1. INTRODUCTION:

Heat treatment are frequently used in food processing in order to obtain safe products with prolonged shelf-life, additionally, heat treatment has a strong effect on the final quality of food[1] . However, many undesired substances are formed during heat processing and/or storage such as 5-hydroxymethyl-2-furfural (HMF).

HMF is formed by direct dehydration of sugars during caramilisation under acidic conditions. HMF is also an intermediate product in the Maillard reaction (series of reactions between reducing sugars and amino acids) [1,2].

HMF is found in bakery products, malt, fruit juices, coffee, and vinegar. Generally, HMF could be used as indicator for quality for a wide range of products like processed fruits, honey and milk .[1]Moreover, HMF is known to be a good indicator to control the heat load of industrial processed foods .[3]

The oral LD50 was shown to be 3.1 g\kg bw in rats .[4] HMF is cytotoxic at high concentrations . Additionally, HMF is irritating to eyes, upper respiratory tract, skin and mucous membranes .[1]

HMF has been shown to be converted in vitro to 5-sulfooxymethylfurfural (SMF), through sulfonation of its allylic hydroxyl functional group, catalysed by sulfotransferases (SULTs)[5] . Humans express SULTs in extrahepatic tissues more extensively than rats, because of that, human may be more sensitive to HMF[6] . Human SULT isoforms have a widespread tissue distribution and are expressed in many tissues including liver, lung, brain, skin, platelets, breast, kidney, and gastrointestinal tract .[7]

SMF reacts with DNA and other macromolecules, thereby results in toxic and mutagenic effects to bacterial and mammalian cells[8] . SMF initiated papillomas, when administered topically to mouse skin[9] , and was found to be strongly nephrotoxic in the mouse[10] .

The Scientific Panel on food additives, flavourings, processing aids and materials in contact with foods (AFC) estimated a dietary HMF intake of 1.6 mg for person per day based on an m TAMDI (modified Theoretical Added Maximum Daily Intake)- approach .[11]

The European Food Safety Authority (EFSA) established an Acceptable Daily Intake (ADI) value of 0.5 mg\ kg for furfural and the furfural component, but no reference value was mentioned for special compound like HMF or others .[12]

coffee is one of the most important contributors to dietary HMF intake[13] because of its high levels of HMF as well as its wide consumption[14] , that shows the importance of this research which focus on the determination of HMF in coffee. HMF is formed during the roasting process applied to green coffee beans where its formation depends on temperature and roasting period[15] .

There are two types of coffee Arabica and Robusta[2] . Arabica and Robusta coffees are different in many things including their ideal growing climates, physical aspects, chemical composition, and characteristics of the brew made with the ground roasted beans .[16]

Coffee brewing methods are important to estimate the exposure to HMF, which vary between countries according to social and cultural habits. There are common brewing procedures for coffee preparation such as filter, plunger, mocha, and espresso coffee brew[17] .

This study aimed to determine HMF levels in roasted ground coffee and instant coffee marketed in Syria (Arabica type), and then to study the effect of storage, sugar addition and brewing process on HMF levels, taking into account the way by which coffee is prepared in Syria.

2. MATERIALS AND METHODS:

Chemicals:

Analytical standard hydroxymethylfurfural ≥ 98.0% (Sigma-Aldrich)

Potassium Hexacyanoferrate (II) 3-hydrate 15% (Carrez solution1) (Riedel-de Haën) zinc acetate 30% (Carrez solution2) (Rectapur).

Methanol HPLC grade (Fisher Chemical).

Samples:

15 roasted ground coffee samples (Coffea Arabica L) and 8 instant coffee samples were obtained from different local markets in Latakia. Ground coffee samples were classified according to the degree of roasting, consequently to the color as dark (hard roasting), moderate (moderate roasting) and light (light roasting).

Preparation of samples:

Roasted ground coffee samples were prepared by heating 3.5 ml of distilled water until boiling, then 0.1g of roasted coffee was added. Heating was stopped as soon as the mixture of water and coffee boiled (B1).

For the preparation of instant coffee samples, 0.1g of instant coffee was weighted in a test tube and 3.5 mL of boiling water was added.

HMF Extraction:

After brewing, coffee was centrifuged at 3400g for 10 min. The supernatant was collected and then 0.5 ml of each Carrez solution (I and II) were added to clarify the extract. After centrifugation at 4000g for 10 min, the supernatant was collected again and filtered through using 0.45 μm filters, then injected into high-pressure liquid chromatography (HPLC) .[18]

Study of some factors affecting HMF levels:

Moderate roasted coffee was selected for these experiments.

Effect of brewing process on HMF:

Roasted ground coffee sample was prepared as described previously in preparation of samples, however the boiling time of water-coffee mixture lasted 2 minutes (B2).

Another sample was prepared as described previously in preparation of samples, however coffee and water were heated together until boiling (B3). All experiments were carried out in duplicate and was expressed as mean value± standard deviation.

Effect of sugar addition on HMF levels:

Roasted ground coffee sample was prepared as described previously in preparation of samples, however 0.1 g of sugar was added after stopping heat treatment (S1).

Another sample was prepared by adding 0.1g of sugar to 3.5 ml water, and were heated together until boiling, then 0.1g of coffee was added. Once the mixture boiled the heating was stopped (S2).

In another experiment, three samples were prepared with different quantities of sugar (0.05, 0.1, 0.3g). Sugar was added to 3.5ml water and heated together until boiling then coffee was added, the mixture (water-sugar-coffee) was boiled for 3 minutes (S3, S4, S5 respectively). All experiments were carried out in duplicate and was expressed as mean value± standard deviation.

Influence of storage on HMF:

Coffee was prepared as described before in preparation of samples then was stored in heat isolator bottle for twelve hours. Aliquots were taken at different times (0, 2h, 4h, 6h, 8h, 12h).

All experiments were carried out in duplicate and was expressed as mean value± standard deviation.

HPLC analysis:

Chromatography was performed on a Jasco HPLC system consisting of a pump (Jasco Pu-2089 Plus), a Rheodyne 7725i injector (Cotati, CA, USA) with a 20μL loop and diode array detector (Jasco DAD-2070). Data acquisition and evaluation was performed using Borwin data acquisition software. The chromatographic column was a BDS Hypersil C18 (250 mm×4.6 mm), particle size 5 μm and pore size 100A◦. The mobile phase consisted of water and methanol (65:35, v/v). The injection volume was 20 μl. The analysis was carried out under isocratic conditions using a flow rate of 0.8 mL/min. HMF eluted after 4.8 min. The UV detector was set at 284 nm. HMF were quantified using the external standard method within the range 0.03–8 µg/ml.

Statistical analysis:

The data obtained in the study were analyzed statistically by student t–test.

3. RESULTS AND DISCUSSION:

Fig 1shows chromatogram of HMF standard while Fig 2 shows chromatogram of HMF in roasted ground coffee sample with the standard. The sample showed polar compounds (eluted before HMF) which could be phenolic compound.

HMF in roasted ground and instant coffee:

The levels of HMF in ground coffee samples were between 9.49 and 212.08 mg/kg.

HMF levels ranged between 9.49 and 74.49 mg\kg with a mean of 26.16 mg/kg in hard roasted coffee, between 30.28 and 148.78 mg/kg with a mean of 76.39 mg/kg in moderate roasted coffee and between 123.79 and 212.08 mg\kg with a mean of 174.93 mg/kg in light roasted coffee. This means that the levels of HMF in light roasted coffee are higher than other types (table 1).

The differences observed between the samples of roasted ground coffee may relate to the thermal treatment (heat load) applied during roasting as well as to the processing type of ground coffee, but there was not a clear criteria. Roasting of green beans is a complex technological process where water loss and final color of the roasted beans reflect the end of the process.

It is found that roasting of coffee at 240°C causes a rapid increases in HMF (up to 900 mg/kg) in the first 3 min. Further roasting leads to decreases in HMF contents probably because of the occurrence of consequent degradation reactions[19] . This explains the decreases of HMF contents, which was noted in our study, in hard roasted coffee (230°C, >10 min) compared to other types.

The levels of HMF varied between samples even for the same type, this due to the differences in roasting process and thermal treatment between coffee roasters in Syria.

Table 1: HMF levels in ground coffee in Syria

Roasted ground coffee type	HMF µg\ml	HMF mg\kg	HMF mean mg\kg	HMF mg per serving (one cup 2g per 75ml)
Hard roasted	1.66 ± 0.03	74.49		0.15
	0.39 ± 0.02	17.54		0.04
	0.35 ± 0.01	15.62	26.16	0.03
	0.3 ± 0.009	13.69		0.03
	0.21 ± 0.004	9.49		0.02
Moderate roasted	2.58 ± 0.04	116.12		0.23
	0.67 ± 0.02	30.28		0.06
	3.31 ± 0.09	148.78	76.39	0.3
	1.89 ± 0.04	85.31		0.17
	0.87 ± 0.01	38.98		0.08
	0.86 ± 0.04	38.87		0.08
Light roasted	2.75 ± 0.04	123.79		0.25
	4.71 ± 0.02	212.08	174.93	0.42
	4.31 ± 0.11	193.81		0.39
	3.78 ± 0.05	170.03		0.34

The levels of HMF in instant coffee samples varied between 526.56 – 1800.9 mg\kg (table 2). It is noteworthy to mention that HMF contents in instant coffee were significantly higher than ground coffee.

Similar results were obtained by Arribas-Lorenzo and Morales, where estimated the levels of HMF as 110, 625, 1734, 2480 mg HMF/kg in natural (obtained from traditional roasting of coffee beans), blend (mixture of natural and torrefacto ground coffee at different proportions), torrefacto (addition of sucrose before roasting) and instant coffee, respectively. Additionally, Arribas-Lorenzo and Morales found that soluble (instant) coffee showed the highest level[20] .

Another study found that HMF content ranged between 594 to 5222 mg\kg in instant coffee[18] .

Table 2: levels of HMF in instant coffee samples

Instant coffee samples	Mean ± SD µg\ml	HMF concentration mg\kg	HMF mg per serving (one cup 2g)
1	15.92 ± 0.04	716.6	1.43
2	13.85 ± 0.13	623.17	1.25
3	40.02 ± 0.03	1800.9	3.6
4	11.7 ± 0.03	526.56	1.05
5	19.82 ± 0.02	892.05	1.78
6	32.09 ± 0.04	1444.13	2.89
7	12.58 ± 0.08	566.4	1.13
8	30.78 ± 0.07	1704.99	3.41

Effect of brewing process on HMF:

HMF content increased about 9.6% by increasing boiling time to 2 min (B2) compared to coffee brewed in normal procedure (B1). Similarly, HMF levels increased about 15.66% by heating the mixture of water and coffee until boiling (B3) compared to coffee brewed in normal procedure (B1) (fig 3). This could be explained by increasing the extent of the Maillard reaction and caramelization because of the applied heat treatment.

There was a significant difference between each of B2 and B3 compared to B1 (first moment of boiling).

Studies investigated how roasting conditions and brewing process affect furan occurrence in coffee and showed that furan concentrations in regular ground coffee obtained by espresso coffee machine were higher (43–146ng/ml) than those obtained by a home drip coffee maker (20 and 78 ng/ml) [15].

Figure 3: Effect of brewing process

Effect of sugar addition on HMF levels:

There was no significant differences between HMF concentrations when sugar was added after coffee preparation (S1) or when sugar and water heated together until boiling, followed by coffee addition (S2) compared to coffee prepared without sugar (B1) (fig 4). This could be interpreted that no enough time for the generation of HMF through caramelization as well as through Maillard reaction, since HMF is formed by the degradation of Amadori product.

However, the levels of HMF increased about 10.84%, 46.99%, 60.24% by the addition of 0.05g (S3), 0.1g (S4) and 0.3g (S5) sugar, respectively, where the highest HMF levels was found in the sample with the highest sugar addition (S5). A significant differences was noted between HMF concentrations in S3, S4 and S5 compared with samples without sugar (B1) as seen in figure 4. HMF increase could be related to direct dehydration of sucrose .[2]

Studies showed that The levels of HMF in traditionally prepared and instant Turkish coffee increased about three times by the addition of 8g sugar compared to samples without sugar[21] .

Another study in Spain, found that the levels of HMF was 110 mg kg−1 in natural (no sugar) versus 1734 mg kg−1 in torrefacto (coffee roasted with sugar addition)[20] .

Figure 4: Effect of sugar addition on HMF levels

Influence of storage on HMF:

Coffee was prepared and stored in heat isolator bottle for twelve hours. The levels of HMF increased about 36% during the first two hours of storage. During the remaining period of storage (10h), HMF levels remained almost stable (fig 5). Acid properties of coffee (pH = 4.89- 5.98) can play a role in increasing HMF during storage, since it induces Maillard reaction and caramelization[2] .

Therefore, HMF can serve as a heat processing index and as indicator of storage conditions[22] .

Figure 5: Influence of storage on HMF

Risk Assessment:

Several studies reffered to coffee as the main source to HMF daily intake. One study estimated the daily intake of HMF in the Norwegian population as 5.56 mg daily and reported that coffee is the most important source for it (63%)[14] . Also researchers estimated a total dietary intake of HMF as 9.7 mg daily for Spanish population in 2006, where the partial contribution of coffee was 50.43% which represents 4.9 mg HMF/day[23] . Another study determinated the daily intake of HMF in Spain related to torrefacto (addition of sucrose before roasting), natural (obtained from traditional roasting of coffee beans), blend (mixture of natural and torrefacto ground coffee at different proportions) and instant coffee, where they found 0.24, 0.59, 4.84, and 2.98 mg\ day, respectively[20] . In another study, it was estimated the daily HMF intake of traditionally prepared (after the brewing process) as 8.14–13.54 and for instant Turkish coffee as 9.36–10.25 μg/kg[21] .

In our study the content of HMF in one cup of instant coffee (one serving) was between 1.05-3.6 mg/2g and in one cup of roasted ground coffee (one serving) was between 0.02-0.42 mg/2g (table 1,2). It is noteworthy to mention that the quantity of HMF in some instant coffee samples were found > 1.6 mg mTAMDI for one cup only.

4. CONCLUSION:

Coffee is known as one of the most important source of HMF. In addition, there is no international or local regulation about the limit of HMF or related substance in coffee. So this study aimed to determine HMF levels in roasted ground coffee and instant coffee marketed in Syria, taking into account the way by which coffee is prepared in Syria.

HMF was found in all samples of roasted ground coffee and instant coffee. the levels of HMF in instant coffee were significantly higher than those in ground coffee. In this research some factors that affect HMF levels were studied. HMF content in ground coffee samples increased after increasing the boiling time or when the roasted coffee and water was heated together until boiling. Also, addition of sugar to roasted ground coffee increased HMF content, where the higher increase was formed by the higher sugar addition. As well, the levels of HMF increased during the first two hours of storage and continued stable during the remaining period of storage.

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Received on 28.03.2019 Modified on 24.04.2019

Research J. Pharm. and Tech 2019; 12(9):4263-4268.

DOI: 10.5958/0974-360X.2019.00733.9