Dusabe and Niyonzima et al doi: 10.32677/ISJ.2021.v01.i01.002

ORIGINAL ARTICLE INES Scientific Journal

Physicochemical and microbiological properties of umurahanyoni, ubuki and ikigage produced in Cyanika sector

Angélique Dusabe1, Innocent Habamenshi1, Emmanuel Munyeshyashaka2, Francois N. Niyonzima1*

1Department of Biotechnologies, Faculty of Applied Fundamental Sciences, INES-Ruhengeri, Rwanda.2Department of Biomedical Laboratory Sciences, Faculty of Applied Fundamental Sciences, INES- Ruhengeri, Rwanda

ABSTRACT

In most Africa countries including Rwanda, sorghum beer is used as a diet. It is also utilized in various festivals like wedding ceremonies. Traditional sorghum beers manufactured in rural areas are poor of hygienic quality and the mode of production is not standardized in the country. The present study was focused on the comparison of umurahanyoni, ubuki and ikigage properties produced in Cyanika sector. Three samples of sorghum beer were collected and analyzed in chemical and microbiological laboratories of INES Ruhengeri. The physico- chemical properties were determined on the 1st and 2nd day and the average was calculated. The pH of ikigage, ubuki and umurahanyoni was 4.8, 4.22 and 4.38, respectively. 1.3, 0.9 and 1.25 g/ml were total acidities for ikigage, ubuki and umurahanyoni, respectively. Degree Brix results of ikigage and umurahanyoni were 0%, whereas 2.5% was observed for ubuki. 0.35, 0.33, and 0.26 g/ml were recorded as the ash contents of ikigage, ubuki and umurahanyoni, respectively. Alcohol content results were 0.5, 4 and 0.75% for ikigage, ubuki and umurahanyoni, respectively. For microbiological analysis, the total coliforms of ubuki, umurahanyoni and ikigage noted were 209.1×108, 15.33×109 and 6.28×109 cfu/ml, respectively. However, 17.9×104, 33.5×103 and 628 cfu/ml were observed as yeast and molds of ubuki, umurahanyoni and ikigage, respectively. Three samples of sorghum beers had normal requirement of physico -chemical specifications of Rwanda Standard Board (RSB). The presence of any coliform was not acceptable by RSB. Ubuki, umurahanyoni and ikigage were not fulfill RSB microbiological specification. Water, malted grains and raw materials used by sorghum beer brewers and sellers should be treated before use and recommend them to ensure that their products meet the standards of sorghum beer. In addition, modes of storing sorghum beers in proper storing facilities with covers, proper handling and hand washing, that will help to prevent the contamination, have to be sensitized to the community.

Key words: Cyanika sector, sorghum beer, physicochemical properties, E. coli

Address for correspondence Dr Francois Niyongabo Niyonzima, Department of Biotechnologies, Faculty of AFS, INES-Ruhengeri, Ruhengeri-155. Email: [email protected]; [email protected]. Mobile phone nb: +250788536610.

1 INTRODUCTION fibres, calcium, pH of 3.9 - 4 and soluble protein which contributes to bubbles and foam stability Sorghum beer is a soft drink produced traditionally (Gupta et al., 2010). In Rwanda, only certain in all African countries with an alcohol content sorghum variety like red variety is utilized to varying between 1- 4%. It is considered as a manufacture sorghum beer known as ikigage fermented food as it is very rich in vitamins and (Lyumugabe, 2013). It is manufactured with malted calories. It also contains starch, antioxidant, dietary red sorghum, water, yeast and sometimes the

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brewer can add optimal ingredients like CO2 or 2.1 Ikigage variants collection from Cyanika lactic acid bacteria (Verma, 2000). The physical, sector microbiological and chemical properties of ikigage were analyzed by Lyumugabe et al. (2010). Cyanika is one of 17 sectors of Burera district and Georgette (2013) studied bacteriological analysis of is the most popular sector with 40 thousands of non-alcoholic sorghum drink commercialized at populations within 30 km of surface. It is Kabaya center. Production of sorghum beer surounded by Kinoni, Gahunga, Kagogo and flavored by ginger was an essay of Maurice (2014). Rugarama sectors and adjacent to Ugandan border, gateway to Kisoro and southwestern Uganda. In Rwanda, traditional beers are popular Muhabura volcan located in Cyanika providing beverages produced in all district with different their soil to be rich and agriculture is very vast names. These are igipende (Nyabihu, Musanze), where beans, irish potatoes, maize and sorghum are amarwa (Rusizi, Nyamasheke, products of agriculture (burera.gov.rw). Nyamagabe), inkangaza (Gakenke), ubushera Umurahanyoni, ubuki, and ikigage were bought (Kicukiro, Musanze), umunini (Burera), umusururu from Cyanika sector and collected in 3 different (Rubavu), munturinde (Nyamasheke, Karongi), labelled sterilized containers. It was analyzed in imbutabuta (Rutsiro), muriture (Bugesera), chemistry and microbiology laboratories at INES umumanurajipo (Nyagatare, Gicumbi), umudindiri Ruhengeri. (Kirehe, Ngoma), ubuki (Burera, Kayonza), umuneza (Rulindo, Rwamagana), bareteta 2.2 Physico-chemical analyses (Gatsibo) and ikigage (Gasabo, Nyarugenge). However, the processing methods may be less 2.2.1 pH measurement attractive because of shorter shelf life, organoleptic variation and poor hygienic quality (Lyumugabe et 50 ml of sorghum beer was transferred in a 500-ml al., 2012). beaker. 125 ml of cold water was added and the mixture was left at room temperature for 30 min Sorghum beer such as umurahanyoni, ikigage, with occasional shakings. 30 ml of the extract was umunini, umudindiri, ubushera, igipende and ubuki transferred in a beaker. Without delay, the pH was are produced in Cyanika sector of Burera district. noted after 30 s by inserting the pH-meter electrode The presence of different variant of ikigage in the extract (model 370). The calibration was suggests the use of different ingredients, and thus done using buffer solution of pH 4, 7 & 9. different properties. Ikigage is healthy and is used in social and cultural activities such as birth, 2.2.2 Total acidity baptism, marriage, dowry and funeral ceremonies (Aka et al., 2014). However, umurahanyoni 50 ml of the sample was added into 200 ml of produced in Cyanika sector causes a lot of troubles neutralized distilled water in a conical flask of 500 to consumers. Indeed, some consumers become too ml. The solution was titrated against 1 N NaOH much drunk and spent their night on the street. and phenolphthalein as indicator.1 ml of 0.1 N Others do sexual intercourses with unknown NaOH solution is equivalent to 0.00375 g of persons because of losing control. tartaric acid. Total acidity was expressed as tartaric acid, grams per 100 litres of absolute alcohol = 0.00375 ∗푣∗100∗1000 ∗2 where v = Volume in ml of Consumption of this beer becomes an addiction, 푉1 which makes people unproductive (https://www.i 0.1N NaoH used for titration; v1 =absolute alcohol, mirasire.com/amakuru yose/amakuru mashya/mu r per cent by volume wanda/article/burera-inzoga-yitwa-umurahanyoni-y adutse-muri-aka-karere-iraza-kurikora). The aim of 2.2.3 Ashes determination the present study was therefore to analyse the physicochemical and microbiological properties of Empty dish was weighted followed by weighing Umurahanyoni, ubuki, and ikigage produced in dish with sample. 100 ml of sample was shaken, Cyanika sector. and evaporated in a drier (BioVac 631/6). Dish with the residue was placed in the muffle furnace 2 MATERIALS AND METHODS (S336RB) and maintained at 45 °C for 1 h. Dish was cooled in a desiccator and weighted. Ash %

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100(푤2−푤1) 50 ml of sorghum beer was filtrated and added in mass by volume = where: w1: weight 푉 50 ml of distilled water. The solution was poured in of the empty dish, w : weight of the dish with ash, 2 volumetric flask with received alcohol to form 100 v: Volume in ml of liquor for ash determination. ml. The solution was kept at 78 °C and 2.2.4 Alcohol content alcoholmeter (India JK) was placed in the solution and the value was recorded.

2.2.5 Sugar degree Brix Sorghum beer is often cloudy with a sour taste. Higher pH levels produce a pink color of the Sample drops were poured on a prism surface of a sorghum beer (Lyumugabe et al., 2010). pH of 3 refractometer (refractive index 1.32000 to samples on 1st day were not statistically different. 1.55700). The demarcation line between the light However, on 2nd day, the pH of ubuki was and dark was focused by recording the value. statistically most acidic since it had lower pH compared to the one of ikigage and umurahanyoni. 2.3 Microbial analyses The pH average of ubuki was also lower than the pH of ikigage and umurahanyoni. There was a Salmonella Shigela agar (SSA), MacConkey agar decrease of pH for all beers on day 2 (Table 1 & 2). (McC), mannitol salt agar (MSA), plate count agar The decrease in pH can be ascribed to the (PCA) and peptone water agar (PWA) were used as continuity of fermentation process (Lyumugabe et microbiological culture media. From each al., 2013). sorghum beer sample, a serial dilution was carried out using peptone water as diluents. After Similarly, a decrease of pH was also observed inoculation, the plates were incubated at 37 °C for by Maurice (2014) on sorghum beer flavored with 48 h. After incubation, the colours and the shapes ginger where pH decreases from 7.4 to 4.3. Kayodé of the grown colonies were written down. Gram (2005) reported a low pH of 3.2 on tchoukoutou staining was followed. For biochemical tests, and chakpalo traditional alcoholic beverages citrate, glucose and lactose fermentation, sulphide produced in Benin and Togo. Sewadogo-Lingani production and motility, and indole and catalase (2007) obtained a pH range of 5.45-5.61. This tests were carried out. The bacteria were then range was higher than the present range (4.18- 4.8) identified based on microbiological analyses. (Table 1). The pH of 3.50 - 4.75 is the range of beer given Rwanda standard specifications. 2.4 Statistical analysis Therefore, the measured pH of ikigage, ubuki, and umurahanyoni matched with RSB standards of Data were collected in duplicates and were beers (RS 344) even if 4.8 was noted for ikigage. analyzed using a Statistical Packages for Social Sciences (SPSS), version 20. 3.2.2 Total acids of ubuki, ikigage and umurahanyoni 3 RESULTS AND DISCUSSION Many traditionally brewed sorghum beers still 3.1 Collection of ubuki, ikigage and contain the last sugar maltotriose fermented by umurahanyoni from Cyanika sector yeast during fermentation (Lyumugabe et al., 2010). The total acids of the 3 sorghum beers were Ubuki, ikigage and umurahanyoni were collected the same because no statistical difference observed from Cyanika sector. The storage containers were between them in 2 days (Table 1). However, dirty and the sorghum beer processing area was not average total acid of ubuki was lower than total cleaned. After sample collection, they were put in 3 acid of ikigage and umurahanyoni (Table 2). The different labelled sterilized bottles for analysis. All similar total acids values (1.7, 0.90 g/ml) of ikigage samples were brown in color. were observed by Lyumugabe et al. (2010). In addition, Orji et al. (2003) showed low total acidity 3.2 Physico-chemical properties of sorghum of 0.12 g/ml for pito, a Nigerian fermented beers alcoholic beverage. Ubuki was the most acidic beer 3.2.1 pH measurement of ubuki, ikigage and than ikigage and umurahanyoni (Table 1) due to umurahanyoni fermentable sugar in honey. Total acid (LAB)

INES Scientific Journal |Vol 1|issue 1|2021 Mansa STM Publishers Dusabe and Niyonzima et al doi: 10.32677/ISJ.2021.v01.i01.002 range of 0.13 to 0.61g/ml reported by Sewadogo- amounts like iron and iodine (Willbourn, 1995). Lingani (2007) was lower than 1.15-1.8 found in The ash content of 3 sorghum beers was identical the present study. Moreover, all beers measured since there is no statistical difference showed were normal regarding RSB specification of beers. among them in 2 days, and average (Table 1 & 2). Higher ash content of 1.7% was reported by 3.2.3 Ashes of ubuki, ikigage and umurahanyoni Chevassus-Agnes et al. (1979) for amgba Cameroonian sorghum malted beer. Furthermore, The ash constituent includes some minerals that are the presented ashes range of 0.28-0.35 is up to present in important amounts like magnesium and standard range while is less than 7% in alcoholic calcium. It also includes other minerals in smaller beverages.

Table 1: Physico-chemicals properties of 3 sorghum beers in 2 days

Properties 1st day 2nd day Ikigage Ubuki Um Ch- Df P- Ikigage Ubuki Um Ch- df P- square value square value pH 4.83 4.26 4.46 6 4 0.19 4.78 4.10 4.3 5.5 4 0.01* Tatol acid 1.7 1.5 1.9 5 4 0.1 0.93 0.8 1.3 6.00 4 0.3 Ashes 0.4 0.36 0.32 6.5 4 0.15 0.3 0.3 0.25 4.5 4 0.19 Alcohol 0 3 0 6.01 4 0.02* 1 5.8 1.5 4.8 4 0.003* content Degree 0 5 0 6.00 4 0.005* 0 3 0 5.6 4 0.001* Brix

*Significant at p < 0.05; df = 4, Um: Umurahanyoni Table 2: Average of physico- chemicals properties of 3 sorghum beer in 2 days

Table 2: Average of physico- chemicals properties of 3 sorghum beer in 2 days

Ikigage 1st day 2nd day Mean P-value pH 4.83 4.78 4.8 0.04 Total acid 1.7 0.93 1.3 0.002 Ashes 0.4 0.3 0.35 0.15 Alcohol content 0 1 0.5 0.003 Degree Brix 0 0 0 0.2 Ubuki pH 4.26 4.10 4.18 0.053 Total acid 1.5 0.8 1.15 0.03 Ashes 0.36 0.3 0.33 0.12 Alcohol content 3 5.8 4.4 0.002 Degree Brix 5 3 4 0.01 Umurahanyoni pH 4.46 4.3 4.38 0.03 Total acid 1.9 1.3 1.6 0.06 Ashes 0.32 0.25 0.28 0.3 degree Brix 0 1.5 0.75 0.4 Alcohol content 0 5 2.5 0.016

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3.2.4 Alcohol content of ubuki, ikigage and effectively. The diluted sample was inoculated on umurahanyoni PCA (37 °C for 48 h). On PCA dish, culture presented an array of color from yellow to white. Sorghum beer is had a low alcohol content, varying The sample was cultured on McC, SSA and MSA from 1 to 4% in general (Agu & Palmer, 1999). No and incubated at 37 °C for 48 h. After 48 h of alcohol content seen in ikigage and umurahanyoni incubation, pink, purples, yellow and white clusted on day 1. On day 2, there was alcohol in all 3 colonies were developed. Rod, spherical and samples with higher production in ubuki (Table 1 curved colonies of microorganisms were grown on and 2). The alcohol content production increase their specific culture media. Georgette (2013) may be ascribed to fermentation continuation over indicated that traditional beer produced at Kabaya time. The higher ubuki alcohol content may be center contain few colored colonies grown on SSA as Salmonella sp. attributed to the honey that may be added during production. In contrast, a decrease in alcohol Ubuki rise many pink colonies on SSA than content was observed by Lyumugabe et al. (2010) ikigage and umurahanyoni. Gram staining was on sorghum beer ikigage from 4.34 to1.49 % (v/v) carried out to identify the types of bacteria present. in 3 days. Ubuki had similar alcohol content (3%) Spirilla, cocci and bacilli were represented in the on day 1 with the one presented by Maurice (2013). sample under the microscope. The findings shapes According to Lyumugabe (2010), the sorghum beer were in accordance with Georgette (2013) and ought to have average alcohol content of 1.49-5.5. Lyumugabe (2010). Biochemical tests were carried However, 0.5 – 4.4% was the range of the present out for identification of several microbes. These study. Ubuki results are up to Rwandan standard tests evaluated the reaction of the sample towards a obligation of alcohol content of beer (2.5-5.5% RS specific substance (Nigatu, 2015). According to the EAS 104), while ikigage and umurahanyoni do not presented biochemical results in table 3, sorghum meet RSB standards, not thus suitable for beer named ubuki contained different species such consumption. as Salmonella species, Shigella sp. and Proteus sp. They were identified based on triple sugar iron test 3.2.5 Degree Brix of ubuki, ikigage and positive citrate and regarding their selective media umurahanyoni used to identify them (Motarjemi et al., 1993).

Total soluble solids presented in sorghum beer However, selective media such as McC (Yadav were in form of amino acids, peptides, starch, et al., 2009) is universally used to isolate total antioxidant, dietary fibres, calcium and soluble coliforms in sorghum beer checking based on protein (Lyumugabe et al., 2010). During 1st, 2nd biochemical characterization of E. coli. Results day and average, ikigage, ubuki and umurahanyoni showed that umurahanyoni sample contain E. coli, were statistical different (Table 1 & 2). Ubuki had and Klebsiella sp. Staphylococcus aureus, Serratia species and Morganella morganii are presented a higher statistical degree Brix than others sorghum microorganisms identified in ikigage sample based beer on 1st and 2nd day and average values. on MSA as selective media and biochemical results Likewise, higher degree Brix of 11.6% was as seen in the table 3. The total coliforms of ubuki, recorded by Lyumugabe et al. (2010). The higher umurahanyoni and ikigage noted were 209.1×108, degree Brix of ubuki was due to the addition of 9 9 15.33×10 and 6.28×10 cfu/ml, respectively. honey during processing. 1.15 -11.6% was degree Ubuki, umurahanyoni and ikigage had higher Brix range reported by Lyumugabe et al. (2010). number of coliforms compare with non-alcoholic This justified 0-4 average degree Brix of the sorghum beer analyzed by Georgette (2013). present study. Hence, ikigage, ubuki, and Stomach cramps, nausea, and vomiting are the umurahanyoni are fitting for consumption based on symptoms caused by Salmonella and Shigella, degree Brix less than 12 as RSB specify. however serious complications can occur (Johnson et al., 1966). The presence of total coliforms 3. 3 Microbiological analysis indicated pathogenic bacteria. E.coli is used as an indicator of poor sanitation measures. Toft et al. African traditional beers may contain (1987) associated E. coli with diarrhea. Therefore, microorganisms if not well handled (Agu & ikigage, umurahanyoni and ubuki were not Palmer, 1999). Ikigage, ubuki and umurahanyoni appropriate for consumption look upon RSB were diluted in peptone water to identify microbes specification (RS ISO 4832).

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Table 3: Gram staining and biochemical results of ubuki, umurahanyoni and ikigage samples

Samples Med Gram Lactose Glucose H2S Citra Motili Catalase Indole

ia staini fermentat fermentat producti te ty test (H2 formati

ng ion ion on test O2) on Ubuki SSA _ _ + + _ _ + ±

Umurahany McC _ + + _ _ + + + oni

Ikigage MSA ± + + _ + _ + _

CONCLUSION brewers have to maintain hygienic conditions to ensure that their products meet the standards of Even if samples of sorghum beers matched sorghum beer. The Cyanika population, especially physico-chemical terms of RSB, microorganisms brewers, sellers and consumers, has to be educated were found in analyzed samples. Sorghum beers about on causes of food borne diseases, modes of are therefore not suitable for consumption as they transmission, and treatment of water, and raw may cause diseases. The ingredients used in materials. Future researchers have to analyze all the sorghum beers production like water and malted physico-chemical properties for a long period. should be treated before use. The sellers and

Acknowledgement: The authors thank INES Ruhengeri for support

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