Effect of Traditional Dried Starter Culture on Morphological , Chemical and Physicochemical Properties of Sweet Fermented Glutinous Rice Products

Sweet glutinous rice products or khao-mak in Thai were prepared by fermenting cooked glutinous rice (RD6 variety) using traditional starter cultures from Samut Sakhon (SSK) and Pathum Thani (PTT) Provinces at room temperature (28±2 °C) for 3 days. Amylolytic activities of microbial excisting in the traditional starter cultures were tested. Meanwhile, types and amounts of microbial growth in fermented rice products were investigated. Both traditional dried starter cultures showed strong amylolytic activity after inoculation for 2 and 3 days in starch agar. Microbiological profiles of rice fermentation were varied in total plate count, yeast and molds over the fermentation period. Different starter cultures resulted in a wide variation of end products with diverse chemical, microbiological and morphology properties; however those changes exhibited similar trends in all treatments. As fermentation time increased, protein content remained unchanged while total soluble solids, pH, acidity, sugar and alcohol content markedly increased compared with unfermented cooked rice. PTT showed products with higher acidity and alcohol content (0.50%, 2.58%) than SSK (0.31%, 1.85%). Morphological examination of rice grains revealed that microbial growth started from the outer surface and then moved inside the rice grains with greater corrosion was observed as fermentation time increased. Pasting and gelatinization properties of the rice flour were also strongly affected by fermentation.


Introduction
Historically, traditional fermented foods have played a very important role in many countries including Thailand, either as main dishes or as condiments.Nowadays, the younger generation has become less interested in these products, which have good impacts on health, due to the influence of Western style diets.Among fermented food products, sweet fermented glutinous rice known as "Amasake" in Japanese or "khao-mak" in Thai is popularly consumed as a sweet dessert or as a seasoning that provides both sweetness and an appetizing aroma of alcohol and acids (Tanasupawat & Komagata, 1995).Campbell-Platt (1994) reported that the fermentation process had several inherent benefits by altering the functional properties of starch embedded in the rice.Generally, final product quality is strongly influenced by the production processes.Khao-mak is a probiotic product (De Llanos, Querol, Pemán, Gobernato, & Fernández-Espinar, 2006) containing viable helpful microorganisms (mold and yeast) which confer health benefits when consumed in adequate amounts (Manosroi, Ruksiriwanich, Kietthanakorn, Manosroi, & Manosroi, 2011).Khao-mak is also used as a dietary supplement to increase and maintain a healthy gut flora by providing food for bacteria (Tongyai, Riebroy, Maneerat, Siriwong, & Chulakarungka, 2012).
Traditionally, khao-mak is usually prepared by washing whole grains of glutinous rice and steeping them 3 h to allow the water to diffuse into the rice kernels; however, some components leach out (Chen, Lu, & Lii, 1999;Phothiset & Charoenrein, 2007).The soaked rice grains are cooked using steam to gelatinize the starch, and then cooled to room temperature prior to brewing with a starter cake containing mold, yeast and bacteria inoculated in rice flour mixed with herbs (Limtong, Sintara, Suwanarit, & Lotong, 2002).The mixture of cooked rice and starter is then fermented at room temperature for 2 or 3 days to obtain the desired khao-mak characteristics as lumps of cooked glutinous rice with soft texture, pale white color, succulent grains, and acidic sweet taste with little alcohol flavor (Wongpiyachon, 1995).Longer fermentation is not acceptable because it creates off-flavors due to excessive alcohol and acid content (Tochampa, Jittrepotch, Kongbangkerd, Kraboun, & Rojsuntornkitti, 2011).Generally, product quality characteristics vary considerably from place to place depending on many factors including traditional starter cultures, environmental conditions and fermentation times.Although khao-mak has been fermented and consumed since the olden days, scientific data concerning changes in rice morphology and properties during fermentation remain limited.Therefore, this research investigated the changes in rice grain morphology and chemical composition as well as physicochemical properties of rice flour during fermentation.Increased knowledge through collecting process practices monitoring data can be used to improve the quality of khao-mak as a healthcare food choice for consumers.

Materials
Glutinous rice, RD6 variety from Nong Khai, a province in northeastern Thailand, was used as raw material for sweet fermented glutinous rice or khao-mak production.Traditional dried starter cultures, or rice cake starter, were collected from Samut Sakhon (SSK) and Pathum Thani (PTT) provinces in the central part of Thailand.

Khao-Mak Preparation
The method for preparing khao-mak in the laboratory was adapted from a local manufacturer, with some modifications.Rice kernels were washed and soaked in water for 3 h.After draining, the soaked rice was steamed for 25 min, then cooled for 1 h at room temperature, and water was added to make the cooked rice less sticky.Cooked rice was mixed with starter culture powder (0.14% w/w of sample), packed in plastic bags (200 g/each) and incubated at 30 °C for up to 3 days to induce fermentation.The fermented products were randomly withdrawn from the incubator every day.One part of the sample was blended and kept at -20 °C for composition analysis, while the other was freeze-dried and used for morphological and physicochemical analyses.Unfermented steamed glutinous rice added with water was used as a control.

Microbiological Characteristic Determination
Amylolytic activity of microbes in the starter cultures was tested following the method of Laloknam, Sirisopana, Attaphinyo, Poohuarai, & Phornphisutthimas (2009).In addition cooked glutinous rice mixed with starter culture was examined for total microorganisms, mold and yeast content during the 1-3 day fermentation according to BAM (Bacteriological Analytical Manual) (1998).Total plate count were performed using plate count agar (PCA) while mold and yeast used dichloran 18% glycerol (DG18) agar, then isolate individual colonies on PDA.

Chemical Composition Analysis
Moisture, protein, titratable acidity (as %lactic acid) and alcohol content of the control sample and khao-mak after fermentation for 1-3 days were determined by the AOAC (Association of Official Analytical Chemists) (2006) standard method.The pH value of samples was measured using a pH meter (Docu-pH Meter ; Sartorius, Goettingen, Germany).Glucose, maltose and total sugar were analyzed using an Agilent 1100 series high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA) following the AOAC method (2006).

Grain Morphology
Freeze-dried sample grains, unfermented and fermented, were placed in a vacuum evaporator attached to an aluminum stub with carbon tape and sputter-coated with gold (Ion Coater IB-2, Eiko, Japan).Samples were characterized by a scanning electron microscope (SEM, JEOL JSM-5600 LV, Japan) operating at an accelerating voltage of 10 kV.

Pasting Properties
Pasting properties of all flours obtained from freeze dried grains (unfermented and fermented) were determined using a Rapid Visco Analyzer (RVA-super 3, Newport Scientific, New South Wales, Australia) with standard profile program following the International Approved Methods of Analysis [AACC] (2000) Method 61-02.01observed parameters were peak viscosity, trough, final viscosity, breakdown and setback.

Thermal Properties
Gelatinization characteristics were determined using a differential scanning calorimeter (DSC) (Pyris Diamond DSC; PerkinElmer, Waltham MA, USA) calibrated with indium.An empty stainless steel pan was used as a jas.ccsenet.

Chemical Composition
Chemical compositions of khao-mak samples obtained from the two different sources of traditional starters are shown in Table 2.During fermentation, many simultaneous changes occurred due to microbial activities.Starch molecules in the rice grains were changed to sugars, alcohols and acids.Enzymes produced by the microorganisms present in the starter (Manosroi et al., 2011) had a major effect on the properties of products obtained at different fermentation periods.After 24 h fermentation, moisture and protein content decreased.As fermentation time lengthened from 1 to 3 days, only slight changes were noted in moisture and protein content.Khao-mak products using SSK and PTT starters showed data with similar trend; however, there were significant differences from the control sample.
Table 2. Moisture, protein, total soluble solids (Brix), pH, total acidity (as lactic acid) and alcohol content in khao-mak using different starter cultures with various fermentation periods Day 0 means unfermented cooked glutinous rice or control.

Brix, pH and Total Acidity
Results in Table 2 also indicated an increase of soluble solids after fermentation due to microbial activities, until reaching a constant of around 44.It can be suggested that damage of rice grain was occurred on the first day fermentation.The pH was sharply declined after 24 h fermentation and then changed slightly.The total acidity, on the other hand, was increased over the fermentation time.This result might be due to the correlation between amounts of weak acid and pH which was factored by the ionic strength of the acid (Gustafsson, Skrifvars, Sandström, & Waller, 1995).All fermented samples had pH values lower than 4.5 which concurred with findings in rice wine production (Dung, Rombouts, & Nout, 2007).In our experiment, the pH of khao-mak prepared from PTT starter was lower than from SSK.This result agreed well with total acidity (as %lactic acid), PTT khao-mak contained relatively high lactic acid content.Decrease in pH with high acidity creates an antimicrobial effect and helps to ensure that fermented food is safe for consumption.

Total Sugar, Glucose, Maltose and Alcohol
Acids and enzymes produced during fermentation were reported to attack the glycosidic linkages of the starch granules (Mestres & Rouau, 1997), splitting starch molecules into soluble products and continuing the process for various kinds of sugar production (Fennema, 1985;Dung et al., 2007) such as oligosaccharides, disaccharides and mono-saccharides.In our experiment, total sugar and glucose contents of the samples significantly increased (p < 0.05) with the extension of fermentation period (Table 3).Maltose, in the opposite, sharply increased on the first day and declined thereafter.Some maltose was converted to glucose by acid and enzyme in the system.In the following, alcoholic fermentation occurred wherein sugar commonly glucose was changed to ethanol by the action of yeast presented in the starter (Dung et al., 2007).Moreover, alcohol might be produced by lactic acid bacteria fermentation with the presence of maltose, especially when the fermentation temperature was higher than 30 °C (Akerberg, Hofvendahl, Zacchi, & Hahn-Hägerdal, 1998).
Increase of glucose, maltose and total sugar was greater in SSK khao-mak.Sugar contributes to the sweetness and improves the taste and oral texture of the fermented product (Liu, Chen, & Lin, 2002).Alcohol content sharply increased after 2 days of fermentation and slightly increased by the third day to 2.15 and 2.58% in khao-mak prepared from SSK and PTT starters, respectively.This demonstrated the ability of microbes in traditional starters, especially yeast, to convert sugar into carbon dioxide and alcohol.In our experiment, microbial activity had more impact on alcohol formation in the PTT starter than in SSK.The presence of alcohol enhanced the acid ability to penetrate the starch granules and caused hydrolysis exclusively inside the granule (Robyt, Choe, Fox, Hahn, & Fuchs, 1996), and this affected the morphology and properties of fermented grains.Note.Different letters in the same column indicate statistical differences (p < 0.05).
Day 0 means unfermented cooked glutinous rice or control.

Morphological Examination
For ease of morphological comparison, all cooked rice grains, both unfermented and fermented as khao-mak product after 1, 2 and 3 days fermentation were freeze-dried and then observed under a scanning electron microscope (SEM).Morphology of the control sample appeared as a smooth surface of gelatinized starch, while the inner grain exhibited a porous gel structure with a dense core (Figure 2).This structural form enhanced the ability of the microflora to attack the grain and provided macro-and micro-environments suitable for its growth.Under SEM examination, all fermented freeze-dried rice grain samples showed dramatic changes on the surface from the first day of fermentation compared with the control.Grains had clearly observable rough and porous surfaces, and the pores noticeably enlarged as fermentation time increased due to exo-corrosion and breakdown of starch granules (Phothiset & Charoenrein, 2007) by enzymes secreted from microorganisms and by acid generated from fermentation.Moreover, the presence of alcohol in khao-mak affected acid properties by enhancing their ability to penetrate the starch granules, causing the hydrolysis of glycosidic linkages exclusively inside the granules (Ma & Robyt, 1987;Robyt et al., 1996), produced changes in grain morphological characteristics.In SSK khao-mak, microbial growth on the outer surface of the rice grain was observed after the first day of fermentation, and then penetrating into the core of the grain on the following days.This caused the grain to soften and release sugar, resulting in hollows inside the grain, as evidenced by the flattened structure on the third day.Similar characteristics were observed using the PTT starter but fermented rice grains showed greater damage and were more flattened.This could be the result of enzymatic activity and relatively high acid level produced during fermentation.Morphology of the grains on the third day showed dense microbial growth on the surface, along with tightly packed mycelium inside.
Day 0 means unfermented cooked glutinous rice

Thermal Properties
Thermal properties of khao-mak flour were analyzed and results are shown in Table 5. Gelatinization enthalpy of fermented flour samples sharply increased after 24 h fermentation.Thereafter, rate of increase slightly declined.
During the initial stages of fermentation, starch was hydrolyzed by acids and enzymes which quickly degraded the amorphous regions.This led to an increase in crystallinity of the residual starch which caused increase in enthalpy of gelatinization in flour fermented for 24 h.after that hydrolysis rate was slower with less degradation of the crystalline regions, leading to a slight decrease in enthalpy of gelatinization after 2 and 3 days of fermentation.Onset temperature slightly decreased, while the end temperature and temperature range increased, especially in the PTT sample.Our results agreed well with rice flour obtained from rice paper production and acid modified starch (Atichkudomchai, Varavinit, & Chinachoti, 2002;Phothiset & Charoenrein, 2007).In this study, gelatinization enthalpy of fermented flour using the SSK starter was higher than when using PTT.Note.Different letters in the same column indicate statistical differences (p < 0.05).

Conclusions
It has been concluded that traditional starter culture had the effects on the morphology, chemical and physicochemical properties of the sweet fermented glutinous rice products.Different source of starter culture used for fermentation, demonstrated the products with different qualities such as amount of microbial count, sugar content, titratable acidity as well as alcohol content.Sweet fermented glutinous rice product at 3 rd day fermentation using PTT starter culture contained yeast count, acidity and alcohol content higher than SSK.The rice grain morphology showed dramatic changes with numerous pores on rice grain surface and pore's size were enlarged as fermentation time increased.The microbial presence in the starter as well as the fermentation time had an influence on the pasting and thermal properties of the rice flour.Research is underway to obtain a more precise understanding of the changes of rice grain and properties during fermentation that might help to encourage the preservation of this traditional and cultural food.Moreover, it can be apply for further study via using mixed or pure starter cultures to ensure the most desirable and consistency products qualities for in enlarge scale-fermentation process.

Table 1 .
Microbiological characteristic determination in unfermented cooked glutinous rice (control) and sweet glutinous rice fermented during 3 days

Table 3 .
Sugar content during khao-mak fermentation using different starter cultures

Table 4 .
Pasting properties of rice flour (RVU) obtained from glutinous rice fermented with different traditional starter cultures

Table 5 .
Gelatinization properties of unfermented and fermented rice flour obtained from khao-mak at different stages of fermentation using SSK starter culture

Table 6 .
Gelatinization properties of unfermented and fermented rice flour obtained from khao-mak at different stages of fermentation using PTT starter culture