THE POTENTIAL OF GESHO ( RHAMNUS PRINOIDES L. HERIT ) AS SUBSTITUTES FOR HOP ( HUMULUS LUPULUS ) IN BEER PRODUCTION

and D (control). The analysis of key brewing variables of the R. prinoides was as follows: total resin (15.96-16.02%), ISO-alpha acid (1.17-1.45 mg/l), alpha acid (1.44-1.92 mg/l), essential oil (3.0-3.07 %), were obtained. Beer type C (7.4+0.01%w/w and 9.5+0.01%v/v) has been shown significantly (p<0.05) greater concentration of alcohol in comparison with other beer types. Real degree of fermentation value of beer type D (66.29+0.03%) was significantly (p<0.05) different compared with other beer types. Bitterness value (2.2 to 2.8) of the beer produced by R. prinoides was in the range of the values (0-100) of common hopped beer. The bitter substance compositions and key brewing variables of beer produced by R. prinoides are comparable with hop. This indicates that R. prinoides can serve as a substitute for hops in the breweries.

in the Dashen Brewery Factory Laboratory. Dashen Brewery Factory is located at 727 kilometers away from Addis Ababa, in North Gondar, North-west Ethiopia.

Raw material
The materials used to run all experiments were: Hops, Barley malt and Yeast (Sacharomyces cervisiae) from Dashen Brewery and gesho (Rhamnus prinoides) from a local market in Gondar Town. Samples were taken at the University of Gondar department of Biotechnology molecular laboratory room for further analysis. Laboratory analysis was performed to determine the R. prinoides constituents for brewing.

Experimental work and beer sample
Analysis was done on beer sample designated as A, B, C and D to test the physicchemical parameters. In this research, all parameters remained the same with the exception of three (A, B, and C) Rhamnus prinoides samples collected from local market and sample D normal beer fermented with a commercial hop as a control.

Sample preparation
Rhamnus prinoides leaf samples were sun dried before being crushed with a mortar and pestle. To remove the moisture content of gesho flour, it was dried in an oven at 60°C for 24 hours. The bittering compounds and other elements of R. prinoides were then identified, and it was utilized to make beer as a hop substitute.

Total resin determination
Twenty gram of the sample was dissolved in 100 ml of cold methanol in a conical bottom flask and the mixture was vigorously agitated by swirling the flask. The solution was then filtered. The filtrate containing the resin was dried and the total resin was calculated as a percentage of the original sample weight (Adama et al., 2011).

Soft resin and hard resin determination
Ten gram of each sample was properly mixed with 10 ml of hexane before being filtered (using watman filter paper). The filtrate was heated to 50 o C and dried to a constant weight. The amount of the original sample weight that was dissolved in the hexane was used to compute the amount of soft resin. Soft resin was subtracted from total resin to get the hard resin (Adama et al., 2011).

Essential oil determination
Twenty grams of the samples were placed in a cap and injected into the soxhlet extractor's internal tube. This equipment was then attached to a flask with a circular bottom that held 200 mL of n-hexane. The arrangement was held to a retort stand and then put on a boiler mantle that was turned on for a 120-minute extraction time at the solvent's boiling point (n-hexane 60 0 C). The vapor rises over the tube, is condensed by the condenser, and then falls into the thimble, slowly filling the soxhlet's body. The solvent siphoned over into the flask when it reached the top of the tube, removing the portion of the samples extracted in the thimble. The operation was repeated automatically for a total of 120 minutes before the equipment was removed. Using the same soxhlet extractor, the solvent recovery process was carried out. In the flask, the solvent and oil combination were heated. When the solvent evaporated it was allowed to condense in the thimble chamber after steady heating. Before it was siphoned back into the flask, the solvent was collected. After that, the oil was recovered and its mass was calculated. After extraction and drying in an oven, the mass of the samples was collected in a sample bottle and recorded (Adama et al., 2011).

Preparation of the water extract
For the preparation of water extract, 0.15% (w/v) solution of the samples was used and the solution was heated for 90 minutes, and allowed to cool. The solution was then filtered using Whatman filter paper. The ISO-alpha acids were the determined using the water extract.

ISO-alpha acid determination
In order to determine ISO-alpha acids 15 milliliters of the sample extract were mixed with 15 milliliters of pure ISO-octane and the it was acidified with 0.5 milliliters of 6 N HCl. Ten milliliters of the ISO-acetone extract were washed with 10 milliliters of a 68:32 (v/v) solution of methanol and 4 N HCl. The absorbance of 5 ml of the washed ISO-octane layer was measured at 255 nm after being diluted with 5 ml of alkaline methanol (60:40, v/v methanol: 0.5 N NaOH). The (AOAC, 2000) method of analysis was used to calculate the ISO-alpha acid (mg/L).ISO-acid (mg/L) =A255 (96.15) +0.4

Beer production
The procedure of beer brewing was carried out using all raw materials (hop, water, Sacharomyces cervisiae) except that of gesho instead of hop as a bittering agent. However, hop (H. lupulus var. lupulus) was used as a control using the same procedure (Kunze, 2004).

Boiling of wort
The wort was boiled and gesho was added as usual used in the beer brewing process (Kunze, 2004). It was stirred until it gets wet. The amount of the gesho used as a hop substitute was (0.5 g/L gesho), while for control (0.15 g/L hop) was used as the factory use for beer production process. The mixture of wort and gesho was mixed and boiled for 15 min to 121 0 C to kill all microorganisms. After that, it was allowed to cool for yeast pitching and fermentation.

Fermentation
The fermentation of sugar-laden wort carried out by the inoculation of S. cerevisiae for fermentation. The yeast was pitched into the propagation flask that containing the same type of wort for fermentation. The flask was closed and cooled to 10 to 11°C. This process kept for one day. The fermentation in the flask was checked by observing the formation of good foam. The fermenter was placed in a protected area to avoid fluctuated environmental conditions. It was placed in an area that is not exposed to direct sunlight.

Specific gravity determination
The specific gravity of the sample was determined by 24 hourly using a digital density meter after 72 h of inoculation of yeast to the wort sugar. To identify the level of fermentation per 3 days, a sample of beer was filtered using filter aids, and specific gravity of the sample was determined using density meter at 20 o C until the extract arrives at 3 and below with the correlation table (EBC, 2008). At the end of fermentation, the specific gravity of the bicarbonate apparent extract, alcohol, and real extract was determined using pyknometer at 20ºC after distillation.

Determination of real extract
Real extract was determined by conversion of the specific gravity of the residue to the corresponding real extract content, Er as % plato (Rosendal and Schmidt,1987). Er (% Plato) =-460.234+662.649 SGER-202.414 (SGER.) 2

Determination of alcohol content
The alcohol content was determined using distillation by direct heating and determining the alcohol % (w/w) from the distillate specific gravity, the alcohol % (v/v) content was determined from the specific gravity of the filtered beer and alcohol % (w/w) (EBC, 2000).

Determination of pH
Two hundred ml of beer samples was filtered by filter paper and excess carbon dioxide was removed by shaking to prevent unstable pH reading. The electrode of the pH meter was inserted into the beer sample and the reading on the screen of the pH meter was observed and recorded (EBC, 2000).

Determination of bitterness in beer
The beer sample was re-filtered and 100 ml was taken after adding 3 drops of octanol. Ten ml of the sample and 1 ml of HCl together with 20 ml ISO-octane was mixed and then shaken with platform shaker until maximum extraction was achieved. Absorbance of ISO-octane layer in 10 mm Cuvette at 275 nm was measured using pure ISO-octane in the reference Cuvette (EBC, 2000). Bitterness (BU) = A x 50 Where A = Absorbance at 275nm

Determination of carbon dioxide in beer
The carbon dioxide content of the beer was determined using titration method. Ten ml NaOH was poured into a 250 ml flask and 200 ml of beer sample was added and it was inserted into the right side of the sample point outlet with the addition of 10ml H2SO4. On the other hand, 25 ml barium hydroxide was taken and inserted in the left side of the apparatus and the two fork tubes was connected with hoses to allow air circulation. After completion of the air circulation 3 drops of phenophtaline indicator were added to the Ba(OH)2 flask and titrated with HCl the excess barium hydroxide up to the end point (EBC, 2000).
Where A, content of bottle or cans in ml B, NaOH added in ml C, sample volume in ml D, 0.100m HCl used for the titration of 25.00 ml Ba(OH)2 (blank value) in ml E, 0.100m HCl used for the titration sample in ml

Determination of total acidity
Ten ml of filtered sample was diluted with 30ml of water and titrated with sodium hydroxide solution using 1ml phenolphthalein; total acidity was recorded as ml of alkali /10 ml of a sample of beer (Adenuga et al., 2010).

Determination of vicinal diketones in beer
Vicinal diketones in beer was determined using the spectrophotometric method. In this case, 100 ml of beer sample was measured using measuring cylinder and added to the distillation flask for direct heating until the sample gives 25 ml of the distillate in the measuring cylinder and thoroughly mixed. Ten ml of the distillate and 0.5 ml o-phenylenediamine was pipetted into a dry test tube and kept in a dark place for 20 min. Lastly, 10 ml of water was added into a 50 ml flask with glass and 500 micro liters of OPD was added to prepare the blank reagent (EBC, 2000).

Test for contamination
Contamination test was done to enumerate the possible presence of wort bacteria, wild yeast and lactic acid bacteria were enumerated by spreading 0.1 ml of the sample plates containing wort agar plus actidione, yeast and mold agar plus copper sulfate and universal beer agar with ABP inhibitor respectively. The expression and definition of "no contamination" was defined as "less than or equal to 1 colony forming units per 0.1 ml for wort bacteria and 0 colony forming units per 0.1 ml for both wild yeast and lactic acid bacteria." (EBC, 2000).

Data analysis
The data were analyzed using SPSS version 20.00. Means and standard deviations of the triplicate analysis was calculated using one-way analysis of variance (ANOVA) to determine the significant differences among variables (p < 0.05) when the F-test demonstrated significance. The statistically significant difference was defined as p < 0.05.

Analysis of bittering agent of Rhamnus prinoides
The most valuable bittering components of R. prinoides like resins, alpha acids, ISO-alpha-acids, essential oils, and beta-acids were studied. For the dosing purpose of beer bitterness, the analyses for determination of content of alpha bitter acids (which are representative for beer bitterness) are important. Some of the components of R. prinoides (total resin and bitter acids) are presented in Table 1.

Analysis of specific gravity of beer that produced using R. prinoides
The specific gravity of the fermentation test for beer brewing with R. prinoides was shown in Table 2. The degree of extract decrease along with fermentation date was used for the immediate control of the fermentation process. When the extract reaches three and below that the fermentation process stopped theoretically According to this finding, beer type designated as B (decrease from 13.46 to 3.02) and D (decrease from 11.68 to 3.05) has relatively good fermentation performance in comparison with other beer type. All beer types at day eighteen shows a similar extract compared with extract at day fifteen, thus the fermentation process stopped at day eighteen.

Analysis of the alcohol content of beer produced by R. prinoides
Ethanol is a major end product of beer fermentation. It forms part of the end byproducts of wort fermentation. Analysis of results showed that beer type C (7.4+0.01%w/w and 9.5+0.01%v/v) has been shown significantly (p<0.05) greater concentration of alcohol in comparison with beer A (6.6+0.01%w/w and 8.5+0.02 %v/v), beer B (6.8+0.01 %w/w and 8.4+0.01%v/v) has been shown low (p ≥ 0.05) alcohol content compared with other beer types. Beer produced with hop has been shown lower (p<0.05) amount of alcohol (5.2+0.01 w/w and 6.6+0.01 v/v) than the rest type of beers.

Analysis of original extract, apparent extract, real extract and the real degree of fermentation of beer produced by Rhamnus prinoides
The value of the original extract of beer type B (21.60+0.01 ) and beer type C (21.62 +0.01) were statistically (p>0.05) similar and are different with the other beer types evaluated. Beer type B resulted in relatively (p<0.05) lower original extract (15.70+0.01) as compared to the other beer types such as A (20.31+0.01), C (21.77+0.01) and D (18.88+0.01) beers. Beer type A has been shown statistically (p<0.05) greater apparent extract (4.80+0.001) in comparison with other beer types. Beer D had been shown statistically (p<0.05) less apparent extract (4.20+0.001 c ) than the other beer types.
The highest (p<0.05) apparent degree of fermentation value (79.72+0.005 a ) was observed by beer type D. Beer type C has been shown statistically (p<0.05) lower apparent degree of fermentation (75.91+0.01) than beer type A (77.50+0.01) and beer type B (77.60+0.01). The highest (p<0.05) real extract value was observed (8.01+0.01) by the beer type A in this investigation. The value of real extract (5.47+0.01) observed in beer type D was statistically (p<0.05) lower than the rest beer types. The highest real degree of fermentation value (66.29+0.03) recorded by beer D was significantly (p<0.05) different compared with other beer types (beer A 64.93+0.01 ) , (beer B 63.22+0.01 ) and (beer C 62.01+0.005). The lowest value recorded was by beer type C in this study. Based on the mean value obtained the total acidity of beer type D (0.42+0.01) was shown less (p<0.05) total acidity compared with other beer types. All other three beer types have been shown statistically (p<0.05) similar values of total acidity. Based on the mean value of CO2 obtained in this study, beer type D (0.25+0.01 a ) had been statistically (p<0.05) greater value of carbon dioxide than the other types of beer. Beer type A (4.2+0.017) and beer type B (4.3+0.005) have been shown statistically(p>0.05) similar pH values. The result obtained by beer type D (4.7+0.02) has been significantly (p<0.05) higher pH than compared with other beer types.

Detection of microbial contaminant in beer produced by Rhamnus prinoides
Availability of microorganisms in beer produced by R. prinoides was evaluated using a standard microbial culture system. Beer samples were speared on universal beer agar medium and incubated for seven days at 25 o C. Microorganism such as molds, wort bacteria and lactic acid bacteria were not observed on cultured beer after seven days of incubation.

DISCUSSION
The research aimed at determining the bittering capacity of gesho (Rhamnus prinoides) as a substitute for hops (Humulus lupulus) used in brewing of beers, the research attempted to determine the key variables identified as necessary in hops. Characterization of the physicochemical characteristics of the R. prinoides extract was done in this investigation to identify any bitter components. The content and quality of the raw materials used are the main determinants of beer quality. The primary brewing ingredient used as a bittering agent is hop. For the quality of the beer and the cost-effectiveness of the brewing process, its chemical composition is very important. In this study important physicochemical characteristics of beer analysis have been investigated to know the bittering potential of R. prinoides on sensory quality of beer. The values of total resin obtained (15.96-16.02%) evaluated for R. prinoides were comparable with Humulus lupulus (16.53%) used as a known bittering agent in commercial beer (Kunze, 1996). The values of hard resin (9.75-9.96%) of R. prinoides was also comparable with Garcinia cola (9.69%) which was used as a tropical hop substitute by Adama et al., (2011), but less than with that of H. lupulus. In the same manner the soft resin (6.02-6.39%) of R. prinoides was greater than Garcinia cola (2.17%), but less than the commercial H. lupulus studied by (Kunze, 1996). In this study, the quantity of total resins of R. prinoides was comparable with other bittering hop substitutes such as Vernonia amygdalina and commercial hop (Adama et al., 2011; Kunze, 1996). The values of total resin, soft resin and hard resin components of R. prinoides were less than the values obtained by Berhanu (2014), who studied the bittering and antimicrobial role of this plant But, the value of oil content was higher than the value recorded by Berhanu (2014), it supports the ideas of hop constituents are place of cultivation dependent (Hieronymus, 2012). The evaluated alpha _ acid (1.44-1.92%) and beta _ acid content (2.07-2.18%) of R. prinoides was in the range of commercial hop plant 0 _ 20% (Hieronymus, 2012). It is the alpha-acid that impacts the bitterness in the beer (Kunze, 1996). The primary elements of hop resins, alpha-acids, beta-acids, and the products of their transformation are known to contribute to beer bitterness (Kunze, 1996). The oils in hop contain fatty acids and esters, which impart the aroma and flavor of beer. The oil component of H. Lupulus ranges from 0.03-3% (Kunze, 2003). In this study, a significant amount of (3.00-3.07%) oil content was obtained and it indicates R. prinoides can be a source of flavor in production of commercial beer. The results obtained from R. prinoides for alpha-acids, betaacids, and essential oil was found to be within the range of dry hops (Hieronymus, 2012). Thus, the value of the plant extract from the analysis performed can be said to be good since there were similarities in the properties of the standard commercial hops and the R. prinoides properties. By measuring the wort's specific gravity as fermentation occurred, the breakdown of the wort components was used as an indicator of the progress of fermentation process. Comparing the post-fermentation specific gravity measurements to their starting values at the wort stage, they were significantly lower. According to this experiment, the profile of the R. prinoides beer was comparable to the profile of the hopped (control) beer (Rourke, 2002). For quality assurance programs and legal reporting requirements, the examination of beer's alcohol content plays a significant role in the brewing process (Kunze, 1996). The alcohol concentrations in this investigation ranged from (5.2 to 6.7% w/w) and (6.6 to 9.5% v/v). The alcohol concentration of the samples used in this investigation, both w/w and v/v, was within the range for string beer. The alcohol content of beer in this study (7.4% w/w and 9.5% v/v) shows that more sugar was fermented in these beer samples than in other samples. The alcohol levels were comparable to those that reported by Okafor and Anichie (1983). In this study, beer type A and B apparent extract capacity were similar but, greater than hooped beer. All beer types in this study showed that apparent and real extract were greater than for apparent and real extract of the given beer (EBC, 2008). The original extract of all beer types was within the range of 15.70 and 20.31 %p. A good alcohol-real extract balance is very important for beer taste (Kunze, 1996). Therefore R. prinoides can be used as a bittering agent for the production of beer in industrial level.
The real degree of fermentation capacity of beer A studied in this experiment agreed with hopped beer, the apparent degree of fermentation of all beer types was lower than the hopped (control) beer. For extra strong beer, the minimum standard values for apparent extract and true extract are 2.50% and 4.42%, respectively (ES 842, 2012). R. prinoides produced beer with a minimum apparent and true extract of 4.46% and 7.62%, respectively. The apparent and real extract percentages for the hooped (control) beer were 4.2% and 5.47%, respectively. Thus, both the apparent and real extract values found in this investigation were significantly higher than the required minimum levels for extremely strong beer. This study demonstrates that the R. prinoides-produced beer exhibits a very good fermentation process for the production of industrially commercialized beer.
The pH values of beer produced were within the range of 4.2 and 4.7. The pH values of beers were within the standard value (3.6 to 4.8) of (ES830, 2012). The pH has a significant effect on the quality of beer (Kunze, 1996). The pH can reduce the possible contamination effect of beer. The total acidity value of beer types A (0.62/10 ml) and beer B (0.61/10ml) was similar to the total acidity value given by Okafor and Anichie (1983) for tropical hop substitute beer. In contrast, the total acidity value for hopped (control) beer was lower than the value recorded by Okafor and Anichie (1983). This indicates that hopped beer has a lower acid level than beer brewed with R. prinoides. Hops are generally responsible for the bitterness of beers, in addition to hops polyphenol can also impart beer bitterness (kunze, 1996). It typically results from the main bittering component of hops, ISO-acids, isomerizing to -acids during wort boiling. Bitterness should be monitored and closely managed to preserve uniformity in quality. IBU ratings for various types of beer typically range from 0 to 100 IBU (Kunze, 1996). The R. prinoides produced beers in this research were significantly less than to the hopped (control). In this investigation, the beer C was shown to have more bitterness compared with other beer type produced by R. prinoides. The degree of bitterness of beer in this investigation was within the range of 2.2 to 25.0 IBU. All beers produced were within the range of the beer types stated by (Kunze, 1996). Vicinal diketones (VDK) provide beer a sweet flavor if their concentration exceeds the limit value and give it a buttery aroma (Fix, 1993). Beer made from R. prinoides had a higher VDK concentration than the controlled beer. In general, the beers made by R. prinoides in this investigation contain VDK values that are relatively higher than the reference value (0.15 mg/L) (ES843, 2012). Generally, the amount of releasing carbon dioxide (CO2) during fermentation is a direct indicator of a good fermentation activity during the brewing process (Kunze, 1996). The CO2 content of beer is one of its most important quality criteria. In this finding, carbon dioxide concentrations of all beer types were shown by far less than the specification in good beers (4.7g/L-5.2g/L) (Fix, 1993). The microbiological profile of beer made from R. prinoides need to be determined in order to assess the quality of beer. It is commonly recognized that beer contaminants could spoil beer, which lowers its quality. Usually, inadequate cleanliness and raw materials cause wort germs to grow in the fermenting vessel. Sterilized wort, pure yeast, and the sterilized and cooled vessel were all free of contamination in this investigation. This might be as a result of R. prinoides's antimicrobial properties (Berhanu, 2014).

CONCLUSION
Rhamnus prinoides can be used as a bittering agent in alcoholic beverages production. The goal of this research was to find R. prinoides bittering agents that could be used to make commercial beer. The beer produced using R. prinoides was comparable to that produced with hops as a bittering agent. According to the findings of this investigation, R. prinoides can be used as a bittering agent as a substitute of hops. The study result can be utilized as a starting point for formulating this bittering substance for commercial beer brewing, reducing the amount of money spent on hops and providing job opportunities for farmers and other members of society who grow gesho plants.