Biochemical Composition Variation among Southern Ethiopian Arabica Coffee ( Coffea arabica L. ) Genotypes

Cofee ( Cofea arabica L.) provides several health benefts to users due to its strong medicinal and nutritional properties and caloric value. Green bean proximate composition diversity is unknown among the cofee genotypes now cultivated in southern Ethiopia. Te study’s major goals are to determine the variability in green bean proximate composition among cofee genotypes and to see if there are any relationships between green bean proximate attributes. Terefore, a nutritional laboratory experiment was carried out at Jimma University College of Agriculture and Veterinary Medicine (JUCAVM). Using the augmented design, a total of 104 entries were examined, including 100 accessions from southern Ethiopia and four standard checks. Each accession had data on 07 proximate composition parameters of green beans. Te presence of signifcant ( P < 0 . 05) diferences among the examined accessions for most of the traits considered was revealed by analysis of variance, and a wide range of variation was detected for several traits, indicating that the cofee germplasm accessions have high genetic variability. According to the fndings, cofee beans have crude protein (6.93 to 10.14%), total lipids (8.89 to 16.08%), crude ash (2.51–5.47%), crude fber (6.79–22.25%), dry matter (89.08 to 91.63%), carbohydrate (40.65 to 59.38%), and caloric value (307.39–382.77k/calories). One hundred four arabica cofee accessions were grouped into ten distinct groups by 20 (19.23%), 21 (20.19%), 39 (37.50%), 12 (11.54%), 04 (3.85%), 03 (2.88%), 02 (1.92%), 01 (0.96%), 01 (0.96%), and 01 (0.96%). Te majority of intercluster distances were signifcantly varied, showing that diversity exists that can be utilized through selection and hybridization. Clusters III and X had the greatest in-tercluster distance ( D 2 � 344.16), followed by clusters II and X ( D 2 � 236.33), VII and X ( D 2 � 199.04), and clusters VI and I ( D 2 � 106.25). Clusters I and IV had the smallest intercluster distance ( D 2 � 10.09), followed by II and IV ( D 2 � 10.66), and I and VI ( D 2 � 11.03). Te frst three principal components with eigenvalues larger than one explained 71.84% of the overall variation. In general, genotypes difered in green bean proximate composition and might be used as gene sources to generate future green bean varieties with appropriate biochemical composition.


Introduction
Cofee (Cofea arabica L.) originated in Ethiopia and there is signifcant genetic diversity in the country. Ethiopia is the highest producer of cofee in Africa and the ffth major exporter in the world next to Brazil, Vietnam, Colombia, and Indonesia, contributing to 4.2% of the total world cofee production [1]. Cofee is one of the most widely consumed beverages on the planet. Te species Cofea arabica (arabica) and Cofea canephora (robusta) are used to make the majority of cofee beverages consumed around the world.
Because of its sensory characteristics, the former is deemed superior and commands greater pricing on the international market [2]. Green cofee beans are mature or immature cofee beans that have not been roasted. Te exterior pulp and mucilage have been removed by wet or dry processing, and the wax coating on the outside surface is intact [3]. Cafeine is primarily responsible for the stimulant properties of cofee brew [4]. However, this beverage contains a vast variety of chemical components, some of which have numerous benefcial properties. Green cofee beans have a diverse spectrum of chemical components that react and interact during the cofee manufacturing process, resulting in a fnal product with even more structure diversity and complexity [3]. Te nutritional contents and characteristics of cofee bean beverages are not well understood. In the literature, there is very little information on these characteristics of cofee's nutritional contents. However, there are still considerable knowledge gaps, and further research is needed to better identify the variation in nutritional contents of cofee arabica genotypes.
As a result, a detailed analysis of the nutritional and biochemical constituent compositions of commercially available arabica cofee beans from southern Ethiopia has been undertaken in this study. Te goal of this study was to determine the proximate and bioactive chemical compositions of 104 cofee accessions collected in Ethiopia's southern regions. Green bean biochemical compounds can be used to forecast arabica cofee biochemical compound variability and provide a foundation for developing a cofee biochemical data library. Te primary goal of this study was to defne cofee accessions based on their biochemical composition and group them into clusters for breeding purposes. Using principal component analysis, the study also seeks to fnd the traits that contribute the most to the variation in the data. As a result, the purpose of this study was to identify the green bean proximate properties of arabica cofee genotypes collected from southern Ethiopia, as well as to assess the extent of biochemical heterogeneity among genotypes.

Description of the Trial Site.
Te study was conducted at Awada Agricultural Research Subcenter. It is located in southern Ethiopia near Yirgalem, 315 kilometers from Addis Ababa. Te subcenter is in southern Ethiopia's moderate to chilly semi-arid mid-highland agroecology. Geographically, it is situated in 6°3′N latitude and 38°E longitude, at a height of around 1740 meters above sea level. With an average precipitation of 1342 mm per year, the area has a semi-bimodal rainfall pattern with double wet and dry seasons. Te average annual minimum and maximum air temperatures are 11 and 28.4 degrees Celsius, respectively, with an annual mean minimum and maximum rainfall of 858.1 and 1676.3 millimeters [5].  Table 1 shows the geographical origins of the genotypes that were gathered.

Trial Management and Experimental Design.
Treatments consisted of 100 cofee accessions and felds established at the Awada Agricultural Research. Moreover, four released varieties (75227, 744, 7440, and 1377) were included as standard checks. Te experiment was laid down in the feld using augmented design, which is used with replicated controls (checks) to assess the performance of nonreplicated accession in complete block designs in fve blocks [6]. A single treatment consisting of ten trees. Te plant-to-plant spacing used was two meters by two meters, while the spacing between blocks was four meters. All the recommended agronomic practices were applied uniformly to all the plots [7].

Procedures for Cofee
Harvesting and Processing. One treatment included ten cofee trees and a total of 5 cofee plants were used to prepare cofee samples for biochemical analysis from each treatment. Green cherries and foreign material were separated from healthy and red ripe cherries before pulping. Te samples were properly processed for biochemical analysis utilizing the wet-processing method (pulping, fermentation, and drying). After the cherries were picked, each genotype was pulped separately using a single disc hand pulper. Pulped cherries were gathered in large plastic buckets, which were then cleaned of pulps and foater parchments. Wet parchment beans were then transferred to the other bucket, which was then flled with fresh water until the parchment beans were completely submerged in the water for fermentation. Te wet parchment cofee was fermented for 40 hours before the frst washing. According to Abrar et al. [8], samples were then immersed for 24 hours before being washed. When the mucilage had completely decomposed, the parchment cofee beans were thoroughly washed to remove all mucilage. Te resulting green parchment beans were prepared and placed on mesh wire in direct sunlight until they were totally dried or their moisture content had reached 10.5-11.5%. Six kilograms of ripe, red cofee cherries from each treatment were used. For each treatment, 1.5 kilograms of clean cofee were made and used as biological samples. Sample parchment green beans were labeled and packed in white perforated plastic bags when they reached the appropriate moisture content.

Laboratory
Analysis. An arbitrary code was assigned to all of the samples that were prepared (an identity letter and number). Green bean samples were labeled with an arbitrary code and brought to the lab. In order to investigate the level of variability among cofee (Cofea arabica L.) germplasm accessions based on biochemical features, a laboratory experiment was undertaken at Jimma University College of Agriculture and Veterinary Medicine (JUCAVM) nutrition laboratory. A total of 2 to 8 grams of dry, powdered cofee were used as technical samples for each treatment in the study of each biochemical parameter. Te following are the biochemical examination methodologies for cofee beans:

Analyzing the Biochemical Makeup of Cofee Bean
Samples. Te procedure was used to assess the approximate composition (moisture content, crude protein, crude fat, crude fber, crude ash, total carbohydrate, and calorie value) of cofee row beans [9].
2.6.1. Moisture Content Determination. Te moisture content of a powdered cofee sample was tested in an oven using the drying method given in Ref. [9]. Weighing 2 grams of sample onto a preweighed dish and drying it in an air pressured draft oven at 105°C until the constant weight of dry matter was reached was used to assess the moisture content of the sample. Te following formula was used to determine the moisture content of the sample:

Crude Protein Determination.
Te crude protein content of the powdered cofee sample was determined using Kjeldahl's method, as defned in Ref. [9], which involves protein digestion and distillation. Digestion of protein: About 2 grams of the material was weighed and placed in 250 ml Kjeldahl fasks with an ashfree flter paper. Ten 15-20 ml of 98% concentration sulfuric acid and 1 gram of digestion mixture (as a catalyst) were added. In the digesting chamber, the entire combination was heated until translucent residue contents were recovered. After that, it is allowed to cool. After chilling, the digest was transferred to 100 mL volumetric fasks and topped up with distilled water before being distilled with Markham distillation equipment.
Protein distillation: Te Markham distillation apparatus was steamed for 15 minutes before use, following which a 100 mL conical fask containing 5 mL of 2% boric acid and 1 or 2 drops of the mixed indicator was placed under the condenser, with the condenser tip submerged in the liquid. A small funnel aperture was used to pipette around 5 ml of the digest into the apparatus's body. After washing the digest with distilled water, 3-4 drops of phenolphthalein and 5 ml of 40% (W/V) NaOH solution were added. Te digest was steamed in the condenser until enough ammonium sulfate International Journal of Agronomy 3 was recovered. Te color of the boric acid plus indicator solution changed from red to green, indicating that all of the ammonia had been released. Te solution in the receiving fask was titrated with 0.1 N hydrochloric acid until it reached a purple endpoint. Along with the sample, a blank was run through. Te percentage of nitrogen was estimated after titration using the formula: Where, Vs � Volume (ml) of acid required to titrate the sample; VB � Volume (ml) of acid required to titrate the blank; M acid � Molarity of acid; W�Weight of sample (g). Ten, the percentage of crude protein in the sample was calculated from the % nitrogen as follows: where, F (conversion factor), is equal to 6.25 [9].

Crude Fat Determination.
Soxhlet extraction for 24 hours was used to evaluate the crude fat content of the powdered sample. A total of 3 grams of materials were correctly weighed into labeled thimbles. Te 250 mL dried boiling fasks were weighed and flled with approximately 150 mL petroleum ether (boiling point 40-60 o C). Cotton wool was stufed into the extraction thimbles. Te Soxhlet device was then put together and allowed to refux for 24 hours. Te thimble was carefully removed, and the petroleum ether from the top container was collected and emptied into another container for reuse. After that, the boiling fask was baked in a hot air oven until the petroleum ether was practically gone. It was dried, cooled in desiccators, and weighed [9].
2.6.4. Crude Fiber Determination. In a fber fask, a 2 grams fat-free sample of powdered cofee was introduced to 100 ml of 0.255 N H 2 SO 4 . Te mixture was then heated for one hour under refux with a heating mantle/layer. A fber sieve cloth was used to flter the heated mixture. Te diference was discarded, and the residue was returned to the fask, which was then flled with 100 ml of 0.313 M NaOH and heated under refux for another hour. To dissolve any organic constituents, the mixture was fltered through a fber sieve cloth and 10 cc of acetone was added. Te residue was rinsed twice on the sieve cloth with 50 mL of hot water before being put into the preweighted crucible. To remove moisture, the crucible with the residue was oven-dried overnight at 105 o C. Te residue-flled oven-dried crucible was chilled in a desiccator before being weighted (W1) and ashed at 550 o C for 4 hours [9]. Te crucible was cooled in a desiccator and weighted to get white and grey ash (free of carbonaceous particles) (W2). Te crude fber percentage was calculated as follows: Where: W1 � Oven dried crucible containing the residue; W2 � Crucible containing white and grey ash.

Ash Content Determination.
After the material has been entirely burned at 550°C in a mufe furnace, ash is an inorganic residue that remains. It is the sum of all inorganic elements that are not volatile. In an ashing mufe furnace, approximately 8 grams of fnely ground dried cofee powder sample was weighed into a porcelain crucible and cremated (burned) at 550°C for 6 hours until ash was recovered.
Desiccators were used to chill the ash before reweighing it [9]. Te following formula was used to determine the percent (%) ash content in the cofee sample: 2.6.6. Total Carbohydrate Determination. Te overall percentage carbohydrate content of the cofee sample was calculated by subtracting 100 from the total values of crude protein, crude lipid, crude fber, moisture, and ash constituents of the sample. Te result is the sample's % carbohydrate constituent [10]. Tus: %carbohydrate � [100(% moisture + %crude fiber 2.6.7. Calculating the Calorie Content of Cofee Samples. By multiplying the protein amount by 4, the carbohydrate content by 4, and the fat content by 9, the calorie value of the samples was calculated [10].

Result and Discussion
3.1. Protein Analysis. Te stated fgures for green cofee protein content are mainly based on determining crude nitrogen and multiplying by 6.25 [11]. For average protein contents, no signifcant diferences (P < 0.05) were found among the 104 genotypes (checks and accessions) evaluated (Table 2). Te protein content of 104 cofee bean samples for diferent cofee genotypes ranged from 6.93% as a minimum value to 10.14% as a maximum value in the current study, with an average of 8.75% (Table 3). Diferences in protein composition in cofee bean samples from diferent cofee genotypes could be attributed to genetic diferences. Santos et al. [12] found that the protein level of several cofee samples ranged from 9.21-14.33%, which is consistent with the current fgure for cofee beans. Alakali et al. [13] revealed the protein concentration of various tea samples ranging from 8.35-10.67%, which is consistent with the current fnding. Te protein level of several cofee bean samples was in the range of 7-16.16% according to Nogaim et al. [14], which agrees with the current data. Awika et al. [15] also recorded the protein level of various cofee samples ranging from 14.00-16.10%. Tessema et al. [16] found that the protein content of various cofee bean samples ranged from 3.69-5.24%, which is lower than the current fgure.

Analyze the Ash (Total Minerals).
Ash is the inorganic residue left after water and organic materials have been removed by heating in the presence of oxidizing agents, and it is used to calculate the total amount of minerals in food. Te notion that minerals (the analyte) may be separated from all other components (the matrix) within food in some measurable way underpins analytical approaches for delivering information about the overall mineral content. Minerals are not damaged by heat and have low volatility compared to other food components, hence, the most generally used methods are based on this. Te total mineral (ash) content of the cofee genotypes difered considerably (P < 0.05) ( Table 2). Te average ash level of 104 cofee bean samples for diverse cofee genotypes ranged from 2.51% to 5.47%, with a minimum of 2.51% and a maximum of 5.47% (Table 3). On a dry basis, mineral content accounts for (4.00 to 5.00%) of cofee weight [17]. Te ash percentage of the present samples was greater than the ash content of cofee bean samples (3.90 to 4.42%) as stated by Risso et al. [18]. According to Santos et al. [12], the average ash content in diferent cofee bean samples is in the range of (4.00 to 4.90%), which is consistent with the current study. Te average ash percentage in all cofee bean samples in this investigation was identical to the ash content in green tea samples (4.79%) reported by Akande et al. [19]. According to Nogaim et al. [14], the ash percentage of several cofee bean samples ranged from 3.40 to 6.51%, which is consistent with the current ash content data.

Lipid Analysis (Crude Fat).
Lipid estimation is one of the most important aspects of any food material's nutritional evaluation [20]. Te amount of lipids in cofee beans from diferent cofee genotypes varied signifcantly (P < 0.05) ( Table 2). Te lipid content of 104 cofee bean samples for various cofee genotypes ranged from 8.89% at the lowest to 16.08% at the highest, with an average of (11.30%) in the middle (Table 3). However, the range of these values was higher than the lipid content of green tea plants, which was reported as 6.09% by Akande et al. [19]. Te present samples' lipid fraction was found to be in agreement with the averaged lipid fraction in cofee beans, which was around 15%, as stated by Ayaz et al. [20]. As reported by Modupe et al., the range of lipid contents of cofee bean samples was also found to be larger than the range of lipid contents of green tea (3.25 to 5.53%) [21]. However, the study sample data are consistent with the lipid content of green cofee beans, which was reported as 2.49 to 13.13% by Nogaim et al. [14]. Cofee has a fat content of 7 to 17%. Green arabica cofee beans have an average lipid content of 15%, but robusta cofees have a substantially lower lipid content, averaging approximately 10% [22]. Te changes in the lipid composition of cofee bean samples from diferent cofee genotypes identifed in this investigation could be related to the efect of genetic composition. Te presence of a signifcant amount of lipids indicates that these beans have the potential to serve as a dietary supplement with promising nutritional properties.

Crude Fiber.
Dietary fber has lately acquired prominence due to its potential to lessen the prevalence of cardiovascular and digestive illnesses. Te World Health Organization   [23]. Te samples studied were found to be signifcantly diferent (P < 0.05) ( Table 2). Te fber content of 104 cofee bean samples for various cofee genotypes ranged from 6.79% at the lowest to 22.25% at the highest, with an average of 16.29% (Table 3). Dietary fbers are nonstarch polysaccharides that bind minerals and speed their passage through the digestive system, reducing nutritional bioavailability and absorption. When fbers work along with other food ingredients like phytate, tannin, or oxalate, the whole process becomes more successful [24].

Carbohydrate Analysis.
Cofee beans in diferent cofee genotypes exhibited signifcant (P < 0.05) variation in the amount of carbohydrate (Table 2) and the carbohydrate content in the study samples was in the range of 40.65-59.38% and the mean carbohydrate content for the cofee beans was 49.78% (Table 3). But, the range of these values was greater than that of carbohydrate contents for green cofee beans as 7.92 to 35.64%, which was reported by Nogaim et al. [14]. As reported by Bhattacharjee et al. [10], the carbohydrate content of diferent onion (Allium cepa L.) bulb samples was in the range of (14.15 to 14.77%) which is still very less than the present result. According to the result of the carbohydrate content in cofee beans, it was possible to conclude that cofee beans can be used as an enormous amount of energy source for consumers.

Caloric Value.
Te inherent chemical energy inherent in the bonds of the organic molecules of foods, such as their protein, carbohydrate, and fat constituents, as well as minor ingredients such as organic acids, are measured by the calorie value of a food. Te quantity of calorifc value in cofee beans from diferent cofee genotypes varied signifcantly (P < 0.05) ( .

Principal Component Analysis (PCA).
Using 104 cofee (Cofea arabica L) genotypes/accessions and principal component analysis for 6 characters, the frst three principal components with eigenvalues larger than one explained 71.84% of the overall variation (Table 5). Discriminatory characteristics such as crude protein, crude fber, and crude ash accounted for the frst main component, which accounted for 31.44% of the variability between accessions. Similarly, variance in crude ash and total carbohydrate accounted for 24.79% of the total diversity among the   (Table 5). Crude ash and crude protein both played a role in the variances in two of the three primary components (Table 5). Te current study found that cofee genotypes/accessions have a lot of variances in the traits they looked at. Tis wide trait diversity among cofee genotypes/ accessions suggests that there are numerous opportunities for genetic improvement through direct selection from genotypes/accessions and/or selection of diverse parents for hybridization programs, as well as germplasm conservation for future use. Te discovery of biochemical compound composition variety in cofee (Cofea arabica L) is in line with previous research [3,16,[26][27][28].

Divergence Analysis (D 2 ) for 6 Quantitative Characters.
Teproc discrim of SAS procedure of pair-wise generalized squared distance was used to examine inter and intracluster distances for six quantitative characters. Te results revealed signifcant and highly signifcant (P < 0.05 and P < 0.01) genetic distances between the majority of clusters, as well as nonsignifcant variation within accessions grouped in the same cluster ( Table 6). Clusters that are divergent in the intercluster distance study are good sources of genotypes that might be employed in the hybridization program to get a wide range of variance in the segregates and maximize heterosis from genetically varied parental lines. Te current study discovered that such information can be used and that there is a group of distantly related genotypes that can be used right away in the hybridization of a hybrid variety generation program. Clusters III and X had the greatest intercluster distance (D2 � 344.16), followed by clusters II and X (D2 � 236.33), VII and X (D2 � 199.04), and clusters VI and I (D2 � 106.25). Clusters I and IV had the smallest intercluster distance (D2 � 10.09), followed by II and IV (D2 � 10.66), and I and VI (D2 � 11.03). The intercluster distance with the highest value suggested that the accessions in these clusters were diferent. Te lowest cluster distance, on the other hand, indicates a close link between the accessions.
Cofee accessions from cluster X and cluster I to XI and VI, as well as cluster XI and cluster I to VII, alongside cluster VIII and cluster I to VII, and cluster VII and cluster I to VI, could be possible parental lines for boosting heterotic value by crossing, based on the fndings. Crossing germplasm accessions from diferent clusters of wide Mahalanobis distance (D2) could maximize opportunities for transgressive segregation, according to Peeters and Martinelli [29], because there is a high probability that unrelated genotypes will contribute unique desirable alleles at diferent loci. Te degree of heterosis between populations, which refects gene frequency diferences, is proportional to their genetic divergence [30]. According to Singh [31], divergence analysis is used to discover varied genotypes for hybridization purposes, with genotypes grouped together being less divergent than genotypes in diferent clusters, especially clusters separated by the greatest statistical distance (D2).
Mean performance of diferent clusters of the 6 traits studied (Table 8) showed that accession in cluster-VII was the high protein value (9.65) followed by cluster-X (9.41) and the least protein value was cluster-VI (7.90). Similarly, an accession in cluster-XI was the high fat value (16.80) followed by cluster-VII (15.80) and the least fat value was cluster-VI (10.21). Besides, an accession in cluster-VIII was the high fber value (20.71) followed by cluster-VII (20.33) and the least fber value was cluster-X (6.79). Also, an accession in cluster-X was the high ash value (4.83) followed by cluster-III (4.56) and the least ash value was cluster-VIII , and I and VI (D2 � 11.03). Te frst three principal components accounted for 71.84% of the overall variation, according to the PCA. Tese genotypes should be adequately preserved and could be utilized as a starting point for improving the genetics of the crop's distinguishing characteristics through selection and hybridization. Furthermore, the majority of the cofee qualities had favorable connections with one another. Te nutritional and antinutritional content of distinct accessions was studied, and it was discovered that cofee genotypes difer signifcantly in terms of caloric value, carbohydrate, crude protein, crude fber, crude fat, and crude ash. Furthermore, cofee could be used as plant food to help those with protein-energy malnutrition by adding essential nutrients to their diet. Furthermore, molecular investigations should be carried out to further characterize the germplasms in order to assure efective usage, conservation, and traceability of the country's vast cofee genetic heritage. As a result, this study ofered quantitative  I  20  19.23  AW-44, AW-54, AW-45, AW-48, AW-34,AW-36,AW-11,AW-47,AW-51,AW-77,AW-70,AW-37,AW-86,AW-76,AW-85,AW-30,AW-31,AW-   data on the biochemical contents of various cofee genotypes based on their inherent features and the alterations that may occur.

Data Availability
Te biochemical data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.