Optimization of Awaze paste formulations: The effects of using spices through a mixture design approach

Previous studies have revealed the microbial quality of Awaze paste. However, limited reports describe the effect of individual spices on Awaze paste quality. A mixture design approach was used to determine the appropriate proportions, with 15 experimental points for independent variables including RP (60–90 %), GA (10–30 %), RO (5–20 %), and GI (5–10 %). The techno-functional properties, particle size, antioxidant activity (DDPH radical assay), proximate composition, iron (Fe), zinc (Zn) content, viscosity, hardness, and microbiological quality of Awaze paste were assessed. The prepared Awaze paste showed a range of characteristics, with antioxidant activity (DDPH radical assay) ranging from 11.86 % to 62.5 %, crude protein content from 6.18 % to 16.22 %, crude fat from 5.7 % to 12.6 %, crude fiber from 16.86 % to 29.06 %, total ash content from 6.32 % to 9.94 %, total carbohydrate from 41.79 % to 60.61 %, energy from 264.3 to 329.2 k cal. , iron (Fe) content from 35.59 to 108.82 mg/100g, zinc (Zn) content from 1.72 to 26.93 mg/100g, viscosity from 65.5 to 125.5 cps, hardness from 8.48 to 55.09 g, yeast and mold count from 0.83 to 2.04 log cfu/g, and total bacterial count from 1.53 to 2.61 log cfu/g. Significant differences (p < 0.05) were observed in proximate composition, techno-functional properties, particle size, antioxidant activity, physicochemical properties, and microbiological characteristics among the formulations of Awaze paste. The selected formula showed a statistically significant difference (p < 0.05) compared to the control sample. The formulation containing 74.79 % RP, 10 % GA, 10.2 % RO, and 5.0 % GI was determined to be the optimal formula with a desirability of 0.73, based on the evaluated parameters. This preferred Awaze paste had a porosity of 28.12 %, particle size of 16.49 μm, antioxidant activity of 63.63 %, crude protein content of 17.28 %, iron (Fe) content of 98.06 mg/100g, and zinc (Zn) content of 15.04 mg/100g. Therefore, this optimal blend of ingredients could be used to produce a consumer accepted Awaze paste.


Introduction
Food formulation is important for developing consumer preferred new food products that offer health and nutritional benefits.* Corresponding author.Faculty of Chemical and Food Engineering, Bahir Dar Institute of Technology, Bahir Dar University, Ethiopia.
Contents lists available at ScienceDirect Food formulation is the process of developing food products with the goal of meeting nutritional and dietary needs [1].Micro-nutrient deficiencies such as zinc (Zn), iron (Fe), and vitamin A pose a significant world health problem.The majority of malnourished individuals worldwide, over 80 %, are found in developing countries [2].Food formulation using staple foods like wheat, maize, and rice in sub-Saharan Africa has been identified as a strategy to address both macro-nutrient and micro-nutrient deficiencies [3].In this region, various approaches are employed to tackle malnutrition, including formulating complementary foods from ingredients like rice, faba bean, peanut oil, and sweet potato, which are rich in vitamin A and iron (Fe) content.In Ethiopia, significant efforts have been made to combat malnutrition [4].The prevalence of chronic malnutrition (stunting) has decreased from 58 % to 38.4 % and wasting decreased from 12 % to 9.9 % between 2000 and 2016.This decrease is due to the use of nutritionally enhanced agricultural products, food formulation, and fortification [5].
Fermented Awaze paste is a spicy Ethiopian traditional cuisine used to make stew (Wot) in rural households and as a seasoning for spicy food.It is prepared mainly from RP, GI, GA, and RO with small proportions of fenugreek, basil, thyme, rosemary, mekelesha, cinnamon, white cumin, black cumin, and rue.These ingredients contain high amounts of antioxidants, minerals, and fiber, including carotenoids.These compounds, with expressed antioxidant and anti-inflammatory activities that improve scar formation, decrease blood cholesterol levels, and improve stamina [6][7][8].The amount and characteristics of flavor, color, and especially pungent behaviour of red pepper are important quality parameters.The pungency, aroma, and availability in different colors, shapes, and sizes are important characteristics of red pepper that make it favourable for use as an ingredient in the production of several foods [9][10][11][12].Garlic is a common food spice used widely for its medicinal value, as a preservative, and for its flavor quality [13][14][15].
Among the main ingredients, red onion has a significant contribution in terms of nutritional value to the human diet as well as improving the taste, flavor, and aroma of food [16][17][18].Ginger is also important in the production of oleoresin and oil due to its high content of bio-active compounds [19][20][21].Spices are well-known food ingredients that enhance the flavor and aroma of foods.Nutritionally, spices play a significant role in reducing fatty acid oxidation and preserving foods, and they also have a vital role in food formulation due to their significant nutritional and sensory quality, as well as their health benefits [22,23].
Previous studies have examined the physicochemical and microbiological quality of laboratory-made Awaze paste.The microbiological quality of Awaze paste during fermentation at different time intervals was reported [24].The behaviour of E. coli under different storage conditions was studied [25].The role of probiotics in lactic acid bacteria isolated from Awaze paste were studied in vitro [26,27].
Mixture design is a crucial tool in the design of formulation of food products, especially for foods prepared with many ingredients, to study the influence of each individual ingredient on the food characteristics.This helps in finding the optimum formula based on the desired quality of the product [28,29].Optimal mixture design is a useful tool for determining the optimum formula by considering the best quality characteristics of foods [29,30].
There is limited research in the literature on the formulation of Awaze paste.Women in Ethiopia prepare Awaze paste at the household level, but the proportion of RP, GI, RO, and GI varies from women to women.There is no standard method of preparation for Awaze paste, which affects the characteristics of the final food product due to variations in ingredient proportions.Therefore, the objective of this work was 1) to formulate a composite powder of spices from RP, GA, RO, and GI for an acceptable Awaze paste and 2) to evaluate the techno-functional properties, particle size, antioxidant activity, proximate composition, iron (Fe) content, zinc (Zn) content, viscosity, hardness, and microbiological properties of enriched Awaze paste.

Materials
The experiment was done in October 2022 at the Holeta Agricultural Research Centre (HARC) in Ethiopia.A sample of RP was obtained from the Bure district in the Amhara region of Ethiopia, due to its potential for red pepper production [31].Additionally, GA, RO, and GI were collected from HARC.The spice ingredients needed in small amounts were collected from the Holeta market in October 2022 in the Oromia region of Ethiopia.These spices were fenugreek, cardamom, black cumin, cinnamon, ground pepper, clove, rue, kemune, gewze, thyme, coriander, white cumin, basil, long pepper, and rosemary.After collecting all the required ingredients, Awaze paste was prepared and techno-functional properties, proximate composition, iron (Fe) content, zinc (Zn) content, viscosity, hardness, and microbiological quality were determined.Analytical grade chemicals and reagents were used for this work.

Experimental design for mixing ratio optimization
The main goal of the experimental design was to maximize the selected dependent variables that serve as the best quality criteria for Awaze paste.These variables include porosity, particle size, antioxidant activity, crude protein content, as well as zinc (Zn) content and iron (Fe) content through optimization of dependent variables.In the experiment, the ratio of ingredients was set according to the household preparation protocol commonly used by many women in the districts where Awaze paste could potentially be utilized.Four independent variables, including RO, GA, RO, and GI, were considered in the experimental design for their proportions.The minimum and maximum ranges for all four parameters are provided to create an experimental design.Based on the mixture design, a total of 15 formulations were obtained.The study included constraints of RP (X1: 60-90 %), GA(X2: 10-30 %), RO (X3: 5-20 %), and GI(X5: 5-10 %).
Numerical and graphical optimization were used to find the best ingredient mixing ratio and predict experimental points for the mixture.The ingredients RO was set to the maximum, while RP, GA, and GI were set to the range for the numerical optimization.Crude B. Kefale et al. protein content, antioxidant activity, zinc (Zn), and iron (Fe) content were set to maximum, while the other parameters were set to in the range.Crude protein content, antioxidant activity, zinc (Zn), and iron (Fe) content were given high relative importance of "5'' in the work sheet.A significant test was conducted using an F-test for the model for analysis.The effects of changing each mixture constituent were visualized through contour plots of the selected model.The formulated Awaze paste was compared to a control sample based on selected parameters.The crude protein content, antioxidant activity, zinc (Zn), and iron (Fe) content of the Awaze paste product were evaluated.
All the formulations were as follows: F stands for formulation, RP stands for red pepper, GA stands for garlic, RO stands for red onion, and GI stands for ginger:

Control sample
The control sample collection area was chosen based on the availability of the product in the area, as well as the frequency of production and utilization of Awaze paste.It was obtained from bure district of Amhara region.It was prepared at the household level using ingredients identical to those used for the formulated Awaze paste.However, the ratio of the ingredients used in the control sample differed from formulated Awaze paste.

Product development of Awaze paste 2.4.1. Preparation of ingredients
Awaze paste was prepared following traditional household methods as described [24].Dry and wet spices were involved in the Awaze paste product development.Dry spices such as fenugreek, cardamom, red pepper, long pepper, cinnamon, clove, kemune, coriander, black cumin, white cumin, and ground pepper were combined with wet spices like ginger, basil, red onion, garlic, rue, thyme, and rosemary to prepare the paste.The dried and cleaned RP was pulverized with a wooden mortar and pestle.Wet spices, including GA, RO, and GI, were washed and peeled.After cleaning, washing, peeling, sun-drying, and lightly roasting of both dry and wet spices, each formulation was done as follows.
The independent variables (RP, GA, RO, and GI) were mixed based on the formula obtained from mixture design.Controlled variables consisted of 5 g of fenugreek, 5 g of cardamom, 2.5 g of basil, 2.5 g of white cumin, 1.25 g of mekelesha, 1.25 g of black cumin, 1 g of rue, 0.5 g of coriander, 0.4 g of thyme, 0.4 g of rosemary, and 20 g of salt, which were constantly added to each formulation.The mixed spices were milled using a laboratory mill (Perten Instruments, Finland).After milling the mixed spices, 200 g of composite powder was added to 300 mL of boiled water and mixed until it became thick consistency.A control sample was used for comparison with the formulations.

Proximate composition
The moisture content, crude protein, crude fiber, crude fat, total ash, total carbohydrate and energy value of each formulation were B. Kefale et al. determined using the official method [32].

Techno-functional property of the composite powder 2.6.1. Water absorption capacity(WAC)
The WAC was determined following the method [32].A 1 g of the sample was mixed with 10 mL of distilled water in a centrifuge tube.The solution was allowed to stand at 30 • C for 1 h, then centrifuged at 2000 rpm for 30 min.The supernatant volume of the composite powder was then measured in a 10 mL measuring cylinder.The WAC was calculated following the equation provided below (1): WAC = 10 − Volume of water left unabsorbed (1)

Bulk density (BD)
The BD of the formulations was analyzed following the method described [33].For each formulation, 50 g of the composite powder of Awaze paste was added to a 100 mL measuring cylinder, and the volume was then measured.The BD was determined following the equation provided below (2): BD = Weight of powder sample Volume of powder sample (2)

Tapped density (TD)
The TD of the formulations was determined following the method [33].The TD of each formulation was determined by adding 50 g of sample to a measuring cylinder and manually tapping the cylinder vertically until the height of the powder in the cylinder did not change.The TD of Awaze paste was calculated according to the equation provided below (3): TD = Mass of taping Volume of taping (3)

Porosity (ε)
The porosity of the formulation was determined using the formula ε (%) [34].The porosity of Awaze paste was determined according to the equation provided below (4): Where BD = bulk density and TD = tapped density.

Hausenr ratio (Hr) and Carr's index (CI)
The HR and CI of the samples were calculated using the following formula [34].The HR and CI of Awaze paste were determined according to the equation provided below (5 and 6): Where BD = bulk density and TD = tapped density.

Particle size
The particle size of the formulated Awaze paste was determined using a laser diffraction particle size analyzer (Master sizer 2000; Malvern Instruments Ltd., Worcestershire, UK) following the method [35].The composite powder of the formulated Awaze paste samples were dispersed in water (ISO 13320-1) using the master sizer instrument.In the particle size distribution curve of the analyzed sample, D 10 , D 50 , and D 90 representing 10 %, 50 %, and 90 % were used to describe the size of particles and D (4, 3) and the specific surface area (m 2 /g) were used to express volume-weighted mean for each formulations.

Physicochemical property of Awaze paste 2.8.1. pH value
The pH value of the prepared Awaze paste was obtained according to the method [36].A 10 g Awaze paste sample was mixed with 100 mL of distilled water, and the pH value was determined using a calibrated pH meter (Mettler Toledo, China).

Titratable acidity (TA)
The TA of Awaze paste was determined following the method [36].A 10 g of prepared Awaze paste were dissolved in 100 mL of distilled water.The mixture was stirred gently and allows to stand for 1 h.A 10 mL of the solution were then taken, and 0.5 mL of phenolphthalein was added.The solution was titrated with 0.1 M NaOH until a pink color appeared.The TA of Awaze paste was determined according to the equation provided below (7): Vol.NoH used(mL)x0.1 M NaoH x milli equvalant factorx100 mass of sample(g) ( 7)

Total soluble solids
The TSS of Awaze paste were obtained following the method [32].A 10 g of Awaze paste were dissolved in 50 mL of distilled water.The mixture was then filtered, and a 1 mL aliquot was placed in a digital refractometer.The result was recorded as o Brix.

Antioxidant activity
The antioxidant activity of the formulation and control samples was determined following the method [37].For each formulated Awaze paste, a 400 mg Awaze paste was placed in a 15 mL centrifuge tube with 5 mL of a solvent mixture MeOH: H 2 O (80:20, % v/v).The supernatant from the centrifuged sample mixture was transferred in to a 15 mL of measuring cylinder.The residue was then re-suspended in 5 mL of MeOH:H 2 O (80:20, %v/v) and gently vortexed for an additional 30 min, followed by centrifugation.The supernatant was combined with the initial extract and the volume of the combined supernatant was adjusted to 10 mL with the extraction solvent.The methanolic extracts of Awaze paste was then tested for antioxidant activity using the 2,2-diphenyl-1-picrylhydracyl (DPPH) radical assay method as described [38,39].For each concentration of methanol extract of Awaze paste (20, 40, 60, 80, 100 μL), 5 mL of a 0.1 m M methanol solution of DPPH radical was added to each formulation and concentration.The tubes containing Awaze paste sample solutions were allowed to stand at 27 • C for 20 min.The sample solution absorbance was recorded at 517 nm in a UV-Vis spectroscopy (Shimadzu UV-Vis Spectroscopy 1900).The IC50 value was determined as the amount of ethanolic extract necessary to decrease the initial DPPH radical concentration by 50 %.Antioxidant activity was determined according to the equation provided below (8):

Iron (Fe) and zinc (Zn) content of Awaze paste
The mineral content (Fe and Zn) of the formulated Awaze paste and control sample were determined using AAS (Model: 240 FS, Agilent Series, America) following the method [32].A 0.5 g Awaze paste sample was digested in a muffle furnace at 550 • C for 3 h until the residue ash turned white.The ashed Awaze paste sample was then digested using concentrated HCl.The digested Awaze paste sample was diluted to 100 mL for iron (Fe) and zinc (Zn) analysis.The Iron (Fe) and zinc (Zn) content of the formulated Awaze paste was obtained following the formula provided below (9): Iron (Fe) and zinc (Zn) content (ppm) = (conc.AAS − blank) × Volume of sample × dilution factor weight of sample (9) Where conc.AAS= Concentration reading of the sample from atomic absorption spectroscopy.
2.9.Rheological and Texture property of Awaze paste 2.9.1.Viscosity A 3 g of Awaze paste sample was dissolved in 5 mL of distilled water.The viscosity of Awaze paste solution was then determined using a Brookfield Viscometer (model Lv-3, Middleboro, MA 02346, USA) set at 60 rpm and a temperature of 25 • C [40].

Texture of Awaze paste
The hardness of Awaze paste sample was measured using TPA (model: TA.XT.Plus, Stable Micro Systems, UK), following the method [41].A P-2 cylindrical probe with a 2 mm diameter was used, and pre-test speed and test speed were set at 2 mm/s and 1 mm/s, respectively.Compression distance was set at 5 mm, with a load cell of 50 kg.Two compression were applied consecutively to the Awaze paste samples, and the hardness was measured.

Microbial profile
The total bacterial count, lactic acid bacteria count, yeast and mold count, and total coliform count were detected using the official method [32].Samples of 25 g of Awaze paste were liquefied with 225 mL of 0.1 % peptone water.Serial dilution was performed by taking 1 mL from the diluted Awaze paste sample, and diluted samples (1 mL) were applied to the media surface using the pouring technique.Duplicate plates were prepared in all cases.The total bacterial count was estimated by applying PCA and incubating at 30 selected and counted using a colony counter (Model: Scan 300, Inter Science).The microbial load was determined following the method [42].The microbial load of Awaze paste was obtained according to the equation provided below (10): Where N = total count of bacteria, yeast and mold, lactic acid bacteria of Awaze sample, n = average number of bacteria, yeast and mold, lactic acid bacteria colony from different dilution, S = volume of sample for plating (mL), and d = dilution factor of Awaze paste sample.

Formula cost and yield percentage of the formulated Awaze paste
The cost and yield percentage of the formulated Awaze paste were determined using the formula [43].The cost of formula and percentage of the formulated Awaze paste determined according to equation provided below (11 and 12): Cost per kg x% in formula 100 (11) Yield percent = (EPQ / APQ)x 100 (12) Where EPQ = edible portion quantity and APQ = as purchased quantity.

Data analysis
Statistical analysis of the formulation and control sample was done using SPSS software (IBM, Chicago, IL, USA).For the formulation experiment Design Expert software version 13.0.1 was used.The individual effects of ingredients (spices) were illustrated using contour plots.ANOVA and the Tukey test were employed to determine the least significant difference among the formulations at (P < 0.05).

Fitting of the model
Table 1 provides details on the mean porosity, particle size, pH, antioxidant activity, crude protein, zinc (Zn) content and iron (Fe) content of Awaze paste influenced by RP, GA, RO, and GI.Residual plots were created to assess the goodness of fit of the model.The best model exhibits an adequacy precision greater than 4, high predicted R-squared (R 2 ), lack of fit (P > 0.05), low standard deviation, and low predicted sum of squares, which is considered desirable for the best fit to the model [44] as shown in Table 1.The linear model was identified as the best fit for zinc (Zn) content, while the quadratic model was identified as the best fit for antioxidant activity and particle size.The special cubic model was found to be the best fit for porosity, pH, crude protein content, and iron (Fe) content with R 2 (0.93, 0.99, 0.97, and 0.99) respectively (Table 1).Therefore, the mixture model could be utilized to explore the design space.
Moreover, high F-values ranged from 1.18 to 56.39 and coefficients (R 2 ) ranged from 0.47 to 0.99 ensuring a satisfactory fit of the proposed model to represent the actual relationship between the dependant and independent variables.The interaction between the amounts of RPxGI, ROxGI, and GAxGI in Awaze paste showed a significant negative effect only on particle size (F = 4.05, p = 0.02) as shown in Table 2. Therefore, this proposed mathematical model is valid and convenient for predicting the porosity, particle size, pH, antioxidant activity, crude protein, iron (Fe), and zinc (Zn) content of Awaze paste prepared under any combination of RP, GA, RO, and GI.

Effect of RP, GA, RO and GI on proximate composition of Awaze paste
The proximate composition of Awaze paste showed a significant difference (p < 0.05) between the formulations for moisture, crude protein, crude fat, crude fiber, total ash, total carbohydrate content, and energy value of the samples (Table 3).The moisture content of Increasing the ratio of RP and GI increases the crude protein content of Awaze paste as shown in contour plot (Fig. 2e).The total ash content of the formulated Awaze paste and the control sample ranged from 6.32 % to 9.94 %.The crude fiber content of formulated paste sample ranged from 16.86 % to 29.06 %.The total carbohydrate content and the energy values of Awaze paste ranged from 40.34 % to 60.61 % and 264.35 kcal.to 329.27 kcal , respectively.The observed differences between the formulations and the control sample for crude fat, crude protein, crude fiber, total ash, total carbohydrate content, and energy value are probably due to the difference in the storage time, raw materials, and preparation method.
A previous study on Korean red pepper paste (gochujang) reported crude fat, crude protein, total ash, total carbohydrate, and energy content ranging from 0.3 % to 3.1 %, 3.4 %-9.3 %, 5.2 %-21.1 %, and 129 kcal.to 247.97 kcal, respectively [45], which was in agreement with the current result for total ash content.However, there were differences in crude fat, crude protein, total ash, total carbohydrate, and energy content, which may be attributed to differences in raw materials and processing methods.The proximate compositions (except moisture and total carbohydrate) of this study were higher than the findings reported in a study on garlic and ginger-enhanced ogi paste [46], The reported values for crude protein, crude fat, crude fiber, total ash, moisture, and total carbohydrate were in the range of 9.83 %-10.92%, 2.34 %-3.06 %, 1.47 %-2.42 %, 0.28 %-1.44 %, 16.32 %-25.67 %, and 59.24 %-66.95%, respectively.A similar study on onion paste reported comparable results to the current study [47].

Effect of RP, GA, RO, and GI on the techno-functional properties of composite powder
The techno-functional properties of the composite powder formulations are presented in Table 4.The water absorption capacity of the formulations ranged from 1.59 % to 2.36 %.Previous studies have reported the water absorption capacity of RP (1.86 %) and GA (2.51 %) [6].Formulations F2 (67.28:19.37:8.28:5.07RP, GA, RO, GI) and F4 (68.84:13.99:12.16:5.01RP, GA, RO, GI) had the highest water absorption capacity, possibly due to the higher proportion of GA and RO in the mixture, while formulation F12 (63.27:24.68:7.03:5.02RP, GA, RO, GI) had the lowest water absorption capacity (1.59 %).Bulk density, tapped density, porosity, and  flowability are important parameters for saving packaging material, transportation, and processing of raw materials during manufacturing [48].The bulk density ranged from 351.9 kg/m 3 to 409.4 kg/m 3 , and tapped density ranged from 436 kg/m 3 to 547 kg/m 3 in the formulations.The highest bulk density (409.4 kg/m 3 ) was obtained for F10 (65.27:10:17.7:7.03RP, GA, RO, GI).The highest tapped density (547 kg/m 3 ) was found for F6 (76.39:10:8.6:5.01RP, GA, RO, GI).These may be due to the highest proportion of GA and RO in the formulation.The porosity of the formulations ranged from 12.9 % to 31.3 %.The lowest porosity was obtained for F3 (70.13:10:9.87:10RP, GA, RO, GI), with the lowest porosity value being 12.9 %.The formulation F6 (76.39:10:8.6:5.01RP, GA, RO, GI) showed a porosity of 31.3 %, which was the highest porosity among the formulations; this could be due to the larger particle size of RP.The results of the current study were similar to the reported result [49].As the proportion of GA and GI increases, the porosity was significantly increased (p < 0.05) (Fig. 2a).As the mixing ratio of RP and RO increases, the porosity significantly decreased.Therefore, we suggest that a significant difference in porosity is due to the difference in the proportion of ingredients (RP, GA, RO and, GI) in the formulation.
Flowability is expressed as the free flow of powders during food processing, handling, and transportation.Flowability of powders can be expressed as Hausen ratio (HR) and Carr's index (CI) [50].Powders having (HR < 1.34) and (CI < 25 %) can be considered as better in flowability.In the current study, F6 (76.39:10:8.6:5.01RP, GA, RO, GI) had poor flow-ability with an HR value of 1.5 and CI value of 31.3 as shown in Table 3. Powder flowability is directly related to the particle size uniformity of powders [51].Powder flowability is an important property in commercial applications such as storage, transportation, manufacturing, and processing of food products [52].Similar results were studied for red pepper paste that was made only from red pepper powder, with values of HR (1.48) and CI (32 %) [53].

Effect of RP, GA, RO and GI on particle size and surface area of the composite powder
The particle size of the formulated composite powder of Awaze paste was reported as D 10 , D 50 , and D 90 percentiles (Table 5).The particle size of the formulated composite powder of Awaze paste showed a wide range, with D 10 ranged from 13.25 μm to 25.05 μm, D 50 from 91.5 μm to 172 μm, and D 90 from 373.5 μm to 558.5 μm.Formulation F2 (67.28:19.37:8.28:5.07RP, GA, RO, GI), had a D 50 value of 172 μm and a D 90 value of 558 μm, showing a higher particle size compared to Awaze paste with lower GA and RP proportions.This difference may be attributed to the larger particle size of GA and RP powders.Significant difference at (P < 0.05) were observed for specific surface area and volume-weighted mean diameter among the formulation, with specific surface areas ranging from 58.45 m 2 /g to 111.8 m 2 /g.The highest volume-weighted mean diameter (230.5 μm) was observed for F2 (67.28:19.37:8.28:5.07RP, GA, RO, GI), while the lowest value (108.5 μm) was measured for F11.This difference indicated that F11 (74.39:13.33:5:7.28RP,GA,RO,GI), had the finest powder among all the formulations.Increasing the proportion of RP and GI led to an increase in the particle size of the composite powder, while an increase in the proportion of GA and RO decreased the particle size (D10) of the composite powder as shown in contour plot in (Fig. 2b).

Effect of RP, GA, RO and GI on total soluble solid, pH and titrable acidity of Awaze paste
Formulations significantly affected the total soluble solid (TSS) of Awaze paste at (p < 0.05).F8 (60:15:20:5.0RP, GA, RO, GI) had the highest TSS value.This may be due to the highest mixing ratio of RO in the formulation.Optimal pH is one of the pre-requisites for the quality of foods and affects the occurrence of several biochemical activities [45].The formulations exhibited pH ranged from 5.15 to 5.75.The lowest pH value was found in the control sample (5.15), while the highest pH value (5.75) were obtained for F3 (70.13:10:9.87:10RP, GA, RO, GI), F4 (68.84:13.99:12.16:5.01RP, GA, RO, GI), and F5 (60:30:5:5 RP, GA, RO, GI).However, the pH value of the formulations and control sample were statistically significantly different (Table 5).Foods with a pH value greater than 4.6 are categorized as low acidic foods, and with a pH below 4.6 are acidic foods [54].All formulations had a pH value lower than 5.2.This may be due to the non-fermented (fresh) nature of the formulation.The low pH value of the control sample (5.15) that was household-made may be due to the fermentation of the product as a result of long storage time.A previous study report showed a pH range of 4.59-4.79for red pepper paste [55].The present study observed similar pH to the report of [56], that was conducted on several laboratory-made red pepper paste.The pH content of Awaze paste was increased with an increase in the proportion of RP and RO as shown (Fig. 2c).
Titratable acidity (TA) indicates the extent of fermentation and acidity in Awaze paste.The titratable acidity of formulations ranged from 0.15 % to 0.32 %.F15 (60:14.81:15.2:9.99RP, GA, RO, GI) had the highest titratable acidity of 0.32 %.TA increased with more GA and less RP.A study on red pepper and tomato pastes reported titratable acidity ranging from 1.12 % to 2.59 % [57], which is lower than the current results.

Effect of RP, GA, RO and GI on antioxidant activity of Awaze paste
The antioxidant activity of the formulated Awaze paste towards DPPH radical was measured at different concentrations (Table 6).There was a significant difference (p < 0.05) between the formulations in antioxidant activity.The antioxidant activity ranged from 11.16 % to 62.5 % for formulations at different concentrations (20,40,60,80, 100 μL), and the antioxidant activity increased with an increase in concentration from 20 to 100 μL (Table 6).The highest percentage of inhibition, 54.13 %, was found for F1 (60: 20.2:12.01:7.77RP,GA, RO,GI), while the lowest percentage of inhibition, 34.24 %, was obtained for F4 (68.84:13.99:12.16:5.01RP, GA, RO, GI) at a low concentration (20 μL).These findings showed that the formulation affects the antioxidant activity of Awaze paste.
The highest percentage of inhibition in F1(60: 20.2:12.01:7.77RP,GA, RO,GI) was because of the high proportion of RP and GA.This finding was in agreement with a study [58] that reported percentages of inhibition for Brazilian spices ranging from 30 to 90 %.Another study reported that the percentages of inhibition of hot red pepper paste ranged from 44 % to 90 % [8], which was higher than the current result.Increasing the proportion of RP, GA, and GI in the mixture enhances the percentages of inhibition of the formulated Awaze paste, as shown in the contour plot (Fig. 2d).
The IC50 value in each formulation gave information regarding the quality and reactivity, indicating the amount of free radical scavenger compounds present in each formulations.The lower the IC50 value, the more potent the substance is at scavenging DPPH radical, indicating a higher antioxidant activity.The IC50 value was ranged from 8.5 μg/mL to 706.78 μg/mL at different concentrations as shown in (Fig. 1).Considering the parameters IC50 value obtained by the DPPH radical assay, F8 (60:15:20:5 RP, GA, RO, GI) had the highest IC50 value 421.26 μg/mL at 80 μL and 706.78 μg/mL at 100 μL respectively; it may be because of the higher mixing ratio of RO in the formulation.On the other hand, F1 (60: 20.2:12.01:7.77RP,GA, RO,GI) had the lowest IC50 value 8.5 μg/mL, 29.88 μg/mL, and 51.11 μg/mL at 60 μL, 80 μL, and 100 μL respectively, meaning that compared to other formulation F1 extract can be considered a fast acting free radical scavenger.Previous study on onion, garlic and ginger reported IC50 value 2224 μg/mL, 41 μg/mL, and 19.1 μg/mL respectively [59].In the other study, the IC50 values were found to range from 694.8 μg/mL to 1153.63 μg/mL for colored bell peppers [60].Notably, both reports indicated higher IC50 values compared to the current study.

Effect of RP, GA, RO and GI proportion on iron (Fe) and zinc (Zn) content, rheological and textural property of Awaze paste
The iron (Fe) content, zinc (Zn) content, viscosity, and hardness of the formulated Awaze paste were significantly different (p <   7).The iron (Fe) and zinc (Zn) content of Awaze paste ranged from 36.54 mg/100g to 108.82 mg/100g and 1.72 mg/100g to 26.93 mg/100g, respectively.F6 (76.39:10: 8.6:5.01RP, GA, RO, GI) had the highest Fe content (108.82mg/100 g) and Zn content (26.93 mg/100 g).This may be attributed to the higher iron (Fe) and zinc (Zn) content of RP and RO [6].The iron (Fe) and zinc (Zn) content of Awaze paste increased significantly, as the proportion of RP and GI increased in the mixture, as shown in the contour plot (2f and 2g).
The viscosity of the formulations and control sample ranged from 71 cps to 125.5 cps.The highest viscosity was found for F7 (64.55:15.46:10.17:9.82RP, GA, RO, GI).The lowest viscosity values were found for formulation F12 (63.27: 24.68: 7.03: 5.02 RP, GA, RO, GI).This could be due to the higher mixing ratio of GI and RO, as GI and RO have higher viscosity compared to RP and GA as reported by Ref. [6].The viscosity in the current results is higher than the reported viscosity values (13.24 cps-85.84cps) of hot pepper-soya bean paste [61].F15 (60:14.81:15.2: 9.9 RP, GA, RO, GI) had the highest hardness value (55.09 g), which was due to the highest proportion of GA and GI.

Microbiological quality of formulated Awaze paste
Microbiological analysis at zero time (without storage period) for the formulations showed that lactic acid bacteria and coliform were not detected in the fresh Awaze paste sample.The tested Awaze paste product, total bacterial plate count was ranged from 1.53 log cfu/g to 2.61 log cfu/g (Fig. 3a), and yeast and mold count in all formulations ranged from 0.83 log cfu/g to 2.04 log cfu/g (Fig. 3b).Considering 6 log cfu/g as the maximum limit of total bacterial count and 4 log cfu/g for yeast and mold, it resulted that the formulated Awaze paste is safe and acceptable according to international standards for food safety [42].
The microbial population in food samples is highly dependent on physicochemical, microbial profile, and the external environment of the raw material.The microbial population in the samples (Awaze paste), especially total bacteria count (<6 log cfu/g) and yeast and mold (<4 log cfu/g), indicated that the raw materials used for formulations were handled and dried in hygienic conditions.The findings from the present study for bacteria count, yeast, and mold were in agreement with the reported results [57,62].Compared to the findings of [45] conducted on fermented red pepper paste (gochujang), reported total bacteria plate count, yeast, and mold were ranged from 2.79 log cfu/g to 8.73 log cfu/g and 1.56 log cfu/g to 7.15 log cfu/g, respectively, which is not in agreement to the current results.

Optimization
The optimum formula was determined by numerical and graphical optimization in Awaze paste samples prepared within the range comprising RP (60-90 %) , GA (10-30 %), RO (5-20 %), and GI (5-10 %).The optimum formula includes all the selected parameters at optimum values (porosity, particle size, pH, antioxidant activity, crude protein, iron (Fe) content, and zinc (Zn) content) as indicated in (Fig. 2h).The proportion of RP, GA, and GI were set to the range, while RO was set to the maximum.The antioxidant activity, crude protein, iron (Fe) content, and zinc (Zn) content were set to maximum, while the other parameters were set within the range.A high relative importance of "5" was given to the antioxidant activity, protein, iron (Fe) content, and zinc (Zn) content of formulations.

Table 7
Mineral content, rheological and textural property of Awaze paste and Household made sample.The results indicated that the optimum formula is better in nutritional value compared to the control sample.

Formula cost and yield percentage of Awaze paste
The formula cost of Awaze paste is shown in Table 8.Accordingly, the cost of the optimized Awaze paste is 301.95 birr per kg (5.25 USD/kg), which is affordable to consumers.Because the cost is low compared to other formulated and manufactured food products available in supermarkets in Ethiopia.The yield percentage for the formulation was also found to be 84.61% by considering GA, RO, and GI as wet spice (washed and peeled spice) from all the spices used to prepare the optimized Awaze paste.During formulation, the wet spices, GA, RO, and GI were washed and peeled, resulting in trim (loss) due to washing and peeling of the cover skin to be edible.Generally, the formula cost and yield percentage indicate a profitable product formulation, and as a result, any entrepreneur can start to manufacture the product using the optimum formula for Awaze paste.

Conclusion
Red pepper significantly increases the total mineral content, iron (Fe) content, and zinc (Zn) content of Awaze paste.Garlic significantly improves the flowability, particle size, and antioxidant activity of Awaze paste.An acceptable Awaze paste was formulated with 74.79 % RP, 10 % GA, 10.2 % RO, and 5.0 % GI.The formulated Awaze paste is rich in antioxidant activity, crude protein, total ash content, iron (Fe) content, and zinc (Zn) content.The paste produced has a low microbial load, indicating that the product is shelfstable.This study shows the potential of utilizing these locally produced spice raw materials and is important for new product development for household producers, cottage industry, and the spice industry.In the future, a morphological study and the influence of storage materials and methods on the physicochemical and microbiological quality of the optimized Awaze paste are highly recommended.

Fig. 3 .
Fig. 3. A) Total bacterial count and B) yeast and mold count of the formulated Awaze paste.
• C for 48 h.PDA was used to isolate yeast and mold, incubating at 25 • C for 48 h.LAB were estimated by using MRS agar and incubated at 30 • C for 48 h.Coliform were estimated by pouring VRBA and incubated at 35 • C for 24 h.Plates containing distinct colonies were B. Kefale et al.

Table 1
Effect of RP, GA, RO, and GI on the physicochemical quality of Awaze paste.Awaze paste ranged from 4.66 % to 12.24 %.The highest moisture content (10.78 %) was obtained from F11 (74.39:13.33:5.0:7.28RP, GA, RO, GI), while the lowest moisture content (4.6 %) was for the control sample.This difference may be due to the freshness of the prepared Awaze paste and the long storage of the control sample.The crude protein of the product ranged from 6.18 % to 16.22 %.The highest crude protein (16.22 %) was observed for F6 (76.39:10:8.6:5.01RP, GA, RO, GI), while the lowest crude protein content, 6.18 %, was for the control sample.

Table 2
Interaction effect of the independent variables (RP,GA,RO, and GI).

Table 3
Proximate composition of formulated Awaze paste and control (household made Awaze paste).
F = formulation, Result = mean standard deviation.Values with different lowercase letters in the same column are significantly different (P < 0.05).B.Kefale et al.

Table 4
Techno-functional property of the formulated composite powder and total soluble solid of Awaze paste from RP, GA, RO and GI.
F = formulation, NI = not important, Result = mean standard deviation.Values with different lowercase letters in the same column are significantly different (P < 0.05).

Table 5
Particle size of the composite powder (D 10, 50, 90 ), and pH, and titratable acidity of Awaze paste from RP, GA, RO and GI.
F = formulation, NI = not important, Result = mean ± standard deviation.Values with different lowercase letters in the same column are significantly different (P < 0.05).B.Kefale et al.

Table 8
Formula cost of the formulated Awaze paste from the optimum formula.= 57.42Birr (current Dollar conversion to Birr in Ethiopia).