Determining Ethanol in Fermented Glutinous Rice

1.0 Introduction
Fermented glutinous rice is a popular dessert among Malaysian consumers. It consist of carbohydrate such as rice and it need to be fermented for a few days until the glucose have been converted into the ethanol, carbon dioxide and energy whereas it signs is when the taste is sweet flavor. Cassava also can be fermented. Both of dishes served mostly in Southeast Asia. When the processes are completed, commonly the fermented glutinous rice will be wrapped in banana leaves. During the fermentation process, it most often done by a product called ‘ragi’. However, the consumption of fermented glutinous rice had rise the halal issue among the Muslim community. It is because the ethanol content that is produced during the fermentation is 5% which is similar to the concentration of ethanol in alcoholic beverages such as beers. As we know, the value of alcohol can be contained in the food that had been stated by JAKIM is 0.01% only. As we know, when a lot of alcohol are consumed, it can affect our health and can caused death. There are many analytical methods that had been studied by researchers to determine the alcohol contained in food. Thus, FTIR spectroscopic had been chosen based on the easiest, low time consuming and low cost for determination of alcohol in fermented glutinous rice. Thus, it can give a contribution by providing the alcohol content for the sake of Muslim.
2.0 Rice
The staple food for nearly two-thirds of the world’s population is rice (Oriza sativa L.). According to Arendt (2013), in 2010, China, India, Indonesia, Bangladesh, Vietnam and Myanmar alone provided more than 75% of the world’s total rice production. As we know, the lowest protein content of all the cereals is rice but it also highly nutritious among others. It is rich in carbohydrates. There are many ways that it can be consumed by the consumers. For instance, in the form of puffed rice, noodles, snack foods and fermented sweet rice. To make bakery products, sauces, infant foods, breakfast cereals, alcoholic beverages and vinegar, it also can be used.
2.1 Fermentation
Fermentation is one of the natural processes. According to Theresa (2014), when organisms convert nutrients for example carbohydrate such as starch and sugar into an acid (lactic acid and acetic acid) or an alcohol, it is called as energy-yielding anaerobic metabolic process which is occurs in fermentation. For instances, to obtain energy, yeast will perform fermentation by converting the sugar into the alcohol. Many people all around the world use fermentation to produce products such as wines, cheese and beers. It is also known as one of the historical biotechnological processes.
2.2 Fermented Glutinous Rice
Tapai or also known as fermented glutinous rice is one of the Malaysian favorite dessert. It is come from Indonesia and Malaysia. Actually it is sour and sweet alcoholic paste, have a very pleasant aroma and it also can be used directly as a food or in traditional recipe. Normally, it is wrapped with rubber tree leaves or banana leaves. It can be made from carbohydrate sources such as cassava, glutinous rice or white rice. It have to undergo fermentation process which is performed by many types of moulds including Rhizopus oryzae, Aspergillus oryzae, Rhizopus oryzae or Mucor species, and including yeast for example Saccharomycopsis fibuliger, Endomycopsis burtonii and others,along with the bacteria. Besides, tapai also can be used to produce alcoholic beverages.

Figure 1: example of fermented glutinous rice
2.3 Preparation Of Fermented Glutinous Rice
To produce fermented glutinous rice, the ingredient needed is glutinous rice, yeast and sugar. The procedures are as follow:

The glutinous rice was washed and soaked it overnight
Glutinous rice will be cooked . After it had been cooked well, we need to cool it.
After it has cooled, the yeast was pounded and then sprinkled it on the top of cooked rice.
sugar was mixed with water and sprinkle each layer of rice with water to start the fermentation
The rice was packed in layers into a tight container. Each of the layer was sprinkled with some sugar water to allow the fermentation proces. It will take about 2 days for fermentation occurred.

After 2 days, there will be a bit liquid in the bottom of the container. At this time, the fermentation is complete. The liquid which collects at the bottom of container is called brem (rice wine). Originally it have a low alcohol content after fermentation in a few days but if it continued fermented, the alcohol content will increase. Yeast will carried out both anaerobic respiration and aerobic respiration. In the fermentation in of yeast, anaerobic respiration is occurred which is the yeast will secrete the enzyme zymase (Ali et al, 2012). According to Ali et al (2012), this enzyme will hydrolyse glucose to form the ethanol, energy and carbon dioxide in the absence of the oxygen
Glucose → Ethanol + Carbon Dioxide + Energy
The enzyme can speed up the fermentatation process and only a small amount of enegy used. Therefore, in ethanol there is a large amount of energy stored as chemical energy due to the incomplete broken down of glucose in anerobic respiration.
3.0 The Importance of Determination of Alcohol
In western countries, there are a lot of alcoholic beverages for example beers, wines and distilled spirit drinks. A drink that typically containing below 60% v/v of ethyl alcohol is known as an alcoholic beverage (Gallignani et al,2005). There are three main classes of alcoholic beverages which are wines, beers and distilled spirit. Ryan (2014) stated that the alcoholic beverages which are made from germinated barley (malt), water, yeast and hops are known as beers while alcoholic beverages that produced by the fermentation of fresh grapes and grains for example rice are called as wines. The distilled spirituous beverages that contained 15% of alcohol or above and made from tubers, grain, fruit or sugar cane are known as spirit. But it is clearly that it is forbidden for Muslims to consume it as we know the alcohol content that exist in the beverages.

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On the other hand, in Malaysia we are not consuming the alcohol in the alcoholic beverages but we are consuming alcohol in the food product without realized about it. As stated above, alcohol can be produced naturally in some product that we consumed in our daily life such as soy sauce, fermented cassava and fermented glutinous rice that also become the Malaysian favorite food especially during the celebrations. Besides, there are also laboratories or specialized factory that produced ethanol artificially for the processing of various consumer products. Generally, the issue of the existence of alcohol in food is still been debated until today in Malaysia. The confusion is not only faced by the society but it also faced by the academicians and scholars. Glutinous fermented rice seems to have ethanol content higher than had been stated by JAKIM. Compared to the others natural product like soy sauce, the alcohol content in the fermented glutinous rice is about 5% of ethanol.
Table 1: Alcohol content (ethanol) in fermented glutinous rice and fermented cassava.
(The Malaysian Agricultural Research and Development Institute (MARDI) , 1983)

Alcohol content (ethanol) %

Day

Fermented glutinous rice

Fermented cassava

3.0

3.4

2.8

5.2

3.3

Based on the table 1, the ethanol content on the day 3 of the fermentation showed that the ethanol content in the fermented glutinous rice is 5.2% which is higher than the fermented cassava which is 3.3%. The alcohol content in fermented glutinous rice is similar to the alcohol content in beers which is 5%. Thus it had raised the halal issues to the Muslim community in Malaysia. Apart from that, consuming too much alcohol can lead to the bad consequences to the consumers especially for the acute chronic consumer. It is the main reason why we need to study the alcohol contents in fermented glutinous rice. The information about the content of alcohol can be such a contribution to the people and for the sake of Muslims all around the world. It also can make them more aware about what they had consumed.
The ethanol content in fermented glutinous rice can increase when we continuing kept it for a longer time. At the certain time, the liquid produced can be categorized as alcohol as it can make us drunk if over consumed. As we know, when we consumed alcohol in a large amount, it can give a bad consequence effect to the consumer. Besides having pervasive psychosocial and economic consequences, alcohol is one of the main contributors to morbidity and mortality (Acharjee et al, 2015). All around the world, the number of deaths and substantial disability from medical and psychiatric consequences of alcohol misuse is increases day by day. Clearly in term of health, it is not good for us to consume alcohol in our life. Alcohol has an adverse impact on human. It has been proven in science, psychology and sociology. Acharjee et al (2015) also stated that large amount of alcohol has harmful effects on our health. For instance, alcohol can effects our cardiovascular such as hypertension, sudden cardiac death, stroke, cardiomyopathy and cardiac arrhythmias. In term of psychology, some crime and immorality in society happened due to the intake of alcohol. Often, drunks do not have a good attitude because when they get drunk, they were not aware of their behavior, which can cause discomfort to the community. This group also can apply chaos in a country because when alcohol addicts exist in a government or private sector, it can lead to abuse of power, property, embezzlement of state funds and breach of trust.
4.0 ANALYTICAL METHODS IN DETERMINATION OF ETHANOL
A number of analytical methods have been employed with the aim of determination of alcohol. Commonly, there are combination of methods in order to achieved better result while analyze the sample. In western countries, various type of analytical method were developed to determine alcohol in alcoholic beverages.
For example, Vonach et al (1998) had presented the coupling of high-performance liquid chromatography (HPLC) with Fourier –transform infrared spectroscopy (FTIR). It can be used to determine the main component of wine such as glucose, fructose, citric, lactic, tartaric acid, glycerol, ethanol and so on. When it was about the sample preparation, it seems that the process was a little bit tedious because when using the internal standard, it need to be dissolved in distilled water and many more process need to carry out. The stationary phase and mobile phase that used in this study is ion-exchange resin based column and sulfuric acid. From the external reference data, an average deviation can be resulted which is 0.16mg/ml (Vonach et al, 1998).
Yarita et al (2002) had proposed HPLC-FID to determine the ethanol content. Pure water had been chosen as a mobile phase in this study. When liquid water was introduced in FOD system, it changes into vapor. Due to limited flow rate of the HPLC eluent that introduced in FID system, capillary tube act as an alternative ways to control the flow rate of the HPLC eluent. Triacontylsylyl (C-30) silica gel is preferred because it show less decrease in the retention time compared with other. The ethanol concentration in the six sample can be detected around 10.2%-40.8% v/v at temperature 150C (Yarita et al, 2002).
Gas chromatography also can be used as analytical tool to determine the alcohol in sample. Wang et al (2004) had proposed direct injection capillary gas chromatography that can be used to detect the methanol in beverages. Megapore capillary column with high polarity had been choosing to get the optimum condition for this study. It is because of it can used to separate the methanol in alcoholic beverages from the other components. At the stable baseline region, according to Wang et al (2004) when using acetonitrile as internal standard, it can perform clear separation from other constituents of sample. The samples studied were wine and whisky. Based on the obtained result, chromatogram give 3.06 min retention time for methanol while for the internal standard, it consumed about 4.21 min.
Gas chromatography coupling with capillary column also has been proposed by Wang et al (2004). The aim was to determine both concentration of methanol and ethanol by using two internal standards which are acetonitrial for ethanol and 2- pentanol for methanol. Wang et al (2004) stated that for methanol, the retention time that revealed by this method was 2.87min while for ethanol, the retention time was 3.41 min. The analysis of both alcohol by using this method is 12 min. Apart from that, the limit of quantitation (LOQ) for methanol was 5 μg/mL and ethanol was 5 mg/mL (Wang, 2004). These methods are assumed to give contribution in order to determine the methanol content in industries due to high accuracy and precision. However, this method is time consuming and tedious.
Vapour-phase FT-IR also can be used as analytical tool in determination of alcohol in beverages. In this experiment, there are aqueous solution of ethanol and methanol in the sample (Garrigues et al., 1997). The alcohol that contained in the sample had determined by using liquid-phase FTIR and vapour-phase FTIR. According to the Garrigues et al (1997), the result that obtained by the liquid-phase FTIR spectra, when pure water is used as a blank, there are a series of overlapping positive and negative peak (Garrigues et al., 1997). On the other hand, when discussing about FTIR spectra obtained by using vapour-phase FTIR after the injection of sample in heated plate, Garrigues et al (1997) stated that both ethanol and methanol provided a well-defined peak in the range of 1500 to 900 cm-1.The absorbance value for liquid-phase and vapour-phase are same but the spectrum for vapour-phase is clearer than spectrum for liquid-phase. Besides, the sensitivity and resolution of the alcohol band also high for vapour-phase FTIR (Garrigues et al., 1997).
For the screening analysis of beverages, NIR can be used as a spectroscopic technique ( Barboza, 2003). The applications range was limited when using NIR because it has low sensitivity (Lanchemier, 2007). According to Chandley (1993), between NIR bitterness value and spectral data, the observation is it has a poor correlations and it needs the evaporation of water (dry extract spectroscopy) to make the determination of bitterness become possible. When mid-infrared combination with FTIR spectroscopy was used, it becomes such a development in the spectroscopic analysis compared with the using of NIR. Compared to the NIR, when using FTIR, we can get better result when analyzing the minor component such as lactic acid (Lanchenmier , 2007). Furhermore,it also can produce high resolution peak and the spectra also clearer when using FTIR ( Gallignani et al, 2003). Liquid Fourier transform-middle infrared spectrometry can be used to analyze wine in a 90 s (Patz et al., 2004). FTIR/PLS have been developed for the multicomponent screening to analyze the alcohol in beverages (Neuwoudt et al, 2004).A lot of information can be obtained when using NMR rather than NIR and FTIR. According to Lanchenmier et al (2005), the distinct signals of the spectra can be produced if NMR was applied. In the calculation of cost-benefit for both NMR and FTIR, Lanchenmier (2007) also mentioned that in contrast with NMR, FTIR can give more advantage for screening method due to the less investment and operational costs. In this study, we want to determine the concentration of the ethanol in fermented glutinous rice with the use of FTIR and combination with Partial Least Square in the context of official food control. FTIR can give us advantages in order to determine and analyse the alcohol in alcoholic beverages because it can help us to reduce the time cost, and at the same time, it is also contribute for a good precision and accuracy when dealing with the parameters (Moreira et al., 2004). Furthermore, according to Lachenmeier (2007), a simple sample preparation is needed when dealing with FTIR spectroscopy compared to the other analytical methods.
Problem Statement
Recently the consumption of the fermented glutinous rice had rise the halal issue to the Muslim community since its alcohol content is similar to beer which is 5%. In order to determine the alcohol contained in the fermented glutinous rice, the government and private sector need the easiest, cheapest and low in cost analytical method for determination of alcohol. So we have come out with FTIR spectroscopy technique as this technique did not require the sample preparation like the other analytical method.
Besides, it is not time consuming method and it has low cost compared to the other method. We also combine the FTIR with combination of Partial Least Square (PLS) in order to achieve better result.
Objectives
This study was conducted based on the objectives stated below: