cereals, lectins, protease inhibitors, saponins and phytic acid are found to be destroyed only by heat treatment of foods (Soetan and Oyewole, 2009). Protease inhibitors, which were found in soybeans and peanuts, prevent proteolysis and subsequent protein utilization. The body reacts to the resulting amino acid deficiency by producing pancreatic proteases. In adolescents, these substances can cause stop in growth and further development (Kvasničková, 1998).
Also, it is importat to report increased risk of food intoxications, which stems from inadequate heat treatment of foods (Cunningham, 2004).
Biogenic amines are one of the substances involved in the food quality. They are low molecular weight organic nitrogen compounds. Biogenic amines exist in living organisms, where they fullfil a number of metabolic and physiological functions (Silla- Santos, 1996

Scientific hypothesis
Biogenic amines can be present in raw bars and their content is variable.

MATERIAL AND METHODOLOGY
Isolation and identification of the microorganisms: Ten grams of the fermented raw food sample ( Figure 1) was weighed out, aseptically removed and put into 90 mL of sterile physiological solution that was subsequently homogenised for 10 min (using a stomacher). The raw bars were then subjected to routine microbiological analysis. The total microorganism counts were assessed according to   Lyophilised raw bar products were used for the biogenic amine (BA) and polyamine (PA) analysis. Triple extraction of BA and PA from the lyophilised samples was carried out using a perchloric acid solution (0.6 mol.L −1 ). Three independent extractions were performed on each raw bar sample. The filtrated extract (filter porosity 0.45 µm) was then used directly for derivatisation and a following determination of BA/PA content (

Biogenic amine detection by HPLC:
The concentrations of eight present biogenic amines, such as histamine (HIM), tyramine (TYM), phenylethylamine (PHE), tryptamine (TRY), putrescine (PUT), cadaverine (CAD), spermine (SPE) and spermidine (SPD), were analysed via high performance liquid chromatography (HPLC) (LabAlliance, USA and Agilent Technologies, Agilent, Santa Clara, California, USA) after derivatisation using dansylchloride. The dansylchloride sample derivatisation procedure was performed according to Dadáková, Křížek and Pelikánová (2009). 1,7−heptandiamine was used as the internal standard. Chromatographic separation (ZORBAX Eclipse XDB−C18, 50 9 3.0 mm, 1.8 lm; Agilent Technologies) and detection (spectrophotometric λ = 254 nm) were performed according to Buňková et al. (2013). Each extract was derivatised twice after cultivation, and each derivatised mixture was applied to the column twice. Each raw bar sample was analysed 12 times (3 extractions, 2 derivatisations, 2 applications to the column). Detection limits for the individual amines were in the range 0.24 -1.39 mg.kg -1 . Given the significance of biogenic amines to human health and food safety, monitoring their content in foodstuffs is very important. Currently, HPLC based methods are the most suitable for the analysis of fermented food (Pleva et al., 2018). The reliability and sensitivity of these methods render them useful as important techniques to determine the concentrations of all biogenic amines in fermented food (EFSA, 2011).

Statistic analysis
The obtained experimental data were analysed using Statistical software Unistat 6.5 (Unistat, London, UK). The significance level of all statistical tests was set at p <0.05. The Kruskall-Wallis and Wilcoxon tests were used to evaluate the data obtained.

Microbial analysis
Raw bars are ideal media for the growth and survival of a variety of fungi and bacteria.
The results of the microbial analysis are given in Table 2. The amount of microorganisms cultured in BHI ranged from 2.9 to 9.2 log CFU.g -1 . Although there is no hygienic limit for this type of product in the current legislation, the log boundary of 6.0 CFU.g -1 is considered to be safe  ( The number of coliform bacteria (VRBA) ranged from 2.7 to 7.3 log CFU.g -1 . However, an increased number of coliform bacteria was observed in samples B3 (7.3 log CFU.g -1 ) and B7 (6.7 log CFU.g -1 ). The presence of coliform bacteria can be caused by fertilization of bio food with faecal matter, insect vector transport or by contaminated water. Červenka, Brožková and Fišerová (2016) reported that the content of coliform bacteria was in the interval from 1.9 to 4.4 log CFU.g -1 in samples of raw food but, in two samples, such increase was not noticed due to the antimicrobial effect of young barley. In their study, Brožková et al. (2016) presented the contents of coliform bacteria in raw materials for the production of raw bars, such as hazelnuts (2.9 log CFU.g -1 ), goji (2.8 log CFU.g -1 ), cashew (<1 log CFU.g -1 ), chia seed (<1 log CFU.g -1 ) and linseed (5.9 log CFU.g -1 ). Although the microbicidal effect of essential oils was described in citruses (Oikeh et al, 2016), these compounds did not have a significant effect on the number of coliform bacteria in B1 and B10 samples. The number of staphylococci (MSA) was found to range from 2.9 to 7.5 log CFU.g -1 , with the highest concentration being detected in samples B3 (7.5 log CFU.g -1 ), B10 (5.3 log CFU.g -1 ) and B2 (5.2 log CFU.g -1 ). Enterococci (SB) were recorded in 8 samples with numbers ranging from 3.0 to 4.13 log CFU.g -1 . Streptococci (M17) and lactobacilli (MRS) were observed in all samples, the number of streptococci ranged from 2.6 to 6.4 log CFU.g -1 and the number of lactobacilli (MRS) ranged from 2.3 to 6.1 log CFU.g -1 .

CONCLUSION
The first part of this study concerned the characteristics of raw food, its microbial quality and the problematics of biogenic amines. 15 types of raw bars with various content composition were selected for this experiment (Table 1). These foodstuffs were subjected to a microbial analysis with a goal to find indicator groups of microorganisms (facultative anaerobic mesophilic microorganisms, enterobacteria, staphylococci, yeasts, moulds, and lactic acid bacteria). The highest concentration of biogenic amines was recorded in the sample of the raw bar containing vegetable components with this product containing, beside others, a biogenic amine tyramine in concentration 42.23 mg.kg -1 and a polyamine putrescine in concentration 53.95 mg.kg -1 . More than a half of the samples did not exceed the limit of concentration of biogenic amines 15 mg.kg -1 ; two thirds of the samples did not exceed the limit 20 mg.kg -1 . Identification of present microorganisms proved that the most represented genus were Micrococcus, Staphylococcus and Bacillus, which a decarboxylase activity was observed in. Taking this fact into account, it is important to consider the content of individual biogenic amines in the tested samples. The achieved results of this study show that raw bars contain various microorganisms according to their content composition. It is necessary to pay attention to the content of individual types of foodstuff and their microflora, especially in relation to human health. Even though it was not a primary goal of this study to focus on presence of

B11
moulds, the occurrence of mycotoxigenic genus Aspergillus and Penicillium in the studied samples is alarming. The presence of mycotoxins is very probable in these products and that is why it would be suitable to focus the studies of raw bars this way. The amounts of biogenic amines in the tested samples were not high. However, it is important to consider a "cocktail effect" of these substances and to consume raw bars in moderate amounts.