Introduction

Betaine is a non-essential nutrient which has been hypothesized to decrease the risk of vascular and cognitive diseases because of its role as methyl donor in the one-carbon metabolism. Betaine is proposed to have many other beneficial effects such as increased body endurance, antiinflammatory effect, reduced risk for neural tube defects, depression, and liver and kidney diseases. Betaine has been recommended as an ingredient in dietary supplements because of its ability to increase nutrient absorption from the digestive tract into the bloodstream, hence increasing the effectiveness of nutrient uptake (Cappello, 2013).

Betaine (glycine betaine) is a small zwitterionic compound (quaternary amine), first isolated from sugar beet. It has dual function in human organism. Firstly, it is an osmolyte which preserves the osmotic equilibrium and tertiary structure of macromolecules in the kidney tissue and allows the passing of waste products against concentration gradient into urine. Secondly, it is a donor of methyl groups required for the remethylation of homocysteine to methionine which mainly occurs in the liver (Craig, 2004). Glycine betaine intake was found effective in lowering plasma homocysteine levels in patients suffering from homocystinuria (Wilken et al., 1983) and chronic renal failure combined with hyperhomocysteinemia (McGregor et al., 2002) as well as in healthy subjects (Brouwer et al., 2000). Mildly elevated plasma homocysteine levels are considered to be independent risk factor for cardiovascular, cerebral and peripheral vascular diseases (Welch and Loscalzo, 1998). Elevated plasma homocysteine concentrations have also been implicated in the development of birth defects and various forms of dementia (Seshadri et al., 2002).

Betaine can be formed endogenously by a two-step oxidation of choline, however, it may not be synthesized in adequate amounts and generally needs to be included in the diet. Humans obtain betaine from foods that contain either betaine or choline-containing compounds (Craig, 2004). Betaine is present in foods in variable amounts depending on the source and processing. Among commonly consumed foods, high betaine foods (>150 mg/g) are rather limited and mainly include wheat bran and germ, spinach, beet, bread and other baked products, shrimps (Craig, 2004). Ross et al. (2014) and de Zwart et al. ( 2003) implicated cereal foods as the major sources of betaine in the Western diet. Plant-based food may contain various glycine betaine analogues such as proline betaine, trigonelline, dimethylsulfoniopropionate (de Zwart et al., 2003). The effects of analogues in the metabolism are not entirely known and glycine betaine was the compound primarily found at higher levels in foods whereas analogues were discovered only in small quantities (Slow et al., 2005).

Betaine can be added to the diet as a supplement. In the US, betaine is self-affirmed as GRAS ingredient whereas in Europe, it received an approval for use in foods by the European Commission (Commision Regulation ЕU 432, 2012) which allows a health claim to be made on food containing at least 500 mg betaine per portion. Health claim is related to contribution of betaine to normal homocysteine metabolism. Betaine is commercially available in three different forms: natural betaine anhydrous, synthetic betaine anhydrous and betaine hydrochloride. On an industrial scale, natural grade betaine is obtained from sugar beet molasses. Betaine is the primary nitrogenous compound in sugar beet and is extracted from molasses by water-based chromatographic separation and crystallization.

In this work, results from the analysis of betaine in a variety of cereal-based foods with an emphasis on wheat and wheat products (aestivum, spelt, durum wheat) are presented. The analysed food items also included gluten-free (GF) ingredients and products. The origin of most analysed items was local or from the wider region (Slovenia).

MATERIAL AND METHODS

Sample collection

Majority of the food samples analysed in this report was randomly obtained from the local market and food stores in Novi Sad (Serbia). Wheat grain (Triticum aestivum), all durum wheat (T. durum), triticale (xTriticosecale), barley (Hordeum vulgare), and rye ( Secale cereale) samples were from the collection of sample reserves of the Laboratory of the Institute of Food Technology. Amaranth grain and related samples were provided from the local producer. Plain GF biscuits and specialty bread enriched with molasses were ex-perimentally obtained and the details of their preparation are described elsewhere (Filipčev et al. 2015, 2010).

Chemicals and reagents

Anhydrous betaine was used as an external standard (98% purity, AlfaAesar GmbH&KG, Germany). All other solvents and reagents were of analytical grade (E. Merck, Darmstadt, Germany).

Extraction and analysis

Betaine is completely soluble in water. The solubility trend is water>methanol>ethanol>n-propanol>n-butanol>2-propanol (Wang et al. 2012). Despite good solubility in water, water was not found appropriate for betaine extraction from the samples because of simultaneous dissolution of impurities which aggravate further chromatographic separation of the analyte. Methanol was found to be more suitable extraction solvent due to better removal of undesirable impurities from the matrix.

Two grams of ground and homogenized sample was weighted and suspended in 25 mL methanol and then it was vortexed for 10 min. After a 30-min ultrasonic extraction in an ultrasonic bath (ATU Ultrasonidos, Spain), the sample was vigorously shaken and centrifuged for 10 min at 5000 r/min (Eppendorf Centrifuge 5804R). Three mL of upper methanol layer was removed and evaporated to dryness. Then the residue was reconstituted in 2 mL of water and filtered through a membrane filter (regenerated cellulose, pore size 0.22 µm, diameter 25 mm, Agilent Technologies, USA).

Betaine is a polar zwitterionic compound. Due to its physicochemical characteristics, it is not suitable to be analysed by conventional reverse-phase HPLC with UV detector. Lee et al. (2012) developed a HPLC method for betaine quantification using ELSD detector. Du Shin et al. (2012) proposed HILIC column in combination with ELSD for betaine analysis.

Betaine analysis was performed using a HPLC system Agilent (Agilent Technologies Inc., USA) equipped with a Kinetex®HILIC (Phenomenex, Germany) column (2.6µm, 100 x 2.1 mm) and ELSD detector. Separation was performed at a flow rate 0.5 ml/min with a mobile phase of acetonitrile and 10 mM acetate buffer at pH 3.7 following a gradient regime: 90% acetonitrile for 3 min, decrease to 60% acetonitrile during 15 min maintaining this proportion for 6 min and then final increase to 90% acetonitrile. Total runtime was 30 min. Injection volume was 5 µL with autosampler injection mode. Injector was at room temperature. Detector parameters were as follows: evaporator temperature 40 °C; nebulizer temperature 55 °C; gas flow rate 1.60 SLM, PMT gain 3.0.

The method of external calibration was applied. The calibration of the chromatograph was carried out by the addition of anhydrous betaine standard within the range expected in the samples. The betaine area and concentrations were considered as the variables to obtain the linear regression equation. Since large betaine variation in samples was expected, low and high concentration calibration curves were prepared. For the betaine range between 0.05-0.2 mg/mL, equation Y=15573 * X-358.74 (r²=0.9859, n=4) was used whereas for the range 0.5-1.5 mg/mL, the equation was Y=19606 * X+3509.1 (r²=0.9821, n=4). All analyses were performed in duplicate.

All values were reported on a dry matter (DM) basis. DM was determined by drying samples in the oven at 105 °C overnight until the constant mass, cooling in a dessicator, and then weighing. All DM analysis was carried out in duplicate.

Statistical analysis

All calculations were performed using Microsoft Excel.

Results and Discussion

The analysed products were characterized by a variable content of betaine. The majority of samples analysed fell into the range expected from previous reports (Tab. 1, 2) but some discrepancies were observed as well. The highest content of betaine was determined in amaranth grain (7420 mg/g DM), wheat bran (5215 mg/g DM) and biscuits enriched with beet molasses (4421 mg/g DM) (Tab. 3, 4).

Betaine in cereal products

In the refined wheat products, betaine amounts ranged from 218 to 529 mg/g DM whereas in the wholegrain products somewhat higher amounts, but lower than expected, were registered (532-675 mg/g DM). Ross et al. (2014) reported 2-4 times higher betaine in wholegrain wheat products but also emphasized that discrepancies among samples may be due to variation of betaine in the field. Slow et al. (2005) noted that betaine level in grain is highly dependent on the stress level of the crop. For example, growth under drought may cause higher grain betaine levels in comparison to wellirrigated crops. Varietal differences may also be the cause of different betaine levels (Corol et al., 2012). There was a wide variation in the betaine contents of durum flour and semolina; durum flour was much lower in betaine (278 mg/g DM) than the semolina sample (1227 mg/g DM) (Table 3). Literature data mainly reports higher betaine contents (700-1400 mg/g DM) in pasta products which are mainly prepared from durum wheat (Tab. 1).

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