Collagenase Production by Immobilized Fungal Association of Aspergillus Awamori 16 and Aspergillus Awamori 22

The highest consumer properties possess meat products, developed from parts of the carcass with a minimum content of connective tissue. At the same time, there is a problem of processing raw meat containing an increased amount of connective tissue characterized by stiffness and dryness. To prevent excessive stiffness in production of meat products different approaches of treatment of raw meat with a high content of connective tissue have been used, e.g. mechanical and biotechnological methods (Bekhit et al., 2014; Sorour et al., 2017; Jatav et al., 2018). In this regards, the use of enzyme preparations that cause proteolysis of connective tissue proteins of collagen-containing raw materials is of particular interest (Alessandro et al., 2016; Lynch et al., 2018; Homaei et al., 2016; Qing et al., 2013; Laishram et al., 2017). The use of enzyme preparations in meat processing makes it possible to rationally use meat raw materials, intensify technological processes, improve quality and expand the range of products.
 
Collagenases are the most important enzymes used for the processing of meat raw materials. Studies in the area of production and use of this enzyme are now focused at searching for microorganisms that  are  capable  of  intensive  synthesis  of  these  enzymes. The producers of these enzymes were found among Actinomyces rimosus, Pseudoalteromonas agarivorans , Pseudomonas aeruginosa etc. (Aguilar et al., 2018; Rani et al., 2018; Rafieenia, 2013; Bhattacharya et al., 2018). The proteolytic enzymes from Bacillus bacteria have been studied extensively (Sharma et al., 2017). Of some interest are fungal collagenases, for example, from Aspergillus terricola, Aspergillus oryzae, Penicillium sp. (Ferreira et al.. 2017; Ida et al., 2017). Despite the fact that among microorganisms that produce collagenase, are bacteria, fungi and actinomycetes, in the recent period micromycets got wide application because of the ease of their cultivation and high productivity. However, great drawback of industrial strains is their low activity, despite the fact that the chief requirement to enzymes is their high catalyst activity which is directly connected with the activity of the microorganism that produces this enzyme (Hassanpouraghdam et al., 2015). Traditionally used enzymatic agent processing technology applies the submerged culture method of micromycetes in periodic conditions. Under such conditions of micromycets growth, their biomass forms a shape of pellets, which during the culture process firms and becomes inaccessible for oxygen and nutrients. The biomass forms the maximum amount of the target product in 3-5 days only once. After that, this culture autolyzes and its enzyme activity decrease.
       
A new method of cultivation of filamentous microorganisms which prolong producers’ cultivation period  up to 60-90 days and creates the opportunity to obtain enzymes repeatedly in every 2-3 days of cultivation has been developed by Blieva R.K. (Patent No. 27164, 2016). This method is based on immobilization of enzyme producers in submerged conditions of growth. Moreover it gives the opportunity to increase the enzymatic activity of immobilized cell culture filtrate in comparison with free cells, growing in periodic culture conditions.
       
In this research active isolate of Aspergillus awamori 16 and Aspergillus awamori 22 in associative culture, using new method of cultivation of filamentous fungi in submerged conditions of growth was selected.

Association of mixed fungi Aspergillus awamori 16 and Aspergillus awamori 22 (own collection) was used in this study. The microorganisms were maintained on potato dextrose agar at 4°C. The initial culture was grown on potato - dextrose agar for 5 days at a temperature of 30°C.
       
For screening of active variant, fungal cells were immobilized in submerged fermentation and cultivated for 30 days at 30° C. For inoculum preparation, 25 ml of sterile distilled water was added to the 5-day-old culture and scraped aseptically with inoculating loop. This suspension with spore concentration of 1.3·10⁵ cells/ml, was used as inoculum for the fungal cultivation. Submerged fermentation was carried out in 750 ml Erlenmeyer flasks with round - shaped carrier for fungal immobilization in optimized Czapek medium: (%): KH₂PO₄ – 0,1; MgSO₄ – 0,05; KCL – 0,05; FeSO₄ – 0,001; peptone -1,0; sucrose – 2,0 (Blieva R.K. et al., 2019). All Erlenmeyer flasks were incubated at 30°C on a rotary shaker (180 rpm) for 36 days. The growth medium was exchanged at 3-day intervals.
       
Collagenase activity was assayed by spectrophotometric method. To 20 mg collagen from bovine tendon (Sigma) suspended in 3.8 ml Tris buffer (0.02 M Tris, 0.005 M CaCl₂, pH 7.4) was added 200 µl collagenase solution (1 mg/ml in Tris buffer) to make a total volume of 4.0 ml. The mixture was incubated at 40°C. for 3 hr or 70°C. for 30 min.  The reaction mixtures were centrifuged in a microfuge for 10 min at 14,000 rpm. 1.5 ml of supernatant was mixed with 4.5 ml of 5 N HCl and kept in a drying oven at 110°C. for 16 hrs (overnight) for complete hydrolysis of soluble peptides. The hydrolysate was then analyzed for hydroxyproline content as follows: the hydrolysate was diluted 25 times with distilled water. To 1.00 ml of diluted hydrolysate 1.00 ml of chloramine-T solution was added and the mixture was allowed to stand at room temperature for 20 min. 1.00 ml of color reagent was added after this period and the reaction mixture is transferred to a 60°C. water bath and incubated for 15 min. Tubes were removed and allowed to cool down to room temperature. Absorbance at 600 nm was measured.
       
The experiments were carried out in triplicates and standard deviation was determined. To determine the significance, the data were analyzed using Microsoft excel software 2010.

Selection of fungal association Aspergillus awamori 16 and Aspergillus awamori 22 for enhanced collagenase production
 
Strain improvement by selection is a highly developed technique and it plays a central role in the commercial development of microbial fermentation processes. The ability to control the enzymes production through the regulation of their biosynthesis and the selection of cultivation conditions allows not only to increase the yield of enzymes, but also to obtain enzyme preparations with certain properties. In this regards, large potentialities are embedded in immobilized cells. Traditionally used enzymatic agent processing technology applies the submerged culture method of micromycetes in periodic conditions. Under such conditions of micromycets growth, their biomass forms a shape of pellets, which during the culture process firms and becomes inaccessible for oxygen and nutrients (Fig 1a). The biomass forms the maximum amount of the target product in 3-5 days only once. After that, this culture autolyzes and its enzyme activity decreases.              
 

       
We propose a new method of cultivation of micromycetes by immobilization of fungal cells. Such method prolongs producers’ cultivation period to 60 days and more and create the opportunity to obtain enzymes repeatedly in every 2-3 days of cultivation. This method is based on immobilization of enzyme producers with solid support in submerged conditions of growth (Fig 1b). Immobilization has been achieved by using a new carrier for absorption of immobilized cultures stores, which is inexpensive and biodegradable. Immobilizing has a range of advantages: decreasing the price of the final product, absence of foreign substances, controlled process of enzyme-genesis, ability of various enzymes simultaneous production, etc. Also, it gives the way to improve quality of filtrates (to make them more clear) and exclude time-consuming processes of recharging fermentative vials, that require manual removing of mycelium.               
       
Cultivation of association of A. awamori 16 and A. awamori 22 was carried out for 30 days (Fig 2). As can be  seen from data presented in Fig 2, enzyme biosynthesis occurs continuously and for a long time. The maximum of collagenase biosynthesis was observed in 9, 18 and 27 days. The enzymatic activity varied from 7.8 U/ml to 17.6 U/ml. Thus, with the immobilization of association of A. awamori 16 and A. awamori 22 active collagenase production was observed throughout the entire cultivation period in submerged conditions of growth.   
 

 
Successful selection depends on the heterogeneous system of the population and the degree of its variability. As soon as the population reaches the highest homogeneity, selection stops before the appearance in the population of different phenotypic variants, i.e. selection depends on the genetic properties of the microbial population and on degree of its variability. Immobilized cells are a convenient tool for  selection of active variants. One of the reasons for increasing the biosynthetic activity of immobilized cells is the variability of the heterogeneous culture. To establish the natural variability of immobilized cells, the isolates were taken at different periods of long-term cultivation, when the enzymatic activity was increased. Three isolates of the immobilized A. awamori 16 and A. awamori 22 associative culture were taken at the beginning of immobilization, i.e. on the 9^th day of fungal cultivation. In the middle of immobilization, on the 18^th day - 4 isolates and at the end of a cultivation period, on the 27^th day - 3 isolates. In total 10 isolates were obtained.
All isolates were cultivated for 72h at 30°C on production medium. After that collagenase activity was assayed (Table 1). As can be seen from the data presented in Table 1, at the first stage of long-term cultivation, 3 isolates were selected. The collagenase activity varied from 6.3 U/ml to 7.1 U/ml, whereas in stage II 4 isolates were selected. The maximal level of enzymatic activity was 8.2 U/ml. At the stage III, the collagenase activity of selected 3 isolates varied from 6.3 U/ml to 7.2 U/ml. Thus, at the beginning of cells immobilization, the isolate 2-3 of fungal association A. awamori 16 and A. awamori 22 was formed with increased enzymatic activity of 8.2 U/ml.
 

        
Submerged cultivation of isolate 2-3 of fungal association of A. awamori 16 and A. awamori 22
 
A submerged cultivation of selected isolate 2-3 of fungal association A. awamori 16 and A. awamori 22 was carried out for 48 days in order to to study the dynamics of collagenase production. Liquid media was exchanged in every three day interval. The data obtained are presented in Fig 3.
 

 
Enzymatic activity was enhanced significantly after 6 days of cultivation of immobilized cells and keeps the same value for 48 days of fungal cultivation. The maximum of enzyme synthesis was in 9, 18, 27, 36 and 45 days of fungal cultivation. The collagenase activity varied from 8.0 to 20.2 U/ml.

It is obvious that proposed method of fungal immobilization is simple and accessible. With its implementation, there are no factors that damage microbial cells and inhibit enzymatic activity, mass transfer is not difficult. It enables the development of continuous biocatalytic technologies. Cells immobilization is also interesting because microorganisms return to their natural attached state, the study of which allows us to come closer to understanding the processes taking place in nature. Enzymatic activity was enhanced significantly after 6 days of cultivation of immobilized cells and keeps the same value for 48 days of fungal cultivation. The collagenase activity increased to 20.2 U/ml.

This study was conducted with financial support from Ministry of Education and Sciences of the Republic of Kazakhstan (Grant # AP05132402).