Determination of Boza's Organic Acid Composition as it is Affected by Raw Material and Fermentation

Abstract

In the current study boza, a low-alcohol-content beverage, is produced from rice, maize, millet and wheat flours by spontaneous fermentation at 24 ± 1°C for 24 h. During the fermentation and storage period, changes in total titritable acidity, pH, alcohol and organic acid profiles, and sensory properties were determined. The major organic acids were found as oxalic, lactic, pyruvic, and acetic acids. Even though the ratings of sensory parameters were not within wide limits, they were significantly affected by storage time. Maize boza scored higher due to the similarity in its colour and flavour to that sold in the market.

Keywords:

• Boza
• Organic acids
• Fermentation
• Sensory properties

INTRODUCTION

The consumption of lactic acid fermented food and beverages is common mid-Asian, Middle East, African, and Balkan countries.[1] One such traditional beverage is boza, which is made through the fermentation of millet, cooked maize, wheat, or rice semolina/flour by natural mixtures of yeast and lactic acid-producing bacteria.2–6 The name boza, in Turkish, comes from the Persian word buze, meaning millet. However, the Turks who lived in Middle Asia named this beverage bassoi. The beverage was known in Central Asia for centuries and from there it was introduced to Anatolia and Europe with immigration. Even though the Turks were the first to produce boza, the subject was significantly suppressed by researchers and boza was introduced into the market by other countries, as their national product.[7,8] In East European countries, the beverage is braga or brascha, in the Balkans it is busa, in Bulgaria bozan, bousa, or bouza in Nigeria, and in Egypt it is bouza.[1,9–13 These vareties contain a high concentration of alcohol (up to 7% by volume).

Boza is regarded as the origin of beer and is considered to be an old drink with a history of 8000–9000 years. It is a thick liquid, with a sweet-sour taste, pale yellow in color with a characteristic acidic–alcoholic odor and is a favourite drink during winter nights.[1,14,15] The steps to produce boza can be summarised as: (i) preparation of raw materials, (ii) boiling, (iii) cooling and straining, (iv) addition of sugar, and (v) fermentation at 30^oC for 24 h.[7,13] According to Turkish Standards, boza is defined as “a product which is made by adding drinkable water to cereals such as millet, maize, wheat, and rice. The sugar is then added to allow alcohol and lactic acid fermentation. Boza can be classified as sweet or sour boza depending on its acid content.”[16] Even though barley, oat, potato or bulghur, in combination with the cereals mentioned in Turkish Standard, can be used for boza production, the millet boza is defined as the product having the most favoured sensorial characteristics.[7, 17]

The cooling effect of lactic acid enables boza to be consumed during summertime; however, the high temperatures during that season result in rapid growth of present microflora, and consequently, in dramatic changes in sensorial attributes. Thus, boza is a congenial beverage for winter.[9,13,14] The sensorial quality of boza may be affected by the raw material, microflora, fermentation process and storage conditions.[18] A quantitative determination of boza's organic acids, the major flavor and aroma compounds of fermented foods, is important for the understanding of the flavor profile and the monitoring of the fermentation process along with microbial activity.[19] The aims of this research were: (i) investigate the organic acid profile of boza during fermentation and storage; (ii) evaluate its sensory properties; (iii) determine its selected characteristics.

MATERIALS AND METHOD

Materials

Raw cereal materials, rice, maize, millet and wheat flours, as mentioned in Turkish Standard TS 9778, and sugar were obtained from local markets. Boza obtained from the local market was used as inoculum. Chromatographic grade organic acid standards were purchased from Sigma (St. Louis, MO, USA), Fluka (Buchs, Switzerland) and Supelco (Bellefonte, PA, USA).

Boza Production

Boza was prepared under laboratory conditions with sugar, and the flours of rice, maize, millet and wheat according to the modified method of Zorba et al.[20] The selected cereal was cleaned of foreign materials, broken into semolina size pieces, and sifted to remove hull and bran. The semolina was mixed in 10 times water (w/v) and boiled in a stainless steel steam-jacketed boiler for 2 h under continuous stirring until a homogenous mixture was obtained. Since the mixture absorbs water, hot water was added during the boiling process. The mashed cereal was filtered through a pulper to separate the starchy part from barks, gums and ungelatinised harsh particles. The filtrate was then termed as unsweetened raw boza. After dilution with water and the addition of 20% (w/v) sucrose, the raw boza was allowed to cool overnight. The sweetened boza was poured into 5 L containers and inoculated with 2% (w/v) boza from the previous batch. Fermentation was carried out at 24 ± 1^oC for 24 h. Following the fermentation process, the boza was cooled to 10 ± 1^oC and stored for 7 days. At predetermined times; samples were taken for the analysis. All experiments were performed in triplicates.

Chemical Analysis

The samples' total titritable acidity and pH values were determined as described by Kozat,[13] whereas the amount of alcohol was evaluated by distillation.[21]

Sample Preparation and Analysis of Organic Acids

Individual organic acids in the boza samples were extracted using the method of Erbas et al.[22] The extracts were analysed using the HP 1100 Series HPLC system consisting of HIChrom column (Hypersil H5 ODS, 25 cm × 4.6 mm) and a G1315A DAD detector at a wavelength of 215 nm with phosphoric acid (0.01%) as the mobile phase. The flow rate was 1.0 mL/min. The injection amount was 20 μL with a total elution time of 10 min. All determinations were performed from three separate extractions of each sample and each was injected in duplicates. The organic acid standards were prepared in the mobile phase and the chromatograph was calibrated using this standard solution. Peaks obtained from the samples were identified by comparing of retention times with standard compounds and the relative percentage amount of separated organic acids was calculated from the peak area.

Sensory Evaluation of Boza

Sensory evaluations were applied using a scoring test modified from Kramer and Twigg[23] with six trained panelists, who are members of the Food Engineering Department. The panelists were selected on the basis of their co-operation and interest in the sensory evaluation of fermented products, These individuals were trained using commercial boza samples. The boza samples were evaluated with a five-point scoring test, where 5 indicated extreme likeness and 1 extreme dislikeness with respect to appearance, taste, color, aroma, odor, mouthfeel, and texture. The experimental vocabulary was similar to that mentioned for organoleptic profiling of boza by Zorba et al.[18] The sensory session was conducted in an air-conditioned room under white light with panelists seated in separate booths. Boza was presented as 100 g samples in glasses, at 10 ± 1°C, along with water and crackers, in order to neutralise the taste from their palates between samples. Total acceptability ratings were taken into account as the sum of evaluated sensory properties. The panelists were asked to note any defects or undesirable characteristics. Sensory evaluations were applied during the fermentation and storage process.

RESULTS AND DISCUSSION

The chemical properties of boza during the fermentation and storage time are significantly affected by the raw material (p < 0.01) (Fig. 1). The compositional differences of boza samples may result from the use of different cereals as the raw material and their amounts in the recipe.24–26 The raw material affects the amount and quality of carbohydrates available as primary fermentation substrates, nitrogen sources, and growth factors for microbial activity.

Figure 1 Changes in (a) total titritable acidity, (b) pH, and (c) alcohol content of boza samples during fermentation and storage made with different raw material; (▄) rice, (▴) maize, (●) millet, and(○) wheat.

Total titritable acidity in terms of lactic acid was found to be lowest in millet with 0.32 ± 0.04% and highest in wheat boza (0.61 ± 0.07%) (Fig. 1a) due to the probable high fermentable carbohydrate content of wheat compared to other raw materials. The pH varied between 3.43 ± 0.08 and 3.86 ± 0.17 (Fig. 1b). The acidity of the samples increased during storage (being highest at 192 h with 0.68 ± 0.06%) in accordance with a decrease in the pH. Furthermore, the alcohol content was lower in wheat boza (0.46 ± 0.04%) (Fig. 1c) and showed fluctuations during storage depending on microbial and enzymatic activity. The acidity and alcohol content depended mainly on the fermentation period. It could be stated that with longer fermentation, the acidity content increases as does the production of alcohol.

Two main types of fermentation occur simultaneously during boza production. First, alcohol fermentation by yeast that produces carbon dioxide and increases its volume. Second, lactic acid fermentation that produces lactic acid and gives boza its acidic character.[1,12,13] The activity of lactic acid-producing bacteria and yeasts present during fermentation may affect boza's composition. These microorganisms compete for the available sugar source in order to produce fermentation metabolites (alcohol, organic acids, and free acids) according to their activity and cell counts.

Patterns of change in acidity and alcohol content were greater than those of Kose and Yucel[27] and Kentel,[28] however, they were lower than the results mentioned by Yavuz.[15] Ustun and Evren[29] investigated the sensorial, chemical, and physical properties of boza produced from bulgur, bread, millet, potato, rice, maize, and wheat. According to their findings, the alcohol content was 0.14–0.53%, titritable acidity (such as lactic acid) was 0.24–0.45%, and the pH was 2.93–3.72. Hancioglu and Karapinar[30] investigated changes during the fermentation of boza made from combinations of cooked rice, maize, and wheat flour, using the traditional method, and found that the pH of the product dropped from 6.13 to 3.48, the total titritable acidity of the samples increased from 0.02 to 0.27%, and the alcohol content increased from 0.02 to 0.79%. According to the Turkish Boza Standard,[16] ethyl alcohol content should not exceed 2% by volume in boza and the total titritable acidity, by means of lactic acid, should be 0.2–1.0%.

Based on the limited information available, during boza fermentation, Candida glabrata, C. tropicalis, Geotrichum candidum, G. penicilatum, Saccharomyces carlsbergensis, S. cerevisiae and S. uvarum as yeasts strains,[1,3] and Lactobacillus acidophilus, Lb. brevis, Lb. coprophilus, Lb. coryniformis, Lb. fermentum, Lb. paracasei, Lb. pentosus, Lb. plantarum, Lb. rhamnosus, Lb. sanfrancisco, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides, L. raffinolactis, L. mesenteroides subsp. dextranicum, Pediococcus pentosaceus, Oenococcus oeni, Weissella confusa, and W. paramesenteriodes as bacterial strains have been identified.[1,3,6,30] The sensorial attributes as well as alcohol and organic acid content of boza were thought to be significantly affected by the strains present and their metabolic activities.

Boza's organic acid content during fermentation are given in Table 1. No relevant research is available concerning the organic acid profile of boza during fermentation and storage. Organic acids are produced during the metabolism of fermentable sugars. In boza, the main organic acids were found to be oxalic, lactic, pyruvic, and acetic acids. In some samples, citric, orotic, and malic acids were also discovered. The production of organic acids in fermented cereals is attributed mainly to the presence of homo- and hetero-fermentative lactic acid bacteria.[22,31] Montanari et al.[32] reported on the possible formation of malic, pyruvic, lactic, acetic, citric, succinic, and fumaric acids by yeasts during alcohol fermentation. Pyruvic acid is produced as a by-product during the conversion of glucose into lactic acid and ethanol, for the duration of metabolic activity EMP (Ebden Meyerhof Parnas).[22] Oxalic acid might be formed during fermentation from the resultant alcohol. The alcohol is oxidized into acetic acid, which is further oxidized to oxalic acid, and finally to carbon dioxide. Changes in citric acid content during fermentation and storage are thought to result from its usage as a substrate in secondary reactions and metabolic activities of lactic acid-producing bacteria and yeasts.

Table 1 Organic acid contents of boza samples during fermentation and storage (mg/kg boza drink)

	N	Oxalic	Pyruvic	Malic	Lactic	Acetic	Orotic	Citric
Treatment
Rice	12	381.55 ± 55.900^b	56.613 ± 53.177^d	8.163 ± 5.500^a	416.860 ± 34.500^a	71.783 ± 4.350^b	0.1633 ± 0.111^b	21.502 ± 4.800^b
Maize	12	186.22 ± 26.300^d	82.607 ± 9.736^a	nd	276.620 ± 20.100^c	49.513 ± 4.810^c	0.4933 ± 0.111^a	43.628 ± 16.000^a
Millet	12	253.78 ± 32.000^c	79.405 ± 8.619^b	nd	197.770 ± 14.500^d	43.827 ± 3.930^d	nd	4.815 ± 3.250^c
Wheat	12	425.50 ± 44.900^a	70.640 ± 8.246^c	nd	322.620 ± 21.200^b	81.902 ± 6.760^a	nd	nd
Fermentation Time (h)
1	8	169.330 ± 21.000^e	76.702 ± 3.100^c	12.245 ± 8.020^a	348.330 ± 43.500^b	46.463 ± 6.730^e	nd	nd
24	8	142.290 ± 20.900^f	72.380 ± 10.587^d	nd	299.520 ± 39.100^d	38.376 ± 3.490^f	0.245 ± 0.161^a	nd
48	8	287.950 ± 46.400^d	81.955 ± 9.835^a	nd	212.840 ± 22.100^f	63.935 ± 6.070^d	0.168 ± 0.110^cd	35.100 ± 5.740^b
96	8	331.700 ± 34.800^c	78.979 ± 9.547^b	nd	222.160 ± 18.600^e	66.808 ± 8.640^c	0.163 ± 0.107^d	25.580 ± 5.750^d
144	8	477.440 ± 52.300^a	59.512 ± 9.089^f	nd	321.450 ± 20.600^c	71.448 ± 4.900^b	0.190 ± 0.131^c	40.270 ± 6.640^a
192	8	461.850 ± 52.900^b	64.370 ± 8.899^e	nd	416.510 ± 41.500^a	83.507 ± 7.950^a	0.220 ± 0.144^b	28.060 ± 4.550^c
ANOVA
Treatment (T)		**	**	**	**	**	**	**
Fermentation Time (FT)		**	**	**	**	**	**	**
TxFT		**	**	**	**	**	**	**
nd: not detected. Values are means of all fermentation time ± standard error; different superscript letters on the same column indicate significant differences; Duncan's multiple range test (**p < 0.01).

Organic acids are known to contribute to the flavor of many foods, including fermented foods. The aspect of flavor most commonly associated with organic acids is a sour taste, although other taste characteristics, particularly astringent and bitter, have also been reported.[33,34] Lactic acid-producing bacteria play an important role in the preservation, microbiological stability, and production of sensorial compounds of fermented foods.[35,36] Organic acids produced during fermentation affect acidity, pH and/or taste of fermented foods, and directly influence sensorial attributes. Fermentation is known to improve a product's organoleptic qualities. The correlations of the different sensory attributes with acceptability during the fermentation of boza are demonstrated in Fig. 2.

Figure 2 Correlations of appearance, taste, color, aroma, odor, mouthfeel, and texture ratings of boza beverage made using different raw material with overall acceptability.

Even though the ratings of sensory parameters were not within wide limits, the length of the storage period affected the properties of boza. Maize boza scored higher for all sensory attributes, most likely due to the similarity of its color and flavor to boza available on the market. For rice, millet and wheat boza, the evaluated properties were highest on the first day of storage, and declined thereafter. Substantial differences in sensory characteristics were noted between boza made with various cereals. It appeared that the panelists preferred boza with a slightly acidic/alcoholic taste and odor, instead of higher acidity, with a yellowish-cream color. Even though the panelists were not ask to compare the boza samples with traditional boza, it is observed that the judges were negatively influenced by color.

According to Turkish Standard, boza should have a pale or dark cream color with a unique flavor.[16] Differences in sensorial properties were significantly associated with the cereal type used. Boza samples made from rice and millet were found to be more fluid than maize and wheat boza. Rice boza was not preferred due to its color. The preparation of boza is normally carried out by spontaneous fermentation with mixed cultures of lactic acid-producing bacteria and yeasts. Interactions between microorganisms are not controlled during the process, which leads to variations in the product's sensory characteristics, composition, storage stability, and fermentation profile.[11]

CONCLUSION

Boza is a traditional beverage made by yeast and lactic acid-producing bacteria through the fermentation of cereals, and is a nutritious beverage, which has positive effects on digestion and intestinal flora due to its microflora. It has been observed that variations occur in the acidity, pH, alcohol and organic acid profile of boza samples made from rice, maize, millet, and wheat during the fermentation and storage periods. Diversity in compositional differences may be due to the raw material, the microflora present, and production processes, which are all thought to have important effects on sensorial attributes and consumer preference. The major organic acids in bozas are oxalic, lactic, pyruvic, and acetic acids. These acids along with the alcohol content influence the sensorial properties. During a storage time of seven days, it was determined that the samples maintain their sensorial properties at acceptable level. This study was one of the first ones to determine the organic acid content of boza produced from different, individual cereals, not in combination with each other. In order to understand the fermentation profile and metabolic pathways that take place, further detailed work is needed to identify the microflora that contribute to the fermentation process.

ACKNOWLEDGMENT

The authors wish to thank the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: TOVAG–105 O 463) for the financial support.