Manufacture of fresh cheese using east African Calotropis procera leaves extract crude enzyme as milk coagulant

Abstract The present work was aimed to develop fresh cheese using the crude extract from calotropis procera leaves and thereby performed evaluating milk‐clotting activity and acute toxicity of crude extract. The extraction of coagulant was performed at 30, 45, and 60℃ using 2, 6, and 10 g of dried calotropis procera leaves, respectively. The highest yield of crude enzyme extract was acquired with 2 g and 30℃, and results were varied significantly (p < .05). The highest cheese yield and fastest clotting time were 17.89 kg cheese/100kg milk and 14:50 min acquired with 10 g C. procera powder at 60℃ extraction temperature, respectively. E. coli, total bacterial count, yeast, and mold microbial load detection were observed below the limit established by codex alimentarius. This study recommends that use of C. procera crude enzyme leaves extract can be used as alternate milk coagulant for production of fresh cheeses. Soft cheeses manufactured with calotropin enzyme as milk coagulant revealed no adverse effects of toxicity tested on albino rates to the highest dose.

the decline in the natural rennet supply (stomach of calves) and the stable increase in world cheese processing in recent years have led to an augmented demand for new rennet substitutes, motivating a search for new sources of proteases with rennet-like properties from plant sources. Numerous attempts have been made to replace expensive animal rennet with low-priced plant substitutes in order to deal with its shortage, but unfortunately most of them are considered unsuitable either due to their excessive proteolytic nature or extreme bitterness in final cheese products (Naz et al., 2009).
Several milk-clotting enzymes of microbial origin have been commercialized and used in cheese processing. Nowadays, plant rennets have become a subject of growing interest in cheese industry, due to their easy availability and simple purification processes (Milica, Lidija, & Marija, 2013). Furthermore, the use of plant proteases in cheese manufacturing promotes the greater acceptability by the vegetarians and may improve their nutritional intake (Duarte et al., 2009). For several years, plant extracts have been widely used in the preparation of various types of cheeses which are mainly produced in Mediterranean countries, Southern Europe, and West Africa (Roseiro, Barbosa, Mames, & Wilbey, 2003).
Crude extracts from fruits and plants have been used for long time as milk coagulating agents. These include extracts from papaya (papain), pineapple (bromelain), castor oil seeds (ricin), the latex of the fig tree, and the plant calotropis procera which grows abundantly in many parts of Africa. The proteases from plant sources offer a high potential as processing aids in production of cheese with characteristic aroma and texture to cheeses. The common plant proteases papain, ficin, and bromelain have a low milk-clotting activity/ proteolytic activity (MCA/PA) ratio and have often been mentioned as the principal obstacle to their utilization in cheesemaking (Feijoo-Siota, Villa, Feijoo-Siota, & Villa, 2011). A broad proteolytic specificity and the nonspecific hydrolysis of caseins affect texture, flavor, and yield of cheese (Jacob et al., 2011). However, some proteases that are highly specific for milk proteins are attractive as milk-clotting and/or ripening agents in cheese production.
The high prices of calf rennet, refusal to accept cheese made from animal rennet in general and porcine rennet by vegetarians and Muslims, respectively, necessitated the need to substitute animal rennet with easily available, relatively cheap, and acceptable source of rennet for cheese preparation. For these reasons, the plant Calotropis procera has been given much attention for alternative vegetable rennet since it has the potential to completely substitute animal and microbial rennet used for commercial cheese production.
Calotropis procera is a wild African bush potential plant, which is rich in bioactive substances (phytochemicals). The plant has high activity of antimicrobial, antioxidant, antifungal, antidiabetics, and anticancer, and it is one of traditional medicinal plant in Ethiopia. The Calotropis procera plant grows in dry deciduous bush lands, plain soil at roadsides, and abandoned residences. The plant grows widely on the altitude around 600-2,300 m in Afar plains, Shoa, Wello, Gojam, Tigray, Harerghe, Arsi, Sidamo, Gamo Gofa, and Iliababra parts of Ethiopia. C. procera is a traditional herbal plant and named in different parts of Ethiopia such as Qimbo, Tobiaw, Ghinda (Amharic), Falfala adal (Oromipha), Akalo, Dinda (Tigregna), Abuwo (Agnuak), and Boha (Somalic), and in English, it is named as Apple of Sodom (Dead Sea fruit) (Bekele, 2007).
Calotropis procera leaves traditionally use as antimicrobial activity in flat bread handling in pastoral and agro-pastoral areas of Ethiopia. The calotropine is a protease cysteine coagulant agent which plays a great role in milk-clotting activity it presents in the C. procera plant parts (Benyahia Krid et al., 2016). Dairy food spoilage is caused by the growth of microorganisms, primarily bacteria and fungi, that convert nutrients into energy which they use for their own growth. Depletion of the nutrient content of dairy food and the secretion of by-products from this biochemical process are two things which contribute to the spoilage of food rendering it inedible.
The most common microbial defects of cheese are the development of early and late gas, but these problems were overcoming by better hygiene in milk production and better quality control in cheese plants (Hayaloglu, 2015). Knowing the microbial quality profile in soft cheese processing is very vital prior to sensory quality analysis in this research. The more control you have over the microbes, the more consistent your cheese will taste.
Calotropis procera is a plant widely distributed in tropical and subtropical regions of Africa and Asia with a long history of use in traditional medicine. The Calotropis procera has been shown to possess some coagulating properties and has been used as a coagulant in soft cheese (ayib) making by the Raya Agro-Pastoralists, particularly in Mehoni, Chercher, Megalle, Harra, and nearby the Kobo and Zoble mountain of northeastern part of Ethiopia.
While the potential exists for using plant proteases more extensively in dairy food processing and other biotechnological processes, information regarding their development as processing aids, particularly their utility in cheesemaking, is scarce. Accordingly, the aim of the present research was to evaluate the milk-clotting activities of C. procera crude enzyme leaves extract in terms of temperature dependence and amount of dried calotropis procera leaves and their performance on coagulating time, cheese yield, and quality characteristics of soft cheese. Besides, toxicity of calotropis procera extract crude enzyme on albino rats was also evaluated.

| Collection of Calotropis procera fresh leaves
The succulent leaves of Calotropis procera plant were collected from Nazareth (90 km southeast of Addis Ababa); it is located 8°33′0″ N Latitude, 39°16′0″ E Longitude at an elevation of 1,627 m with an average annual rainfall of 808 mm. The collected C. procera leaves were, identified by Botany Department, cleaned very well and dried by setting the temperature at 45 ± 1°C according to the method described by Netoa et al. (2013). Then after, dried leaves of Calotropis procera ground into powder by using an electric blender and kept the powder under airtight container until next use.

| Coagulant extraction
The C. procera leaves powder was weighed in portions of 2, 6, and 10 grams using the method described by Chikpah et al. (2014), by increasing the sample dose based on the sideline laboratory toxicity test. The extraction was assessed by the method described by García et al. (2012) and Shobowale, Ogbulie, Itoandon, Oresegun, and Olatope (2013). The extraction temperature was modified from constant temperature to 30, 46, and 60°C for each sample dose, to analyze crude extract on clotting activity and cheese manufacturing process.

| Processing of soft cheese
The development of soft C. procera cheese was prepared following the general soft cheese production procedure described by   (Figure 1). Each cheese sample was made from 2,000 ml pasteurized cow milk with protein and fat content of 3.5% and 2.7%, respectively. Cheese vat temperature was set at 32°C, and the probiotic starter culture was added at a rate of 1.5 ml/100 ml at set temperature. Then, it was incubated for about 4-5 hr. When the pH reached to 6.1-6.3, the prepared crude calotropin was added to the milk and stirred to enhance distribution of enzyme throughout the cheese vat and kept until curd formation was apparent. After checking curd strength by using spatula, the curd mass was cut using stainless steel cheese knife at pH 4.8 to remove the whey and drained further using cheese cloth and mesh sieve. After whey was drained, the curd was molded in shapes and finally molded soft cheese was dipped in a brine solution of 12% NaCl for about 12-15 hr, which was performed to impart a good test for the cheese and as a preservative. Samples coded (C1, C4, C7), (C2, C5, C8), and (C3, C6, C9) are cheese samples made at concentration (dose) of 2, 6, and 10 g of C. procera powder extract enzyme, respectively. C1 to C9 are cheese produced by C. procera crude extract enzyme.
Finally, different analysis for developed soft cheese samples was performed comprising yield, moisture, texture, color, compositional, microbial, and sensory quality attributes using standard procedure.
The following flowchart was generally employed to produce soft cheese production.

| Acute toxicity evaluation of crude enzyme extract and soft cheese
Acute toxicity of C. procera extract and cheese developed by crud extract were performed on albino mice as method described by OECD guideline 420 (OECD, 2001) and according to Netoa et al. (2013).
Experimental animals used in this study were thirty-five healthy female mice (weighing 20-25 g) and were obtained from animal house of Ethiopian Public Health Institute. They were kept under standard conditions (at a temperature of 21 ± 2℃, with 12 hr' light/12 hr' dark cycle) and provided with free access to standard pellet laboratory diets and drink tap water unlimitedly. Before the experiment, they were grouped randomly into 7 groups (n = 5/group) and then kept in their cages for 5 days to allow acclimatization to the laboratory conditions. F I G U R E 1 Flow diagram of soft cheese production according to  After acclimatization, all groups were fasted. Doses and the vehicle were calculated based on their body weight. Conversion of human to animal dose calculation was performed according to Shin, Seol, and Son (2010) and Nair (2016). The C. procera extract was then administered orally, using oral gavage, at a single dose of 26.84, 46.97, 67.1, 93.94, and 134.2 mg/kg (with conversion factor) for albino mice in the test group of 1, 2, 3, 4, and 5, respectively.
The control group (7) received distilled water, and the other group (6) were fed with cheese made using C. procera extract instead of pellet (standard laboratory diets) for 14 continuous days. The observations were performed during the first four hours after the oral treatments to determine LD 50 and to assess the effect on behavior.
After that, clinical observations were made once every 24 hr for the next 14 days for mortality, behavior (piloerection, tremors, sedation, loss of corneal reflex, motor activity), body weight, and gross physical changes. Finally, on the 15th day, their final weights were measured, and then, they were sacrificed to examine gross pathological changes in their organs.

| Measurement of milk-clotting activity
The method of Berridge (1952) was used to measure the clotting activity in rennet unit (RU). The milk-clotting activity of C.procera crud enzyme extract was measured for nine different extracts (E1-E9) at various temperatures (32, 50, and 60℃). The standard substrate was prepared by reconstituting skim milk powder at 10% (w/v) solution of 0.01 M CaCl 2 and warmed at 32℃. The prepared extracts were added at a proportion of 1 ml/10 ml of standard substrate, mixed manually, and incubated in a water bath at 32°C. After thoroughly mixing three times, the "zero" clotting time started. The milkclotting activity of each extract was measured, with the assumption that all the soluble proteins from the extract were enzymes which coagulate milk at 32, 50 and 60℃. The clotting activity equation as reported by Berridge (1952) in rennet units (RU) was used.

| Compositional analysis of milk
The crude fat, protein, and lactose of the pasteurized milk were determined by using milk scan instrument, while the ash content was determined according to AOAC (2000) official method no.923.03.
The pH of the pasteurized milk was determined based on the standard methods of AOAC (2000).

| Proximate and physicochemical analysis of soft cheese
Protein Protein content of cheese samples was determined according to the method described by Ogunlade, Oyetayo, and Ojokoh (2017).

Fat
Fat content determination was carried out by Soxhlet extraction method based on AOAC (2000), Method no. 933.05.

Ash
Ash content determination was performed according to AOAC where dry matter coefficient = % solids/100.

| Mineral composition
The mineral composition of developed soft cheese was analyzed according to the method described by Hui (2006) using the atomic absorption spectrophotometry.

| Color analyses
The color in cheese was measured using color flex spectrocolorimeter after being standardized using a Hunter Lab color stabilizer. The unripened cheese samples were placed beneath the optical sensor at the tip of the instrument and allowed for postprocessing. The parameters recorded were L*, a*, and b* coordinates of the CIE scale was performed according to Netoa et al. (2013).

| Titratable acidity
For determination of titratable acidity, about 10 g cheese was weighed and crushed with 105 ml water. This solution wasLLac filtered, and 25 ml of filtered solution was used for titration. Three drops of phenolphthalein were added and titrated with 0.1 N NaOH until the first permanent pink color was appeared it was performed according to Endale, Eshetu, and Zelalem Yilema (2016).
(3) Ash (%) = wt after ashing − tare wt of crucible original sample wt * dry matter coefficient * 100 (4) Lactic acid (%) = ml N 10 NaOH * 0.009 ml of sample used 100 2.8 | Cheese yield, clotting time and texture profile Cheese yield, clotting time, and texture profile analyses were performed for ten cheese samples produced using commercial rennet and C. procera crude extract enzyme.

| Cheese yield
Cheese yield was calculated according to the method described by Akinloye and Adewumi (2016)  where Ya = actual cheese yield.
Ya expressed in kg cheese/100 kg milk.

| Clotting time for cheese production
The time taken for the pasteurized cow milk to make a first clot formation was observed carefully and recorded. The average time taken for coagulation was taken as a clotting time for each samples according to the method described by Chikpah and Teye (2015).
Milk-clotting activity of crude enzyme extract was assessed at different temperatures. The clotting time was determined by dipping a clean spatula or glass slide into the milk. When coagulation has occurred, flecks of curd were appeared in the milk film on the slide.

| Texture profile
Texture profile analysis (TPA) of soft cheeses was performed according to the method described by García et al. (2012). Texture Analyzer (LLOYD Instruments, TA plus Ametek, UK 2007) was used to determine the hardness of soft cheeses. The sample was wrapped and equilibrated at 20℃ for 30 min before testing. The cheese was placed in the center of the plate, and then, cutting jig probe was applied at constant crosshead speed of 21 mm/s.

| Microbiological quality of soft cheese
Microbiological analyses of fresh cheese samples were conducted based on Adetunji and Babalobi (2011).
It was performed for four cheese samples, where sample 1, 2, and 3 are produced at minimum, medium, and maximum concentrations of coagulant and extraction temperature whereas sample 4 was a control cheese produced by commercial rennet. Each four samples of cheeses were analyzed in triplicate at initial day of production. E. coli, yeast, mold, and total aerobic plate counts (TPC) were determined using the method NMKL No. 125 (2005) and NMKL No. 98 (2005) and NMKL No. 86 (2006, correspondingly. The number of colonies on the tested sample was described as colony forming unit cfu/g.
where N = the average colonies per ml or g of product; C = sum of all colonies on all plates counted; n 1 = number of plates in the first dilution counted; n 2 = number of plates in the second dilution counted; d = dilution from which first count was obtained.

| Sensory attribute analyses of fresh cheese
Three soft cheese samples of equal weight were designed to sensory evaluation using 35 unexperienced panelists based on a five-point hedonic scale from 1 to 5 (1-"disliked" to 5 -"excellent") according to Adetunji and Babalobi (2011). The sensory quality attributes considered taste, flavor, color, texture, and overall acceptability of products.

| Experimental design and statistical data analysis
This design of the research experiment used was completely randomized design. ANOVA of 95% confidence interval and Design Expert software version 7.0 were used. In addition, the data obtained were statistically analyzed by using SPSS software version 20.

| Acute toxicity of crude extract and manufactured fresh unripen cheese
In toxicity study, the extract of leaves up to the highest dose of 134.2 mg/kg did not show any significant changes on behavior (such as alertness, aggressiveness, and irritability), gross physical appearance (condition of fur, general cleanliness) compared with the control (Figure 2). The gross pathological examination on the organ of treated albino mice showed no significant changes in color, size, shape, and texture compared with the control group based on laboratory observations (Figure 2). The extract of C. procera and cheese developed by C. procera crude extract did not induce changes in the behavior of mice. No death was recorded during the 14 days' test.
Crude C. procera aqueous extract and the cheese LD50 could not therefore be estimated and are possibly higher than 134.2 mg/kg.
Treated albino mice organs (liver and kidney) were not significant (p > .05) statistically as compared with the control groups feed with (5) Ya = grams of cheese produced grams of milk used + starter culture * 100% distilled water as control (Table 1). There was a gradual increase in the body weight of both the treated and control mice. At the end of the experiment (after 14 days), the mean body weight gain for mice treated with different doses, control diet, and soft cheese was presented in Table 1.

| Crude C. procera extract yield and pH
The yield of crude C. procera extract as coagulant ranged 85.04-57.74 ml at extraction temperature of 30°C and pH value of crude C. procera extract arrayed between 6.69 and 6.41 (Table 2). Results of C. procera extract pH value were moderately close to the finding reported by Akinloye and Adewumi (2016). According to the research findings reported by Akinloye and Adewumi (2016), the Calotropis procera plant coagulant yield of extract was 666 ml which is more value compared to the current findings (85.04 ml). The pH and yield higher values for Calotropis procera extract were 6.71 and 85.04 ml, respectively. In general, the crude C. procera extract yield can depend on the origin of the plant material. extraction temperature, environmental condition, moisture content, rain fall pattern, plant management, and dose applied.

| Milk-clotting activity of crude enzyme extract
The milk-clotting activity of crude C. procera extract is shown in   (Table 4).

| Compositional analysis of fresh cheeses
Sample coded with C3, C6, and C9 shows the highest fat and protein content, which is high dose treated with high temperature.
Compositional analysis of the unripened cheese obtained for all samples in the current study was more or less in conformance with the findings of Uaboi-Egbenni et al. (2010). Where the moisture content of 64%, 12.86% protein, 13.4% fat, and 0.60% ash was reported for a cheese made with the extract of Sodom apple (Calotropis procera) leaf extract and lactic acid.
Other findings on wara soft cheese produced by C. procera extract were reported by Adetunji and Babalobi (2011)  Note: All values are means ± SD. Values with the same subscripts of the column are significantly(p < .05) different, where samples coded (C 1 ,C 4 ,C 7 ), (C 2 ,C 5 ,C 8 ), and (C 3 ,C 6 ,C 9 ) are cheese samples made at concentration (dose) of 2, 6, and 10 g of c.procera crude extract enzym,; respectively. C 1 to C 9 are cheese produced by c.procera crude extract enzyme.

TA B L E 4 Compositional analysis of fresh cheese based on dry weight basis
Generally, C. procera coagulant significantly (p < .05) affects the compositional analyses of cheese, which in turn indicates that as the dose of C. procera increases, the content of protein and fat also increases too. Although ash content also increased in the manufactured soft cheese products which in turn indicate C. procera cheese might be rich source of minerals.

| Mineral composition
The processed soft cheese contains essential minerals such as calcium, iron, and zinc. From Table 5, it is evident that calcium content of the soft cheese ranged from 6.78 to 312.07 mg/100 g. Similarly, zinc and iron contents were ranged from 1.41 to 2.50 mg/100 g and 0.07 to 1.85 mg/100 g, respectively.
Fresh cheese developed by C. procera had increased mineral content comparing to control cheese. This is due to the presence of minerals in the plant coagulant. Thus, the calcium is an important for osteoporosis and bone disease, as it is the major building block of human bone tissue. Recommended daily average intake of calcium is 1,000-1,300 mg/day stated by international osteoporosis foundation (IOF). Thus, the required amount of calcium can be obtained from fresh cheese of C. procera coagulant.
The calcium content obtained in this study was in agreement with the findings of Roseiro et al., 2003, in which calcium contents at two dairy plants were 13.11 and 12.48 g/kg. The finding of iron content was moderately higher compared with research findings of Malomo, Faduola, Adekoyeni, and Jimoh (2013) and which was 32.7 ppm.
Although it is more likely close with the findings of Adetunji and Babalobi (2011). Moreover, control cheese sample mineral composition was less value compared with the soft cheese samples prepared using crude enzyme extract of C. procera.

| Color and titratable acidity of soft cheese
Titratable acidity and color indices (L*, a*, and b* values) and pH of the three cheese samples were presented in Table 6. Result of pH varied from 6.12 to 6.35, titratable acidity varied from 0.104% to 0.108%, and color indices L* (88.26-84.1), a* (0.78-2.33), and b* (25.68-18.98) as shown in the Table 6. All values were differed significantly (p < .05).
Actual cheese yield (kg cheese /100 kg milk) result of cur-  (2015) were for soft cheeses produced at concentrations of 2, 5, and 7 g, respectively. Results of cheese yield vary compared to other Extraction temperature Samples Ca (mg/100 g) Fe (mg/100 g) Zn (mg/100 g) Note: All values are means ± SD; values with the same subscripts of the alike column are significantly (p < .05) different. Where T 1 , T 2 , and T 3 are extraction temperature of crude extract enzyme at 30, 45, and 60°C, respectively; where C 10 (cheese 10) represents control sample produced using commercial rennet and the rest cheese 1-9 (C 1 -C 9 ) were cheeses produced by c.procera crude extract; and where samples coded (C1, C4, C7), (C2, C5, C8), and (C3, C6, C9) are cheese samples made at concentration (dose) of 2, 6, and 10 g of c.procera crude extract enzyme, respectively.

TA B L E 5
Mineral concentration of soft cheese expressed in mg/100g researchers findings, whereas the current study soft cheese yield was expressed in actual yield which includes amount of starter culture used for manufacturing.
The clotting time for C. procera dried leaves with 2-7 g dose was ranged from 27 to 29 min (Chikpah and Teye (2015). The shortest clotting time was observed while used starter culture for cheese production. The clotting time differed significantly (p < .05) among different temperature (30, 45, 60°C) and different concentration (2, 6, 10 g) treatments. Generally, the coagulating time reduced with increasing concentration of C. procera extract as well as extraction temperature.
The texture of dairy foods is as important as flavor and color.
The dairy manufacturers are now using texture as a key differentiating quality to improve a product's overall consumer appeal.
The texture profile of soft cheese prepared from C. procera coagulant has harder texture profile compared with control sample. The val- ues of hardness of soft cheese prepared from C. procera coagulant obtained in this study were higher compared with the findings of García et al. (2012), who reported hardness of 9.51N and 7.99N for a cheese made from the plant extracts of Cynara Cardunculus subspecies Cardunculus and Cynara cardunculus sub species Flavescens, respectively. The difference was being by the type of plant origin, differences in environmental conditions, and biochemical composition of extract.

| Microbiological quality of fresh cheese
The three cheese samples (1, 2, and 3) show reduced amount of microbial load comparing to sample 4 ( All results are means ± SD. Values with the same subscripts of similar column are significantly (p < .05) different, where C 10 (Cheese 10) represents control sample produced using commercial rennet.
C 1 , C 2, and C 5 are selected cheeses made at lowest, medium, and highest extraction temperature and various dose of crude calotropin extract.
In this count, <1 × 10 1 is the standard reporting format for plates from all dilution of the sample which has no colonies.
Yeast and mold counts of cheese samples produced by C. procera coagulant and commercial calf rennet were superior than sample 3, sample 2, and sample 1, correspondingly (Table 8).
This could be because of the sanitation adopted during milking, transportation, storage, and processing of cheese production. Even though the results for E. coli for all samples were detected below 1 * 10 1 .
Yeast and mold counts of the three cheese samples were in conformance with the findings of Seifu, (2013) who reported yeast and mold counts of metata ayib are ranging from 1.2 × 10 2 to 6.1 × 10 3 cfu/g, whereas TPC of metata ayib are ranging from 2.5 × 10 5 to 6.9 × 10 7 cfu/g and 6.35 log10 cfu/g which is much higher than reported by the current study. In the current research findings, the TPC values of soft cheese samples were lees than that of the international standard set by Codex Alimentarius (1968).
Microbial load of cheese samples (1, 2, and 3) were significantly (p < .05) affected by C. procera crude extract. Furthermore, samples 1, 2, and 3 revealed reduced TPC comparing to sample 4 which was a control cheese sample processed by commercial rennet. This is because the antimicrobial property of plant extract inhibits the microbial load of the cheese. In a nutshell, the results of microbio- logical quality characteristics of soft cheese samples obtained in this study were in agreement with the standard set by codex alimentarius commission.

| Sensory quality evaluation of soft cheeses
The sensory evaluation of the cheese produced from the different concentration of crude enzyme extract of C. procera is shown in Figure 3. Taste, flavor, color, texture, and overall acceptability of all the cheese products were significantly (p < .05) affected by the coagulants, whereas texture of cheese samples was not significantly different. The cheese sample produced at low dose (2 g) and extraction temperature (30°C) revealed better sensory quality acceptability. Generally, overall acceptability of the products reduced when the coagulant concentration amount increased. Sample 1 had better taste, flavor, texture, and color, as well as high overall acceptability. A good milk-clotting enzyme is characterized by a high specific caseinolytic activity and a low general proteolytic activity, since the proteolysis strongly affects the sensory properties of cheese.

| CON CLUS IONS
The research focuses on the use of the enzyme calotropin extract,

| S TUD IE S IN H UMAN S AND ANIMAL S
This research does not involve the use of human subjects performed by the any of the authors. All mice experiments comply with the guidelines and carried out in accordance with the U.K animals (Scientific Procedures) Act, 1986, and associated guidelines for the F I G U R E 3 Radar plot of sensory quality evaluation of soft cheese. Where sample 1 (C1) was cheese produced at low dose and extraction temperature (2 g at 30°C), sample 2 (C5) was cheese produced at medium dose and extraction temperature (6 g at 45°C), sample 3 (C9) was cheese produced at high dose and high extraction temperature (10 g at 60°C) care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). The authors indicate that informed consent was obtained for experimentation with animals. Overall, for toxicity test of the extract and manufactured cheese products a total of thirty-five healthy female mice (weighing 20-25g) were obtained from animal house of Ethiopian Public Health Institute. They were kept under standard conditions (at a temperature of 21 ± 2℃, with 12 hours' light/12 hours' dark cycle) and provided with free access to standard pellet laboratory diets and drink tap water unlimitedly.

| COMPLIAN CE WITH E THIC AL S TANDARDS
The study conforms to the declaration guideline reviewed and approved by the research ethics committee of the Addis Ababa Institute of Technology. Informed consent was waived by the board, and the research is in line with the international recommendations for the ethical standards.

ACK N OWLED G M ENTS
The authors would like to thank the Ethiopian Public Health Institute and Ethiopian Institute of Agricultural Research for permitting to use their key research laboratories.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. The author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported.