Production of a novel milk-clotting enzyme from solid-substrate Mucor spp. culture
Abstract
Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk-clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk-clotting activity under a solid-state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal-sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost-effective production, milk-clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese-making industry.
Practical Application
The milk-clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers.
1 INTRODUCTION
Cheese has been manufactured using calf rennet for thousands of years. Tremendous research has been made with great efforts to explore alternative sources for the natural calf rennet to meet the increased demand for different varieties of cheese (Hsu et al., 2005). Chymosin (E.C. 3.4.23.4) has a remarkable potential for milk coagulation such as having specific cleavage on a substrate, good flavoring (sweet) properties (Mamo and Assefa, 2018), and helps with ripening and (smooth) texture of cheese , making it the best milk coagulant in cheese manufacturing (Shankar et al., 2010).
Obtaining calf chymosin is limited by various ethical and economic reasons. This has led to alternative methods to produce adequate chymosin to meet the high global demand. The most common method is to produce recombinant chymosin via cloning method in various microorganisms such as Aspergillus species, Bacillus species, and Escherichia coli. However, there is a huge group of opponents of GMO (Genetically modified organisms) technology that rejects the food in which GMOs are involved in any way (Wei et al., 2016).
Therefore, to find a suitable alternative that rules out the GMO issue, extensive research has resulted in the fungal acid protease produced by different Mucor species such as Rhizomucor miehei ((Ha et al., 2009), Mucor circinelloides (Fernandez-Lahore et al., 1999), and Mucor mucedo (Yegin et al., 2011).
Some other fungi have also been reported to produce microbial milk-clotting enzymes such as Amylomyces rouxii (Hsu et al., 2005) and Aspergillus oryzae (Vishwanatha et al., 2009).
However, various plant-derived milk-clotting enzymes are also available for cheese-making (Nicosia et al., 2022). Aspartic proteases, commonly known as acid proteases or aspartyl proteases, are classified as hydrolases (group 3) within subgroup 4, which can hydrolyze peptide bonds. They are diversely found in living organisms as well as in viruses (Norero et al., 2022). They are endopeptidases that use an activated water molecule bound to one or two aspartate residues for catalysis. In the active site, these Asp residues are exceptionally conserved (Asp32, Asp215) due to their essential role in catalysis. Most of the aspartic proteases are active at acidic pH of 3–4 and have an isoelectric point in the range of 3–4.5. Their molecular masses are in the ranges of 30–45 kDa (Shankar et al., 2010). Many microorganisms are being studied in detail to investigate the production of aspartic proteases and different fungal and microbial strains are reported as excellent producers of milk-clotting enzymes such as Talaromyces leycettanus (Guo et al., 2020), Rhizomucor miehei (Wang et al., 2021), R. pusillus, Endothia parasitica (Thakur et al., 1990), Mucor circinelloides (Fernández -Lahore, et al., 1997), Aspergillus oryzae (Vishwanatha et al., 2009), Amylomyces rouxii, (Hsu et al., 2005), Rhizopus microsporus (da Silva et al., 2020), Mucor mucedo (Yegin et al., 2010), Mucor pusillu (Arima et al., 1970), Mucor racemosus (Bernardinelli et al., 1983), and Iprex lactis (Bailey and Siika-aho, 1988).
Rennin produced by microbes possesses high milk-clotting activity, and the low proteolytic activity makes it able to be used as an excellent alternative to calf chymosin (Lee et al., 1986). Increased demand for the enzyme in the cheese industry and religious and ethnic regulations against the use of animal-derived enzyme have stimulated the research and interest in microbial rennins (Shankar et al., 2010). Several reports explain the production of aspartic protease by different Aspergillus spp. and especially Mucor spp (Fraile, E. R., Muse, J. O., and Bernardinelli, 1981). R. miehei and R. pusillus belong to the Zygomycetes fungi, which are well-known species to produce a milk-clotting enzyme called Mucor rennin (Thakur et al., 1990). The enzyme has high milk-clotting activity and less proteolytic activity, which makes it an excellent alternative to the calf chymosin (Andrade et al., 2002) that has been accepted widely in the cheese industry (Silveira et al., 2005). It has been reported that the cheese produced by the protease obtained from the Rhizomucor miehei was found the same as that from the calf rennin in terms of yield and quality, and it also ripened the cheese more quickly, having no bitter flavors (Wynne and Yada, 1991). The fungi are characterized by their tendencies to produce and secrete enzymes in the external environment. The species belonging to the genus Mucor are a wide group of fungal strains that are the major producer of industrial enzymes used in the pharmaceutical, baking, dairy, and detergent and cosmetic industries with biotechnological importance (Hsu et al., 2005). Mucor racemosus and Mucor racemosus f. racemosus both belong to the class Zygomycetes, having also the capability to produce milk-clotting enzymes (Bernardinelli et al., 1983).
Mucor racemosus has a potential role in the food industry, especially in cheese production. It has been used globally as a painkiller against minor injuries as well as to get brief comfort in joints and muscle pain. The fungus has been given a GRAS (Generally recognized as safe) status by the FDA (Food and Drug Administration). It does not cause any infection in human beings except for hypertensive human beings. Some allergic responses have been reported (Soeria-Atmadja et al., 2007).
In recent years, solid-state fermentation (SSF) has emerged as a potential technology for the production of various industrially high value-added products such as enzymes, secondary metabolites, pharmaceutics, feed, fuel, and food (Rajkumar et al., 2011). Using the solid-state fermentation technique, several types of aspartic proteases have been produced. Previous studies indicate that Mucor spp. strains give high yields of aspartic protease in solid cultures (Kumar et al., 2005). It has been reported after screening different fungal strains that Mucor racemosus produced 6500 U/g of the milk-clotting enzyme in solid culture (Higashio, K., and Yoshioka, Y., 1981). It has also been observed that the quality of the milk-clotting enzyme is much improved; the one obtained through SSF is better as compared to submerged fermentation (SmF). The influence of several media on the production of aspartic protease by M. bacilliformis was intensively studied (Thakur et al., 1990; Fernández Lahore et al., 1997). Durum wheat bran was found the best substrate after testing sunflower hill, rice bran, durum wheat bran, and bread wheat bran. Maximum milk-clotting activity of 7500 U/g was obtained after 72 h of cultivation at 24°C, using wheat bran wetted with 120% with 0.2 M HCl inoculated with 5×105 spores per gram of wheat bran (Bernardinelli et al., 1983). Aspartic protease shows maximum milk-clotting activity of 7100 U/g on the fourth day of cultivation of M. circinelloides (M-105) on wheat bran as a solid substrate (Fernandez-Lahore et al., 1999). This research aims to find a microbial as well as a cost-effective alternative to animal rennin.
An additional aim is to enhance the production of the milk-clotting enzyme. Numerous reasons have led to the research on the production of microbial rennin such as the global increase in cheese production, shortage of calf rennet, rearing the calf to maturity to fulfill the increased demands of meat, finding an economically reasonable alternative to calf chymosin, and to find a vegetarian source for cheese making. To meet these major aims, two different microbial strains Mucor racemosus f.racemosus CBS 381 and Mucor racemosus DSM 62760 were evaluated. For Mucor racemosus f. racemosus CBS 381, it was reported that it has the potential to produce a milk coagulant.
2 MATERIALS AND METHODS
2.1 Microorganisms and culture conditions
The microorganisms used for the study were Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae var. oryzae CBS 570.65 (control) obtained from the German Collection of Microorganisms and Cell Cultures-DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). The strains were maintained by subculturing on molasses–agar slants at 24°C. Spores were harvested after 4, 7, and 10 days of cultivation by suspension in sterilized water. Inoculum size adjustment was done by manual counting of spores in a Thoma chamber.
2.2 Chemicals
All the chemicals and materials were purchased from Applichem GmbH (Darmstadt, Germany), whereas casein and gelatin were purchased from Sigma-Aldrich Chemie GmbH (Hamburg, Germany). Hannilase and Naturen were from Chr. Hansen (Hørsholm, Denmark).
2.3 Growth medium for solid-state fermentation
Experiments were carried out in Erlenmeyer flasks (250 ml) containing sterilized wheat bran as a basic medium for growth. Experiments were done to study the effect of different parameters such as carbon source, nitrogen source, temperature, incubation time, inoculum size, moisture content, and age of the spore on the production of milk-clotting enzyme. Combinations of the different parameters were proposed by the software Design of Experiment (DoE) (MODDE; Umetrics, Sweden). The growth media consisted of 1% of carbon source and 0.5% of nitrogen source. Glucose, fructose, lactose, and molasses were tested as carbon sources while peptone, yeast extract, skimmed milk, casein, urea, and sodium nitrate were tested as nitrogen sources. Every flask containing 10 g of the wheat bran including the nitrogen and carbon sources was wetted with 0.2 M HCl considering the desired moisture content and was autoclaved at 121°C for 20 min for sterile conditions. Inoculation was done with the different spore suspensions and the flasks were incubated at different temperatures. Manual shaking was done twice on the day of incubation and day 1. Flasks were kept in dark to avoid any kind of effect of light on the production of the enzyme.
2.4 Enzyme leaching
The enzyme extract was recovered by adding 100 ml of distilled water to the flasks, and was placed on the shaker for 60 min at 250 rpm at 24°C to detach all the mycelia from the media. Filtration of the extract was done using cotton cheesecloths, and the extract was centrifuged at 3220 g for 30 min at 4°C to settle down the media particles. The supernatant was collected for further analysis.
2.5 Analytical methods
2.5.1 Statistical analysis
Data was expressed as mean ± standard error of the mean. Qualitative and quantitative data analysis was done and the statistical differences are indicated in tables and graphs.
2.5.2 Assay for milk-clotting activity
2.5.3 Assay for proteolytic activity
2.5.4 Protein determination
Protein was determined according to the Bradford procedure with bovine serum albumin as the standard (Bradford, 1976).
2.5.5 Determination of molecular weight
The molecular weight of the enzyme was estimated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) by the method of Laemmli. By using 12% acrylamide, electrophoresis gels were prepared and protein bands were observed by applying Coomassie blue R250 staining (LAEMMLI, 1970). Zymography was done in two steps. For native–PAGE, 12% polyacrylamide gel was prepared without SDS using the method described by Perera (2017) with some modifications. Samples were loaded without boiling. The gel was later soaked overnight in a 5% casein solution in order to confirm the enzyme activity (Perera, 2017).
2.6 Design of Experiment software
Design of Experiment was used in this study to discover the effect of different factors (inputs, qualitative or quantitative) such as temperature, carbon and nitrogen source, incubation time, and moisture content on responses (outputs) that are supposed to be measured (e.g. milk-clotting activity, specific activity, proteolytic activity). DoE is a multivariate analysis software, which helps a researcher to make changes to factors either in terms of combination or level to enhance the values of responses over a few runs of experiments. Thus, the amount of information obtained from the study is enhanced while the amount of data to be gained is decreased. Not only every possible combination of each factor is tested with the other factors but also their levels have been tested. It is a systematic, organized, and statistical approach to testing and analyzing different experimental tests to evaluate the factors affecting the response variables (Eriksson et al., 2008). Predictive models can be determined by a variety of different factors, which are independent of each other.
Screening and optimization of the experimental procedure in this study were done using DoE. Screening is a process that is done during the initial experimental procedure to investigate the effect of different factors on responses. It helps to determine the correct and appropriate ranges and the combination of the factors that can enhance the response values. The significance of the screening design is to run a few experiments concerning several factors.
Optimization was done after the screening design to predict the response values for all the combinations of factors and ranges, and it provided with an optimal experimental point by giving detailed information regarding how the factors combine to influence the responses. It is difficult to recognize a single experimental point at which the targets of all the responses are achieved. That is why the final result is most of the time is depicting a compromise between partially conflicting targets. This happens when several responses are investigated at the same time (Eriksson et al., 2008).
2.7 Characterization of the milk-clotting enzyme
Partial characterization of the enzyme was done in terms of pH stability, thermal stability, and lyophilization as all three parameters are quite crucial and influential in the process of cheese manufacturing. The pH stability of the enzyme was studied by diluting the samples 10 times in 20 mM citrate-phosphate buffer and phosphate buffer having desired pH values ranging from 4.6 to 7. Incubation of the samples was carried out at 25°C for 10 min–2 h. Samples were tested for the milk-clotting activity after incubation, and commercial preparation Hannilase was taken as control. Thermal stability was determined by incubating enzyme samples at temperatures ranging from 35 to 55°C for 10, 20, and 30 min. Before the determination of milk-clotting activity, samples were cooled in an ice bath. The enzyme was tested for lyophilization and before that, it was subjected to dialysis to remove all the salts present in the crude extract. The sample was lyophilized after dialysis and the milk-clotting activity was determined after the rehydration of the lyophilisate to investigate the loss of activity after freeze-drying.
3 RESULTS AND DISCUSSION
3.1 Strain selection
Erlenmeyer flasks were used to cultivate the two different Mucor strains via solid-state fermentation. A decrease was observed in the protein content for the first 3 days of fermentation and then a gradual increase is observed. Microorganisms utilize the protein content for the biomass production present in the media and after 3 days, the strain secrets extracellular protein into the media, which consequently enhances the protein content. Both of the Mucor strains have the same trends in terms of milk-clotting activity, proteolytic activity and so on; however, Mucor racemosus f. racemosus CBS 381 showed high milk-clotting activity as shown in Figure 1a for all the days as compared to Mucor racemosus DSM 62760 depicted in Figure 1b.
A gradual increase in the enzyme activity of the Mucor racemosus f. racemosus CBS 381 was observed from day 2 to day 4, while there is a slight decrease in the activity on day 5 and then it reached a maximum on day 7. Both of the Mucor strains have approximately the same trend in terms of the decline in milk-clotting enzyme activity except that the decline in activity was observed on day 3 and day 4 for Mucor DSM 62760 and only on day 5 for Mucor CBS 381, while the maximum activity was achieved at day 7 for both the Mucor strains. It is important to note that for both the strains, all the fermentation parameters such as fermentation time, substrate, moisture content as well as temperature were kept the same. In contrast to these, other strains belonging to the Mucor species have different trends for example, M. bacilliformis and R. miehei show maximum activity on day 4 and day 5 (Thakur et al., 1990; Fernández Lahore et al., 1997).
The milk-clotting activity for Mucor racemosus f. racemosus CBS 381 and Mucor racemosus DSM 62760 at day 6 was 2919 and 2443 U/g, respectively. The enzyme produced by all the investigated fungi on day 6 of fermentation was analyzed by sodium dodecyl gel electrophoresis (SDS-PAGE) and as a control, commercially available enzymes Naturen (Chymosin) and Hannilase (Rhizomucor miehei) were utilized. Figure 2 depicts the SDS-PAGE profile, where a strong band was observed for Mucor CBS 381 and Mucor DSM 62760 although the band for the Mucor CBS 381 is sharper than for the other Mucor strain, which also explains the high milk-clotting activity of this strain over the other. At the same time, Aspergillus oryzae (control) has several sharp strong bands, which show that it has produced some different proteins other than aspartic protease. The molecular weight of ∼30 KDa was assigned to the bands shown by the Mucor species and it corresponds to the band of Naturen (Chymosin). This value is in the range of molecular weights normally determined for other aspartic proteases such as values from 30 and 35 KDa have been reported for fungal protease (Fernández-Lahore et al., 1998).
To determine if there is a difference in the molecular weight of the protein produced by this Mucor strain on different days of fermentation, SDS-PAGE electrophoresis was run as shown in Figure 3a. However, the produced protein by Mucor strain CBS 381 always showed the same molecular weight of ∼30 KDa as shown in Figure 3a. Additionally, it can be noticed from the SDS-PAGE profile in Figure 3a that Mucor CBS 381 is producing aspartic protease in large quantities besides some other extracellular proteins. It can be considered as an additional characteristic of this strain that the enzyme produced by it is almost pure. Based on these results, Mucor racemosus CBS 381 was selected for further research. Gel zymography was done in order to study the enzyme activity of the enzyme produced by Mucor CBS 381 depicted in Figure 3b. Crude extract from day 7 was allowed to run on a Native-PAGE; sample blank was used as a negative control. Aspartic proteinase activities were detected as clear areas on the gel.
3.2 Media optimization
3.2.1 Screening
Design of Experiment (DoE) was used for media optimization and the first step was to perform the screening. In total, 26 experiments were suggested by the software making every possible combination of the different factors and it also included replicate experiments which are the center point of the whole experiment.
According to the results shown in Table 1, there is no growth of the strain at all in the flasks where casein, skimmed milk, sodium nitrate, and urea are used as nitrogen source, while lactose, glucose, and fructose were poor carbon sources with few exceptions, for example, experiment number 1 and 15. The highest activity was achieved by the extract in flask number 10, where the carbon source was wheat bran and the nitrogen source was casein and is almost close to the value reported by Bernardinelli et al, which is 4000 U/g for M. varians Pispek. The overall analysis of the results shows that molasses and casein are the most significant carbon and nitrogen sources, respectively.
FACTORS | RESPONSES | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Exp No | Carbon Source | Nitrogen source | Temp (°C) | Time (Day) | Inoculum Size | Moisture content (%) | Spore age (Day) | MCA (U/g) | Specific activity (mg/g) | Proteolytic activity (PU/g) |
1 | Glucose | Peptone | 20 | 10 | 1.00E+08 | 70 | 4 | 2869 | 730 | 267 |
2 | Fructose | Peptone | 20 | 10 | 1.00E+04 | 150 | 10 | 501 | 112 | 55 |
3 | Molasses | Peptone | 30 | 4 | 1.00E+04 | 70 | 4 | 2687 | 740 | 230 |
4 | Wheat bran | Peptone | 30 | 4 | 1.00E+08 | 150 | 10 | 0 | 0 | 0 |
5 | Lactose | Yeast extract | 30 | 4 | 1.00E+04 | 150 | 4 | 584 | 389 | 11.00 |
6 | Fructose | Yeast extract | 30 | 4 | 1.00E+04 | 70 | 4 | 1890 | 883 | 103 |
7 | Molasses | Yeast extract | 20 | 10 | 1.00E+08 | 150 | 10 | 0 | 0 | 0 |
8 | Glucose | Casein | 30 | 10 | 1.00E+04 | 70 | 10 | 0 | 0 | 0 |
9 | Lactose | Casein | 20 | 10 | 1.00E+04 | 150 | 4 | 1141 | 275 | 146 |
10 | Wheat bran | Casein | 20 | 4 | 1.00E+08 | 70 | 10 | 3750 | 1638 | 204 |
11 | Lactose | Skimmed milk | 30 | 10 | 1.00E+08 | 70 | 10 | 0 | 0 | 0 |
12 | Sucrose | Skimmed milk | 20 | 4 | 1.00E+04 | 150 | 4 | 0 | 0 | 0 |
13 | Molasses | Skimmed milk | 30 | 4 | 1.00E+08 | 70 | 4 | 1941 | 551 | 115 |
14 | Glucose | Sodium nitrate | 30 | 4 | 1.00E+04 | 150 | 10 | 0 | 0 | 0 |
15 | Lactose | Sodium nitrate | 20 | 4 | 1.00E+08 | 70 | 10 | 2188 | 0 | 0 |
16 | Sucrose | Sodium nitrate | 30 | 10 | 1.00E+08 | 150 | 4 | 0 | 0 | 0 |
17 | Wheat bran | Sodium nitrate | 20 | 10 | 1.00E+04 | 70 | 4 | 0 | 0 | 0 |
18 | Fructose | Urea | 30 | 4 | 1.00E+08 | 150 | 4 | 0 | 0 | 0 |
19 | Sucrose | Urea | 20 | 4 | 1.00E+04 | 70 | 10 | 0 | 0 | 0 |
20 | Wheat bran | Urea | 30 | 10 | 1.00E+04 | 150 | 4 | 0 | 0 | 0 |
21 | Glucose | Wheat bran | 20 | 4 | 1.00E+08 | 150 | 4 | 0 | 0 | 0 |
22 | Sucrose | Wheat bran | 30 | 10 | 1.00E+08 | 70 | 10 | 0 | 0 | 0 |
23 | Molasses | Wheat bran | 20 | 10 | 1.00E+04 | 150 | 10 | 1040 | 377 | 79 |
24 | Wheat bran | Wheat bran | 25 | 7 | 5.00E+07 | 110 | 7 | 634 | 167 | 78 |
25 | Wheat bran | Wheat bran | 25 | 7 | 5.00E+07 | 110 | 7 | 635 | 167 | 83 |
26 | Wheat bran | Wheat bran | 25 | 7 | 5.00E+07 | 110 | 7 | 529 | 125 | 76 |
Figure 4 shows the contour plots of carbon and nitrogen sources for all the possible combinations and the optimal points for milk-clotting activity. The horizontal axis in the first row shows the nitrogen source such as wheat bran, casein, and peptone and its vertical axis shows glucose, molasses, and wheat bran as carbon sources. For example, the contour plots in the first row describe the optimal value for the nitrogen source (wheat bran) with all the carbon sources (glucose, molasses, and wheat bran). The x-axis on the individual plots shows the incubation time, whereas the y-axis shows the temperature. The two factors, inoculum size and the age of the spores, were set to the center point as 5×107 and 7 days, respectively, whereas the moisture level to as low as 70%.
According to the plots, lower temperature and incubation time can give high milk-clotting activity with different carbon and nitrogen sources. Considering all contour plots, the combination with casein as nitrogen source and molasses as carbon source gives the best milk-clotting activity. Other factors' values were taken from low to high such as moisture content 70–120 %, temperature 15–25°C, and carbon and nitrogen source from 5–15%.
3.2.2 Optimization
For the optimization, 29 experiments were conducted in different combinations of factors as indicated in Table 2, and the highest milk-clotting activity was obtained from the first experiment with 3445 U/g using molasses 5%, casein 0 %, moisture content 70 %, temperature 15°C, and incubation time of 10 days. The second highest value was found as 3364 U/g for experiment number 9 using the same values for all the parameters except that the incubation time was 4 days. The third highest milk-clotting activity 2963 U/g obtained in experiment number 10 had the same levels for all the parameters as in experiment number 1, only with higher molasses content of 15% and a higher temperature of 25°C. Considering all the three values for milk-clotting activity from the above-mentioned experiments, it is obvious that moisture content and the carbon source of molasses have a very strong effect on the production of the enzyme. It is being observed that all the experiments with 70 % of moisture content have resulted in good milk-clotting activity.. This was also found by Thakur et al. (1990) who used 70% moisture content for Rhizomucor miehei in a solid-state fermentation system (Thakur et al. 1990). While Fernández-Lahore et al (1997) achieved high milk-clotting activity at 120% moisture content for Mucor bacilliformis in solid-state culture (Fernández Lahore et al., 1997).
Factors | Responses | |||||||
---|---|---|---|---|---|---|---|---|
Exp No | Molasses (%) | Moisture (%) | Casein (%) | Temp (°C) | Incubation Time (Days) | MCA (U/g) | Specific activity (mg/g) | Proteolytic activity (PU/g) |
1 | 5 | 70 | 0 | 15 | 10 | 3445 | 609 | 1175.3 |
2 | 15 | 70 | 0 | 15 | 4 | 917 | 251 | 2514.9 |
3 | 5 | 120 | 0 | 15 | 4 | 2637 | 639 | 6392.1 |
4 | 15 | 120 | 0 | 15 | 10 | 0 | 0 | 0 |
5 | 5 | 70 | 10 | 15 | 4 | 1989 | 460 | 4599.8 |
6 | 15 | 70 | 10 | 15 | 10 | 1057 | 62 | 207.6 |
7 | 5 | 120 | 10 | 15 | 10 | 0 | 0 | 0 |
8 | 15 | 120 | 10 | 15 | 4 | 575 | 134 | 1335.7 |
9 | 5 | 70 | 0 | 25 | 4 | 3364 | 698 | 6975.9 |
10 | 15 | 70 | 0 | 25 | 10 | 2963 | 496 | 180.7 |
11 | 5 | 120 | 0 | 25 | 10 | 0 | 0 | 0 |
12 | 15 | 120 | 0 | 25 | 4 | 849 | 203 | 2029.3 |
13 | 5 | 70 | 10 | 25 | 10 | 0 | 0 | 0 |
14 | 15 | 70 | 10 | 25 | 4 | 2257 | 478 | 4777.8 |
15 | 5 | 120 | 10 | 25 | 4 | 0 | 0 | 0 |
16 | 15 | 120 | 10 | 25 | 10 | 0 | 0 | 0 |
17 | 5 | 95 | 5 | 20 | 7 | 1760 | 145 | 309.1 |
18 | 15 | 95 | 5 | 20 | 7 | 711 | 63 | 26-Sep |
19 | 10 | 70 | 5 | 20 | 7 | 2697 | 300 | 179.2 |
20 | 10 | 120 | 5 | 20 | 7 | 0 | 0 | 0 |
21 | 10 | 95 | 0 | 20 | 7 | 2791 | 286 | 240.4 |
22 | 10 | 95 | 10 | 20 | 7 | 0 | 0 | 0 |
23 | 10 | 95 | 5 | 15 | 7 | 2677 | 270 | 46.3 |
24 | 10 | 95 | 5 | 25 | 7 | 0 | 0 | 0 |
25 | 10 | 95 | 5 | 20 | 4 | 2209 | 442 | 4415.4 |
26 | 10 | 95 | 5 | 20 | 10 | 0 | 0 | 0 |
27 | 10 | 95 | 5 | 20 | 7 | 1494 | 218 | 394.2 |
28 | 10 | 95 | 5 | 20 | 7 | 2118 | 331 | 228.5 |
29 | 10 | 95 | 5 | 20 | 7 | 1027 | 120 | 118 |
The incubation time has some different trends as high activities can be seen for day 4 and day 10. Fernández -Lahore et al. (1997) and Thakur et al. (1990) have observed the highest activity on day 3 and day 5, respectively.The highest activity on day 5 was also observed by Sankararajan et al. (2012), who further reported that an increase in incubation time reduces the production of aspartic protease, which is due to the inactivation of protease by another constituent protease. The center points in the optimization show good activity also on day 7, which is why finding an optimal point in all three incubation times is difficult. Mucor racemosus is a mesophilic fungus and grows rapidly at room temperature ranging from 20°C to 25°C and shows good milk-clotting activity at this temperature unlike the other thermophilic fungi such as Rhizomucor miehei, which gives high milk-clotting enzyme production at 42°C (Thakur et al., 1990).
The optimal experimental range to achieve high milk-clotting activity is shown in Figure 5. The contour plot describes that maximum response can be obtained by setting casein to 0%, molasses should be used up to 11 %, the temperature can range from 23.5°C to 25°C, and the moisture content should be 70%.
The Optimizer (prediction) of the software was run to give the optimal point of five possible settings; the one with low proteolytic activity was chosen considering good milk coagulation. An incubation time of 8 days is suggested by the software as investigated before in the worksheet since the high values were achieved for all three different incubation times. Values of the factors based on the conclusion of the Optimizer are for the carbon source (Molasses) 11 %, nitrogen (Casein) 0%, incubation time 8 days, incubation temperature 25°C, and moisture content 70%. The final fermentation (optimized run) was run in triplicates based on the experimental optimal point and the enzyme was harvested on day 8. The crude extract was tested for milk-clotting activity, specific activity, and proteolytic activity. The expected values were obtained from DoE and the measured value was obtained after the optimized run. Milk-clotting activity and specific activity are quite close to the predicted values as shown in Table 3. The little variation in the values can be justified by considering the humane error while inoculating the flasks and adding or wetting media. But the achieved values are quite considerable, especially the proteolytic activity is even less than the predicted one, which is very important during cheese formation. A milk coagulant is considered perfect and attractive if it has less proteolytic activity as compared to the milk-clotting activity.
Responses | Predicted results | Measured results |
---|---|---|
Milk-clotting activity | 3348 U/g | 3090 ± 12 U/g |
Specific activity | 727 mg/g | 631 ± 12 mg/g |
Proteolytic activity | 173 PU/g | 153 ± 7 PU/g |
3.3 Characterization of the milk-clotting enzyme
3.3.1 pH stability
Samples were incubated for 10 min–2 h to test for pH stability ranging from pH 4.6 to pH 7 as shown in Figure 6 and it was observed that the enzyme is stable and active at pH 4.8 since it is also known from the literature that aspartic proteases work more efficiently in an acidic environment. The pH plays a critical role particularly in cheese texture as chemical changes in the protein network of the cheese curd are directly dependent on the change in pH (Gunasekaran and Ak, 2002). During cheese manufacturing, before milk coagulation, the pH of the milk is lowered so that the milk-clotting enzyme more actively coagulates the milk.
The trend is similar for Mucor mucedo (Yegin et al., 2010), R. pusillus (Venera et al., 1997), and A. oryzae (Sankararajan et al., 2012), which hasve the highest milk-clotting potency at pH 5.0. Evaluation of data at different pH conditions depicted that pH ranging from 4.0 to 5.0 is suitable for protease production by different filamentous fungi and revealed that fungi produce large amounts of extracellular protease (Sankararajan et al., 2012).
3.3.2 Effect of temperature on the milk-clotting activity
The milk-clotting activity was studied as a function of the temperature ranging from 35°C to 55°C. It is interesting to know that the highest activity was achieved at 45°C, which is an optimum temperature for the coagulation of milk in cheese manufacturing while unstable performance was observed at 50°C and the total activity was lost at 55°C indicating pronounced thermal sensitivity as shown in Figure 7. This is similar to the behavior of chymosin reported by Fazouane-Naimi et al. (2010). On the other hand, for R. pusillus and R. miehei, the highest milk-clotting activity was observed in the range of 55–60°C (Martin et al., 1980). This can be also be related to a certain extent to the activity of the enzyme produced by M. racemosus since it was active for the 10 min of exposure even at 55°C (Fazouane-Naimi et al., 2010).
Thermal sensitivity is a very conspicuous criterion in the manufacturing of cheese. Thermal stability is also another important criterion for the definition of a good milk coagulant depending on the cheese type. If the enzyme is heat-sensitive, unspecific proteolytic activities could be avoided during the cooking process. During the first stage of coagulation, the temperature was adjusted from 40 to 45°C for 20–30 min. Then, the curd formation temperature was raised from 50 to 56°C to inactive the enzyme to avoid further unspecific proteolytic activity, which can lead to the bitterness and undesired texture of the cheese (Martin et al., 1980). The sensitivity of the aspartic protease produced by Mucor CBS 381 at 55°C can make it attractive in the cheese-making industry.
3.3.3 Lyophilization
Before lyophilization, the enzyme was subjected to dialysis to remove all the salts present in the crude extract. The sample was lyophilized and the milk-clotting activity was determined after the rehydration of the lyophilisate to investigate the loss of activity after freeze-drying. Half of the milk-clotting activity was lost after dialysis. Presumably, the dilution of the crude extract has led to the loss in activity since water enhances the volume of the extract as it was increased up to 2 ml from the initial amount. The enzyme is also not stable upon freeze-drying as 3-fold activity has been lost compared to the original activity of the enzyme.
4 CONCLUSIONS
Mucor racemosus f. racemosus CBS 381 was the most promising strain due to the enzyme production as well as the high activity of the milk-clotting enzyme. Molasses and casein were the best carbon and nitrogen sources, respectively, for the production of milk-clotting enzyme via solid-state fermentation The optimal moisture content was 70% whereas 25°C was the optimal temperature for enzyme production. The high ratio of milk-clotting to proteolytic activity for the aspartic protease of the optimized culture of M. racemosus f. racemosus CBS 381 makes it suitable for cheese production. Cheese trials would be done in future on a laboratory as well as pilot scale.
ACKNOWLEDGMENTS
The authors are immensely thankful to Jacobs University Bremen and the Downstream bioprocessing lab for providing scientific and technical support for the study.
Open Access funding enabled and organized by Projekt DEAL.
AUTHOR CONTRIBUTION
Farhat Qasim: Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Software; Visualization; Writing – original draft. Sonja Diercks-Horn: Conceptualization; Data curation; Methodology; Software; Supervision; Validation; Writing – review & editing. Doreen Gerlach: Conceptualization; Methodology; Software. Anna Schneider: Writing – review & editing. Hector Marcelo Fernandez-Lahore: Conceptualization; Funding acquisition; Supervision; Writing – review & editing
CONFLICT OF INTEREST
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.