Application of Milk-Clotting Protease from Aspergillus oryzae DRDFS13 MN726447 and Bacillus subtilis SMDFS 2B MN715837 for Danbo Cheese Production

Microbial, Cellular and Molecular Biology Department, Addis Ababa University, Addis Ababa, Ethiopia Department of Biology, College of Natural and Computational Science, Debre Berhan University, Debre Berhan, Ethiopia Centers for Food Science and Nutrition, Addis Ababa University, Addis Ababa, Ethiopia Food Science, Nutrition and Technology Department, College of Agriculture and Veterinary Science, University of Nairobi, Nairobi, Kenya


Introduction
Cheese is one of the most important components of dairy industries and more than 1000 different varieties are produced throughout the world [1]. Generally, different varieties of cheese are industrially produced by using calf rennet [2]. Among which, Danbo cheese is semihard cheese characterized by surface ripening with few round pea-sized holes and distinctive flavor and is consumed after ripening for 2-3 months, but a few variants are allowed to mature for up to 12 months [3]. e flavor of Danbo cheese originates from smear bacteria that develop on the surface during the first weeks of maturation [4]. It is originated and most commonly consumed cheese in Denmark and comprises about 13.2% of total Danish cheese production [4,5].
However, the ever-increasing demand for cheese, shortage of calf rennet, and religious restriction on the consumption of calf rennet-based cheese are major challenges for the dairy industries. is necessitated a search for alternatives to calf rennet [6]. With this regard, microbial proteases have become popular and substituted the traditional trend of production. Hence, today only 20-30% of the world demand for milk-clotting preparation is derived from calf rennet [6,7]. Milk-clotting proteases with high activity and stability at acidic pH have important industrial applications, specifically as milk-coagulating agents for cheese processing and as flavor enhancers in other food industries [8]. Microbial rennets are replacing calf rennet because of their low production costs, greater biochemical diversity, and easier genetic modification [9]. Accordingly, many fungal and bacterial proteases are widely used for cheese production. For instance, about 60% of cheese in the USA is manufactured using fungal enzyme sources [9].
However, as to our knowledge, there are limited studies in evaluating the biochemical composition and sensory quality of cheese produced using microbial enzymes.
erefore, this study aimed to investigate the efficiency, biochemical composition, and sensory quality of Danbo cheese produced using proteases derived from the fungus and bacterium compared to the commercial product.

Microbial Strains Used for Aspartic Protease Enzyme
Production.

Solid-State Fermentation and Enzyme Extraction from
Fungi. Enzyme production was induced by growing the micro-organisms using solid-state fermentation [10]. Briefly, 10 g of the durum wheat bran substrate (purchased from an open market) was transferred in a 250 mL Erlenmeyer flask and moistened with 12 mL of (0.2 M) HCl. en, 0.5 mL of 0.5 × 10 6 spore suspensions of A. oryzae DRDFS13 were inoculated into SSF media and incubated at 30°C for 5 days. e nutritional composition of the wheat bran is listed in Table 1.
For enzyme extraction, the fungal culture was dispensed in 100 mL distilled water (1 : 10 ratio) and shaken on a rotary shaker (MaxQ 2000 Open-Air Platform Shaker, ermo Fisher Scientific, USA) at 240 rpm at room temperature. e mixture was centrifuged (Heraeus Pico17/21 centrifuge, ermo Electron Led, Germany) at 10000 rpm at 4°C for 10 min to recover the culture filtrate to be used as crude enzyme source [11].

Submerged Fermentation and Enzyme Extraction from
Bacteria. e medium used for submerged fermentation for bacteria contained (g/L) the following: glucose, 16 [12].
For enzyme extraction, samples were filtered through a Whatman No 1 filter paper (90 mm) after 72 h of cultivation, centrifuged at 10000 rpm for 10 min at 4°C, and then the filtrate was used for the enzyme assay [11].

Detection of Aflatoxins in Fungal
Extract. Aflatoxins B1, B2, G1, and G2 were detected by methanol extraction methods (methanol/water 80/20 (v/v) + NaCl) at Eurofins Analytik GmbH, Neuländer Kamp 1, 21079 Hamburg, Germany. en, the extract was diluted with the Tween solution and applied on the immunoaffinity column, followed by washing the column and elution with methanol, and finally diluted with water. e measurement was taken using high-performance liquid chromatography-fluorescence detector (HPLC-FLD) with cobra cell postcolumn derivatization.

Partial Purification of the Enzyme.
e clarified culture supernatant was subjected to dialysis against 20 mM phosphate buffer (pH 6.0) utilizing a 10 kDa cutoff membrane. After dialysis, a crude enzyme preparation was concentrated by contacting with carboxymethyl cellulose overnight (4°C). e crude enzyme was resuspended in sodium phosphate buffer (20 mM, pH 6.0) [13].

Assay for Milk-Clotting Activity.
e milk-clotting activity of the enzyme was undertaken according to Arima et al. [14]. Accordingly, 0.1 mL of the crude enzyme was added to 1 mL of reconstituted skimmed milk (Nestle ™ ) in a 10 mL test tube preincubated at 35°C for 10 min. e reconstituted skimmed milk (Nestle ™ ) solution consisted of 10 g dry skimmed milk/100 mL and 0.01 M CaCl 2 (AppliChem ™ ). e appearance of the first clotting flakes  Journal of Food Quality was visually evaluated and quantified in terms of Soxhlet Units (SU). e endpoint was recorded when discrete particles were discemible. e clotting time T (s), the period of time starting from the addition of the crude enzyme to the appearance of the first clots and the clotting activity, was calculated using the following formula: where T = clotting time (s) and D = dilution of the crude enzyme.
One SU is expressed as the quantity of the enzyme required to clot 1 mL of the solution comprising 0.1 g skimmed milk powder and 0.01 M CaCl 2 at 35°C within 40 min.

Protease Activity Assay.
e proteolytic activity was assayed according to Arima et al. [14]. Briefly, 0.5 mL of the enzyme extract was added to 2.5 mL of 1% (w/v) soluble casein in 20 mM potassium phosphate buffer at pH 6.5, and the mixture was incubated in a water bath at 35°C for 10 min. After the addition of 2.5 mL of 0.44 M trichloroacetic acid to terminate the reaction, the mixture was filtered through the Whatman No.1 (90 mm) filter paper. e filtrate was then mixed with 1 mL volume of three times diluted 2N Folin/ phenol reagent and 2.5 mL of 0.55 M sodium carbonate solutions and incubated at 35°C for 20 min to detect color development, and optical density (OD) was measured using a spectrophotometer (UV-vis, Liantrinsat, and Model-CF728YW-UK) at 660 nm. One unit (1 U) of enzyme activity was defined as the amount of enzyme that liberated 1 μg of tyrosine per 1 mL in 1 min: where PA is the protease activity, µTry is the µ g of tyrosine equivalent released, V t is the total volume of the assay in mL (5 ml of the substrate plus 1 ml of the enzyme plus 5 ml of TCA), V s is the sample volume (i.e., the volume of protease used for the assay in mL), T is the reaction time (i.e., time of incubation in minutes, 10 min), V a is the volume assayed (i.e., final volume of the product used in calorimetric determination).

Protein
Determination. e protein content in the dialyzed enzyme was determined according to the Kjeldahl digestion, distillation, and titration method [15].

2.9.
Danbo Cheese Production. Danbo cheese was produced in a dairy pilot plant at the University of Nairobi, College of Agriculture and Veterinary Science, Food Science, Nutrition, and Technology Department. e product was made according to the method described by [16]. Cow milk (30 L) was standardized by skimming (fat removal) and pasteurizing by indirect heating at 62°C for 10 min. A total of 30 L of cow milk divided into three equal portions (10 L each). All the three portions were inoculated with 0.6% fermented milk with mesophilic Streptococcus lactis (MicroKwik Culture ® , Carolina). en, the first portion was renneted using commercial rennet (rennin-coagulate (2 g/100 L) as a control (C)). e second and third portions were renneted using the applicable ratio of dialyzed fungal and bacterial enzymes, 5.0% each, and labeled as E1 and E2, respectively. After treatment with a 40% CaCl 2 solution (10 mL/100 mL), all treatments were kept for 30 min for clotting. After this, the curd was cut using a cheese knife at a size of 4-6 mm and stirred for 30-40 min in order to get better cheese grains. After removing the whey, the curd was cooked with hot water at 65°C-39°C for 15-20 min, stirred for 30-40 min for precipitation. en, the cheese was molded for 20-30 min and pressed for 2-3 h by using a metal weighing 30-35 kg/kg of cheese. Finally the pressed cheese samples were salted by dipping into 20% NaCl for 48 h and ripened at curing room for 2 months [16]. e whole production design is illustrated in the flowchart in Figure 1. e fresh Danbo cheese was weighed immediately and the yield was calculated as follows [17]:

Sensorial Analysis of Danbo Cheese and Rating
Its Acceptance. e Danbo cheese was subjected to sensory tests using 13 panelists (8 men and 5 women) from dairy pilot plant and staff members of the Food Science, Nutrition, and Technology Department, University of Nairobi. e rating acceptance sensory test was scored for attributes of color, flavor, aroma, texture, and overall acceptability on a 7point hedonic scale where 7 is extremely like and 1 being extremely disliked. e order of product presentation to the panelists was randomized [15]. In brief, sliced cheese (about 10 g) of each product was provided to the panelists randomly. en, after 3 min, they were provided with the next sample following the same procedure.

Determination of Moisture, Ash, and Fiber Contents.
e moisture content was determined according to [15]. Ash content was determined by burning 5 g of the sample in a muffle furnace at 550°C for 3 hours, as per the [15] protocol.

Determination of pH and Titratable Acidity.
About 10 mL of the Danbo cheese sample was dispensed into a conical flask to determine the pH using the pH meter (ABS accumet, Fisher Scientific, Singapore). e titratable acidity (TTA) was determined by direct titration with 0.1 M NaOH. e results were converted to lactic acid concentration [6]: where titre = 25 mL-sample of titre.

Determination of Crude Protein and
Fat. e crude protein was determined according to the Kjeldahl digestion Journal of Food Quality and titration method [15]. e protein content was calculated from the relationship: total protein(%) � titre * 6.25 * normality of NaOH(0.1N) * 0.014 1000 * sample weight * 100, where protein conversion factor � 6.25 for milk, protein (%) � % nitrogen * 6.25, normality of acid (HCl) � 0.1 N, and sample weight � 1.0 g. Fat content was also determined by the Soxhlet extraction method according to [15].

Determination of the Carbohydrate Contents and
Caloric Value. Total carbohydrate content was determined by difference (i.e., subtracting the sum of the percentage moisture, ash, protein, and fat from 100%). Energy value was quantified indirectly considering the three groups of nutrients, which provide the body with energy carbohydrates, fats, and proteins. One gram of carbohydrate (C), protein (P), and fat (F) will provide 4, 4, and 9 Kcal energy, respectively. erefore, the total caloric values are calculated as follows: where P � protein content (%), F � fat content (%), and C � total carbohydrate (%).

Determination of Mineral Content.
e concentration of minerals was determined using an atomic absorption spectrophotometer (Analytik Jena Nov AA350, Germany) by the method of Osborne and Voogt (1978) [18]. Briefly, 2.5 g of samples was transferred to a porcelain dish and heated at 120°C for 4 h on a hot plate until the entire content had become carbonized. e samples were then heated in a furnace at 530°C until free of carbon; the residue appeared grayish/white after 8 h. en, the crude ash was dissolved in 5 mL of 6 M HCl on a hot plate for 2 h. Subsequently, 7 mL of 3 M HCl was added and heated on a hot plate until the solution boiled. e digested sample was cooled and filtered. en, 5 mL of 3 M HCl was added to the dishes and heated the extract to dissolve the residue. For calcium determination, lanthanum chloride (10% W/V) was added to both standards and samples to suppress interference from phosphorus. en, the concentration of the minerals sodium, potassium, magnesium, zinc, manganese, iron, and calcium was analyzed using an atomic absorption spectrophotometer against calibration curves prepared by plotting the absorption or emission values against the metal concentrations in mg/100 g using the following formula: where W = weight of samples (g), V = volume of extract (mL), A = concentration of sample solution (µg/mL), and B = concentration of blank solution (µg/mL).

Properties of Partially Purified Microbial Enzymes from
Aspergillus oryzae DRDFS13 and Bacillus subtilis SMDFS 2B. e physical and chemical properties of the enzymes derived from bacterial and fungal strains in Ethiopia are reported in Table 2.
ere was no significant difference in titratable acidity (TTA) and total protein content (TP %) between the enzymes extracted from the two sources (p < 0.05). However, the bacterial enzyme had a 2.5-and 7.0-fold higher in milk clotting and protease activities than the fungal enzyme (p < 0.05), respectively. In contrast, the MCA/PA ratio was significantly higher (p < 0.05) in the fungal enzyme than the bacterial enzyme (Table 2).

Properties of Danbo Cheese and Its Properties.
In this study, Danbo cheese was successfully produced using the enzymes from the two sources. e desirable characteristic of the different cheese products was reported in Table 4.
ere was no significant difference in the cheese yield upon the three treatments (p < 0.05). e cheese made with commercial rennet was firm and acceptable. Similarly, the cheese made with the fungal enzyme was slightly firm and acceptable.
However, the bacterial enzyme cheese was watery. In fact, the whey yield was significantly different (p < 0.05) upon the three treatments, in the order of CR > DFE > DBE.

Sensorial Analysis of Fresh Danbo Cheese.
e result of acceptability on the basis of the 7-point hedonic scale of the sensory test of the Danbo cheeses was reported in Table 5.
e color score was in the accepted order of CR > DFE > DBE. ere was a significant difference in flavor among the treatments. However, there was no significant difference in odor and texture scores between the three kinds of cheese.

pH and TTA Liquid Samples Produced during Danbo Cheese Production.
e pH and titratable acidity (TTA) of the three liquid products are shown in Table 6. e inoculation of the raw milk with mesophilic S. lactis (LAB) reduced the pH of the milk by 0.7-1.0 units. e renneting with the crude fungal and the commercial enzymes slightly decreased the pH (0.8-1.0). Also, there was a significant difference in titratable acidity (TTA) among the treatments ( Table 6).

Proximate Composition of Liquid Samples Produced during Danbo Cheese Production.
e addition of the LAB (Streptococcus lactis) drastically decreased the crude fat content by 12-30%, while the crude enzymes from the fungus and bacterium slightly decreased the crude fat content after renneting. e renneting treatment also  Journal of Food Quality decreased the carbohydrate and energy contents of the samples, where the decrease in the latter was higher with the commercial rennet than the crude enzyme treatments (Table 7).

pH and TTA of Danbo
Cheese. e fresh Danbo cheeses produced in all the three treatments were salted by dipping into 20% NaCl solution for 48 h and ripened in the curing room at 10°C for 2 months. In all cases, pH and TTA significantly increased in the ripened cheese irrespective of the treatments (Table 8).

Proximate Composition of Fresh and Ripened Danbo
Cheeses. Moisture content decreased in salted and ripened cheese, in which in the former the rate was higher. Among the three fresh cheese types, CR had the highest protein content followed by DBE and DFE, respectively. e crude protein content in the ripened cheese was in the order of CR > DFE > DBE. In contrast, there was no significant difference in crude protein content among the three salted kinds of cheese. Among the three cheese types, CR ripened cheese had the highest crude protein content (Table 9). Salted CR and DFE cheese types had higher crude fat content, while the DBE salted cheese had the lowest amount. ere was no significant difference in the crude fat content among all the three ripened cheese types (p < 0.05). e ash content in the fresh DBE was significantly lower than the amount in the other two cheese types. In all the cases, the salted cheese had higher ash content than the fresh and ripened cheeses (i.e., CR > DBE > DFE) except for DFE. e ripened cheese types had shown the highest carbohydrate contents in all the three treatments. e salted cheese types had the highest total caloric value compared with the fresh and ripened cheese.

Mineral Contents of Ripened Danbo Cheese.
e ripened cheese products showed a significant difference in their mineral composition except for sodium (Table 10). Potassium and zinc concentrations were the highest in DBE while calcium and manganese concentrations were the highest in DFE cheese. Zinc and potassium concentrations were higher in the microbial enzyme-made cheeses than in the control.
Similarly, calcium content was higher in the DFE-and DBEmade cheese than in the control. Manganese was not detected in the control cheese, but significantly higher concentration was found in the DFE and DBE cheese, respectively. e magnesium content in CR cheese was the highest. Iron was not detected in all the samples.

Discussion
e aflatoxin content of the crude enzyme from A. oryzae DRDFS 13 used for Danbo cheese production was below the standard limit set by the European Union [19]. is indicated the safety of the crude enzyme for application in food production. In fact, other studies also marked that enzymes extracted from Aspergillus species are generally recognized as safe (GRAS) [20][21][22][23]. us, it can be used as a rennin substitute in the coagulation of milk and manufacture of cheese [2]. Similarly, the milk-clotting enzyme obtained from Rhizomucor miehei NRRL 2034 used for white soft cheese was free from aflatoxins B1, B2, G1, and G2 in the study by [2]. Fazouane-Naimi et al. [24] also reported that the culture supernatants from A. niger FFB1 used for cheese production were free from ochratoxin A (OTA). e raw cow milk used for the Danbo cheese making had pH, TTA, water, crude protein, crude fat, total ash, and total carbohydrate values of 6.65, 0.12%, 88.93%, 2.96%, 4%, 0.48%, and 3.64%, respectively. Similar chemical composition of cow's milk used for the production of white pickled and Prato cheeses was reported by Çepepoglu and Güler-Akın [25,26], respectively. Substantial volumes of whey were discarded during the Danbo cheese production. e highest whey yield was obtained from commercial rennet followed by fungal and bacterial enzymes, respectively. e physico-chemical characteristics of the whey in the present study were similar, with whey samples reported by Omole et al. [27]. Similarly, the highest amount of cheese was yielded from the commercial rennet enzyme as compared to fungal and bacterial enzymes. In contrast, a higher yield of UF-Domiati cheese was obtained by the M. mucedo KP736529 enzyme (E-cheese) than control cheese produced by commercial calf rennet [28]. e yield of  Journal of Food Quality semihard cheese produced using commercial chymosin was slightly higher than the report in the present study [29]. As per the rating acceptance sensory test on the Danbo cheeses, the color and flavor score was in the accepted order of CR > DFE > DBE. However, there was no significant difference in odor and texture between the three kinds of cheese. e less rating acceptance score for the DBE could be due to the nonspecific catalytic activity of the bacterial     DBE salted cheese DBE ripened cheese pH 5.39 ± 0.00 g 5.77 ± 0.01 e 7.31 ± 0.00 b 5.22 ± 0.02 h 5.61 ± 0.01 f 6.56 ± 0.00 c 5.63 ± 0.02 f 6.06 ± 0.02 d 7.34 ± 0.00 a TTA 0.05 ± 0.00 e 0.03 ± 0.00 f 0.11 ± 0.00 c 0.06 ± 0.00 d 0.02 ± 0.00 g 0.27 ± 0.00 a 0.05 ± 0.00 e 0.03 ± 0.00 f 0.16 ± 0.00 b Data are expressed as mean ± SD (n � 3). Mean values within a row with different superscripts are significantly different at p < 0.05. TTA, titratable acidity; CR, commercial rennet; DFE, dialyzed fungal enzyme; DBE, dialyzed bacterial enzyme. enzyme on casein as compared to the fungal enzyme [30]. e nonspecific activity and heat-stable property of proteases led to the development of bitterness in cheese [31].
In previous studies, white soft cheese produced using a milk-clotting enzyme from M. miehei NRRL 3420 [2], UF-soft cheese produced by the Rhizomucor miehei NRRL 2034 coagulant [7], fresh cheese manufactured with the A. niger FFB1 enzyme [24], and fresh goat cheese produced by the M. miehei microbial coagulant [32] had similar organoleptic characteristics with control cheese produced by calf rennet. Also, Çepepoglu and Güler-Akın [25] reported higher sensory acceptability of fresh Turkish white cheese made using A. niger var awamori recombinant chymosin than commercially made cheese. us, the fungal enzymes might be considered as potential substituents for the commercial rennet.
e results also showed that, the titratable acidity of Danbo cheeses decreased after salting but increased after ripening in all the experimental and control treatments. e decrease in titratable acidity after salting could be associated with the diffusion of lactic acid from the cheese into the brine [25]. Similarly, the titratable acidity of all Turkish white cheeses produced using different coagulants decreased during a storage time of 30 days and then increased [25]. Similarly, the total acidity of experimental Domiati cheese manufactured using the M. mucedo KP736529 enzyme and control cheese was gradually increased during ripening for 60 days [28].
Among the three fresh cheese types, CR had the highest protein content followed by DBE and DFE, respectively. e crude protein content in the ripened cheese was in the order of CR > DFE > DBE. Similarly, higher total nitrogen content was reported in a control cheese (Domiati cheese manufactured by calf rennet) than experimental cheese (Domiati cheese manufactured with the M. mucedo KP736529 enzyme) [28]. On the other hand, the UF-white soft cheese manufactured by the B. stearothermophilus coagulant [33], fresh white soft cheese made with Rhizomucor miehei NRRL 2034 rennet [2], and fresh goat cheese produced by the M. miehei coagulant [32] revealed comparable protein content with control cheese produced by calf rennet.
Salted CR and DFE cheese types had higher crude fat content, while the DBE salted cheese had the lowest amount. e ash content in the fresh DBE was significantly lower than the amount in the other two cheese types. In all the cases, the salted cheese had higher ash content than the fresh and ripened cheeses (i.e., CR > DBE > DFE). e salted cheese types had the highest total energy compared with the fresh and ripened cheese. is could be due to the removal of moisture content during salting which leads to an increase in crude fat and crude protein contents of the cheese.
In the present study, the crude fat contents were increased after salting but decreased after ripening for Danbo cheeses produced from commercial rennet and the dialyzed fungal enzyme. However, the crude fat content was continuously decreased after salting and ripening for Danbo cheese produced using the dialyzed bacterial enzyme. e highest crude fat was noticed from ripened Danbo cheese produced by the fungal enzyme, whereas the lowest crude fat was recorded from ripened Danbo cheese produced by a bacterial enzyme. e lipid contents (5.5-10%) noticed from cream cheeses produced using the purified milk-clotting enzyme by Bacillus sp. P45 were lower than the present study [6]. e low lipid content noticed in cream cheese could be attributed to the high moisture content of the cream cheese as compared to Danbo cheese.
In contrast to the present study, the fat contents of Turkish white cheeses remained steady in all treatments throughout the ripening period [25]. In other studies, a higher fat content was detected in control cheese than cheese produced by the fungal enzyme [28]. However, the fat contents of UF-white soft cheese produced using the B. stearothermophilus coagulant [33], Prato cheese produced by the ermomucor indicae-seudaticae N31 enzyme [26], white soft cheese produced by the Rhizomucor miehei NRRL 2034 rennet enzyme [2], and fresh goat cheese produced by the M. miehei coagulant [32] were similar with control cheese produced using the commercial enzyme.
e total carbohydrates contents of Danbo cheese were decreased after salting and increased after ripening, whereas the total caloric value (Kcal/100 g) produced from Danbo cheeses was increased after salting but decreased after ripening for all the three treatments. e increase in energy after salting could be due to the increase in crude fat content due to the removal of moisture content. e highest carbohydrate content was detected from ripened Danbo cheese produced using the bacterial enzyme in comparison with the fungal enzyme and commercial rennet. However, the highest caloric value in Kcal was obtained from ripened Danbo cheese made by the fungal enzyme. e carbohydrate contents of 5.34 to 9.01% and caloric value (113.14-139. 16 Kcal/100 g) obtained from cream cheeses produced using the purified milk-clotting enzyme by Bacillus sp. P45 are lower than reported in the present study [6]. e low nutrient noticed in cream cheese could be attributed to the high moisture contents of the cheese.
In the present study, both the experimental and control Danbo cheeses showed variable ash contents. However, the highest ash content was recorded from fresh salted cheese produced using commercial rennet. In contrast to this study, higher ash content was noticed from Prato cheese produced by the ermomucor indicae-seudaticae N31 protease as compared to control cheese produced using a commercial coagulant [34]. e crude fiber was not detected in both experimental and control Danbo cheeses. e absence of fiber in the Danbo cheese could be associated with the limited use of only milk and enzyme as ingredients for Danbo cheese production rather than different flours. In contrast, the fiber content of 3.00 to 4.96% was detected from cream cheeses produced using the purified milk-clotting enzyme by Bacillus sp. P45 [6].
e concentration of sodium was the highest in all three types of cheeses. is may be due to diffusion of sodium into cheeses during dipping of cheese into 20% NaCl.
e Zn content noticed from double crème white brined cheese (2.0 mg/100 g) was comparable with this study [38]. On the other hand, the Zn (31 mg/kg) and Fe (3.0 mg/ kg) contents recorded from ripened Prato cheese are higher than the present study, while the Mn content (0.26 mg/100 g) is lower than this study [40]. Different from the present study, 0.7 mg/100 g of Fe was noticed from full-fat cheese produced by a high-pH method [35].

Conclusion
erefore, the results obtained from the body property, organoleptic characteristics, proximate composition, and partly from mineral composition revealed that the fungal enzyme from Aspergillus oryzae DRDFS 13 is more appropriate for Danbo cheese production than the bacterial enzyme from Bacillus subtilis SMDFS 2B.

Data Availability
e data used in this study are publicly available wherever possible-as open as possible and as closed as necessary.

Conflicts of Interest
e authors declare no conflicts of interest.