ISSN 2308-4057. Foods and Raw Materials, 2016, vol. 4, no. 2

STUDY OF TECHNOLOGICAL PROPERTIES OF MILK-CLOTTING FROM IRPEX LACTEUS (Irpex lacteus (Fr.) Fr.)

L. R. Lebedeva, Т. А. Kosogovab, Т. V. Teplyakovab, A. V. Krigerc, V. V. Elchaninovc,*, А. N. Belovc, and А. D. Коval'c

a Medical Biotechnology Institute of the State Research Center of Virology and Biotechnology “Vector”, Khimzavodskaya Str. 9, Berdsk, 633010 Russian Federation

b State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk region, 630559 Russian Federation

c Siberian Scientific Research Institute of Cheesemaking, Sovetskoy Armii Str. 66, Barnaul, 656016 Russian Federation

* e-mail: [email protected]

Received April 28, 2016; Accepted in revised form June 26, 2016; Published December 30, 2016

Abstract: Due to the scarcity of natural rennet, in this study we have considered an option of using in cheesemaking a milk-clotting enzyme produced by the Irpex lacteus fungus. We describe main properties of I. lacteus coagulant: milk-clotting activity (МА), overall proteolytic activity (PA), thermal stability, MА dependence on рН level and calcium ions content. Partially purified preparations of I. lacteus milk-clotting enzyme was obtained by salting out and gel-filtration. Technological properties of the I. lacteus coagulant were compared to natural calf rennet and cow (CP). МА of I. lacteus enzyme amounted to 29.1 ± 0.7 RU/ml, with protein content of 23 mg/ml. The coagulant was completely inactivated at 60○С. Thermal stability of I. lacteus milk-clotting enzyme, MA sensitivity to pH variations and Ca2+ content were comparable to respective parameters of calf rennet and CP. Overall PA of the I. lacteus coagulant exceeded CP and calf rennet activity by 33 and 220 times, respectively. As for enzymatic specificity, the following order was observed: calf rennet (100%) > CP (14.9%) > I. lacteus coagulant (0.5%). These findings suggest that there is a need to increase the MA of I. lacteus coagulant in order to be able to use it in cheesemaking. We have considered chemical, biochemical, and genetic corrective actions applicable to technological properties of microfungal milk coagulants.

Keywords: milk-clotting , rennet, rennet substitutes, microfungal coagulants, milk-clotting activity, proteolytic activity, thermal stability, cheesemaking, mucorpepsins

DOI: 10.21179/2308-4057-2016-2-58-65 Foods and Raw Materials, 2016, vol. 4, no. 2, pp. 58–65.

INTRODUCTION In the second part of the XX century, cheesemaking A key step in cheesemaking is milk coagulation and industry faced a challenge of rennet deficit [1], as obtaining a milk clot. This was conventionally volumes of rennet-based cheese production kept achieved with rennet: a complex preparation from growing, causing a severe shortage of calf and lamb abomasums of nursing calves and lambs. Rennet abomasums [2–4]. This deficit initiated a search for contains two milk-clotting enzymes (ME): rennet substitutes, but a valid equivalent of cow (EC 3.4.23.4) and pepsin (EC 3.4.23.1). Chymosin is chymosin was not to be found. In 1990–2006, the considered to be the standard enzyme for problem of rennet shortage was partially resolved when cheesemaking, due to the combination of its process- recombinant chymosin (rCh) of cow [5] and camel [6] related properties: high specificity for cleavage the were developed, however, there are some factors that prevent their wide application. In a number of Phe105-Met106 bond in κ-casein molecules, milk-clotting activity (MA) within the mild acidic pH range, low countries, use of rCh is limited in production of cheese overall proteolytic activity (PA), and thermal lability. subject to national certification of origin. Besides, a Many prokaryotic and eukaryotic certain customer segment is opting for cheeses demonstrate MA, however, due to the high level of produced with natural rennet, not a recombinant one. In nonspecific proteolysis and thermal stability, that the end of the 20th century, the problem of rennet reduce product output and result in cheese taste and deficiency was exacerbated due to a scrapie epidemic: texture defects, these are not used in cheesemaking, or a prion cattle disease [7]. This epidemic devastated the else their application is very limited. feedstock of natural ME and triggered a new – and

Copyright © 2016, Lebedev et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/ ), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. This article is published with open access at http://frm-kemtipp.ru. 58 ISSN 2308-4057. Foods and Raw Materials, 2016, vol. 4, no. 2 currently ongoing – phase in the search for rennet shaking baths at 26 ± 2○С. Liquid culture used for substitutes among animal, plant and microbial milk- inoculation was obtained by fungal cultivation on a clotting proteases [2, 3, 8–14]. This search is aimed at liquid medium inoculated with culture blocks grown on the diversification of feedstock in natural milk agar medium. Fungal biomass suspension was frozen coagulants, and the selection of enzymes that might be at –30 ± 2°С [22]. adapted for cheesemaking after certain chemical, Samples of milk-clotting enzyme were obtained in biochemical, or genetic "adjustment". the nucleic acids and recombinant proteins laboratory Due to the rennet shortage, starting from the 1970s, in the Medical Biotechnology Institute of the State modern cheesemaking uses microfungal coagulants: Research Center of Virology and Biotechnology mucorpepsin (E.C.3.4.23.23) and endotiapepsin (EC VECTOR. Frozen I. lacteus biomass suspension 3.4.23.22): aspartic proteinases Rhizomucor miehei (~850 ml) was thawed in a funnel with gauze filter. (Cooney & R. Emers.) Schipper (less often Rhizomucor The filtrate was made up with dry ammonium sulfate pusillus (Lindt) Schipper) and Cryphonectria (AS) to the final 70% saturation, stirred, and then left parasitica (Murrill) M.E. Barr (synonym: Endothia to sediment at 4○С overnight. The mixture was parasitica (Murrill) P.J. Anderson & H.W. Anderson). centrifuged at 8000g and 4○С for 15 minutes. The Microfungal coagulants have the following advantages: supernatant was rejected, and the sediment dissolved in low cost of production, compliance with natural origin approx. 25 ml of distilled water, and then desalted by criteria and with vegetarian requirements, conformity gel-filtration on Sephadex G-25 column (gel volume – with kosher and halal eating principle. Their main 60 ml.). Six fractions of desalted protein material disadvantages are: low specificity (МА/PА ratio), and (15 ml each) was obtained, these were combined into high thermal stability [2]. two samples: А (fr. 1–3) and B (fr. 4–6). А and B Object of this study is a fungus Irpex lacteus samples were sedimented again with AS at 70% (synonym: Polyporus tulipiferae (Schwein.) Overh.), saturation, precipitate dissolved in 20 ml of distilled which belongs to higher basidiomycetes and is known water, and dialysed for 8 hours against two 1 l charges to produce milk-clotting [15]. Milk coagulant, of 20 mM Tris-HCl, 50mM NaCl (рН 7.5). Total produced by I. lacteus, is registered in the IUBMB protein level in A and B samples was determined by enzyme nomenclature database as polyporopepsin (EC the Lowry method. A and B samples were poured in 3.4.23.29), which is related to the aspartic 1 ml microflasks and lyophilized. endopeptidases family (the family of chymosin and Technological properties of A and B samples were pepsin А). assessed in the biochemical laboratory of the FSBSI Data on process-related properties of I. lacteus Siberian Scientific Research Institute of coagulant are scarce and fragmented. We should Cheesemaking. Milk-clotting activity, overall PA, mention a series of studies by K. Murakami et al. thermal stability, МА dependence on рН and Ca2+ [15–18] that presented a partial description of the concentration were assessed in accordance with the enzyme and obtained its kDNA sequence, and a previously published techniques [14, 23]. Along with number of works carried out in the Donetsk National the I. lacteus coagulant samples, we also studied University (Ukraine) on some aspects of the I. lacteus natural ME of animal origin: an industrial control strain productivity, and studies of thermal stability of sample of calf rennet (ICS CR) with approx. 80% of crude milk-clotting preparations of this fungus [19–21]. chymosin content, and 100% cow pepsin (CP). Nowadays, I. lacteus coagulant is not used in dairy Standard milk substrate, ICS CR, and CP were industry as a rennet substitute. Meanwhile, biochemical provided as a courtesy of the "Moscow Rennet Plant" properties of polyporopepsin indicate that its JSC. derivatives might be successfully used in Milk clotting activity was assessed by mixing cheesemaking. solutions of dry standard substrate (DSS) and test ME The objective of this work is to diversify the heated up to 35○С, mix ratio 10 : 1. The result was feedstock of natural milk-clotting enzymes (ME). The expressed in reference units (RU), with MA-certified ensuing task of this study is to assess process-related ICS CR used as reference. properties of the milk-clotting enzyme derived from the For assessment the overall PA, we used I. lacteus basidial fungus, from the point of view of Hammersten grade casein solution (1%) as a substrate potential applicability in cheesemaking. in 20 mМ sodium phosphate buffer (рН = 5.6). ICS CR and CP activities were normalized as per MA OBJECTS AND METHODS OF STUDY demonstrated by the I. lacteus coagulant. Test ME were introduced in the substrate solution at 1 : 4 mix A pure culture of the I. lacteus fungus was derived ○ from a spore print in the Republic of Altai (Russian ratio and incubated at 35 С for 6, 60, 90, 180 minutes. Federation) in 2008. The fungus strain No. 2265 is The reaction was interrupted by adding trichroloacetic stored in the Collection of Basidiomycetes Cultures in acid (5%), then the samples were filtered, and the the Botanical institute named after V.L. Komarov resulting filtrate was assessed for optical density at (Saint-Petersburg), and in the Mycology Laboratory of λ = 280 nm (D280). Specificity was determined as the State Research Center of Virology and МА/PА ratio. PA was assumed as D280 of samples Biotechnology VECTOR, where the fungal biomass for incubated for 180 minutes. this study was produced. Cultivation was performed in Thermal stability. Aliquots of milk-clotting 0.75 l flasks, with 0.15 l of tryptone glucose nutrient enzymes were heated up for 30 minutes within the ○ medium (рН 6.0), for 7 days, in temperature-controlled temperature range of 30–70 С, and then assessed for

59 ISSN 2308-4057. Foods and Raw Materials, 2016, vol. 4, no. 2 residual MA. МА values obtained in the samples S.М. Boyko and I.N. Ivanov used a specially selected heated at 30○С were assumed to be 100%. and highly productive strain of I. lacteus Fr., BN-3 DSS samples with рН 5.5, 6.0, 6.5, 7.0 were (stored in a specialized collection of cultures at the prepared, and their coagulum formation time was Institute of Botany named after N.G. Kholodniy NAN measured, in order to determine MA dependence on the of Ukraine, Kiev) and, as they modified the surface pH level. Clotting time at рН 5.5 was assumed to be conditions for fungus cultivation, obtained a cultural 100%. filtrate with overall МА of approx. 600 RU/ml, and МА dependence on Ca2+ content was determined by with specific activity of 186 RU/mg. In deep introducing CaCl2 into the DSS samples, making up to cultivation of the BN-3 strain, activity of the cultural the 1–5 mM concentration; clot formation time was filtrate increased up to 900 RU/ml. After partial then measured. Values obtained in CaCl2-free samples purification of the coagulant, the authors obtained a were assumed as 100%. preparation with specific MA of 1463 RU per 1 mg of protein [21], which fully complies with the activity RESULTS AND DISCUSSION requirements for dry commercial ME. Protein concentration in A and B samples amounted Overall proteolytic activity. High overall to 23.6 mg/ml and 29.0 mg/ml, respectively. proteolytic activity of a ME is an extremely negative Milk-clotting activity. Specific МА of А factor for cheesemaking. Non-specific proteolytic sample was 29.1 ± 0.7 RU/ml, which amounted to activity of a ME results in considerable losses of 1230 ± 30 RU/g in protein equivalent. Sample B proteolysis products with whey, and cheese output demonstrated residual MA (< 1 RU/ml) and was is significantly reduced. Coagulants of R. miehei, removed from further assessment. R. pusillus and C. parasitica might result in 0.5–1.2% Activity of dry commercial milk-clotting enzymes decrease in cheese output in comparison to calf used in cheesemaking amounts to 100–200х103 RU/g, rennet [9]. If the remaining within granular curds milk- of liquid agents, 50–100х103 RU/ml. Thus, MA found clotting enzyme is not inactivated by heat treatment, in sample A is about 103 times lower than the required then cheese with a long ripening process and shelf life value for commercial ME. Low МА values might shall develop texture and taste defects (bitterness). An result from low productivity of the I. lacteus strain active proteolytic enzyme with low specificity affects 2265, suboptimal cultivation conditions, inadequate process-related properties of cheese whey, which is purification of the milk-clotting enzyme, and/or used as feedstock for various dairy products and presence of inhibitors. derivatives [3, 13]. Yet another possible explanation is partial The I. lacteus milk coagulant has high PA inactivation of the coagulant in the very process of (Fig. 1a). Overall PA of the I. lacteus coagulant production, for example, due to the рН level of the exceeds this parameter in ICS CR by approx. final buffer (рН 7.5), which exceeds the pH stability 220 times, and in CP, by approx. 33 times. Proteolytic limits for polyporopepsin (рН 3–6). Pepsin A, activity of the I. lacteus coagulant is so much above the belonging to the same aspartic proteinases family as control enzymes values that within the scale of Fig. 1a, polyporopepsin, is known to be irreversibly inactivated the curves built for ICS CR and CP almost merge with at рН > 7.0. the horizontal axis. In order to demonstrate differences MA efficiency is considerably affected by I. lacteus between ICS CR and CP, we provided a bigger scale strain selection and its cultivation conditions [20]. graph (Fig. 1b).

(a) (b)

Fig. 1. Assessment of overall proteolytic activity in milk-clotting enzymes: (a) Overall proteolytic activity of I. lacteus coagulant, ICS CR, and CP; (b) Overall proteolytic activity of ICS CR and CP. (Please note: in Fig. a and b, Y-axis scales are different). Text boxes show equations for polynomial trend lines (with factor 2) and approximation probability (R2).

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Position of microfungal coagulants among ME for might develop in cheese certain texture and taste commercial use is determined by M. Harboe et al. [3] defects. Therefore, cheesemakers try to stick to the 2+ classification, which suggests that, along with the minimum CaCl2 concentration. Low sensitivity to Ca specificity decrease (calculated as MA/PA ratio), milk- content in the milk mixture is a valuable factor, clotting enzymes are arranges as follows: rCh because it provides an opportunity to vary the (camel) > rCh (cow), calf chymosin > cow pepsin > introduced CaCl2 concentration, without worrying too mucorpepsin XL > mucorpepsin L > endotiapepsin. much about significant MA and PA changes. We have performed a similar arrangement, assuming Whenever preparations with high sensitivity to Ca2+ are the МА/PА ratio of calf rennet as 100%. We therefore used, the effect of MA and PA increase should be obtained the following sequence aligned with accounted for. specificity decrease: ICS CR (100.0%) > CP (14.9%) > In comparison to natural ME of animal origin, I. lacteus coagulant (0.5%). clotting activity of the I. lacteus preparation is the least One of the possible causes accounting for low dependent on the Ca2+ content in the DSS (Fig. 2). specificity of the I. lacteus coagulant might be its At CaCl2 3 mМ (most common concentration in insufficient purification. Ammonium sulfate in 70% cheesemaking), clotting activity of the I. lacteus saturation sediments the milk-clotting enzyme together preparation is increased by 27%, and respective with most proteins present in the cultural liquid. activities of ICS CR and CP, by 55% and 70%, “Ballast” proteolytic enzymes without MA or respectively. The biggest differences in clotting time inhibitors of specific clotting activity are also likely to decrease are observed when CaCl2 is increased from be present. Desalting allows to eliminate electrolytes 0 to 3 mМ. Within the range of CaCl2 3–5 mM, MA and low molecular weight organics, however, it does dynamics (inclination angles of all curves) is similar in not provide considerable purification of the resulting all tested enzymes. enzyme. MA of the I. lacteus might have probably It should be remembered that ICS CR sensitivity to been affected by a high pH level of the final buffer. We Ca2+ content is partially determined by 20% pepsin suggest that, once the purification procedure is admixture. In modern high-quality natural rennet improved and more homogeneous products are preparations for commercial use, chymosin content obtained, we might achieve certain increase in МА and reaches up to 96%. Along with the pepsin percentage specificity of the I. lacteus coagulant, which would decrease, rennet sensitivity to Ca2+ shall diminish as allow for its application in cheesemaking. This well and approach the respective values of the hypothesis is based on data reported in literature. In I. lacteus coagulant. 1988–1989, K. Murakami et al. reported a series of These findings allow us to conclude that the experiments on using the I. lacteus milk-clotting I. lacteus coagulant sensitivity to Ca2+ ions content is in enzyme in production of soft fibrous cheese without conformity with the cheesemaking requirements. ripening, and also such varieties as Gouda and Cheddar Thermal stability. In cheesemaking, thermal [16–18]. However, these works were discontinued, and stability of the milk-clotting enzyme is one of the the practical results of using the I. lacteus coagulant in regulating factors for proteolysis intensity and cheesemaking were never confirmed by other studies. specificity during cheese ripening. The substrate for Based on the collected data, we can conclude that ME remaining in cheese mass are α- and β-caseins. cheesemaking applications for the Irpex lacteus milk- Degree of enzymatic hydrolysis of these proteins clotting enzyme preparations seems unlikely, unless its determines the level of non-specific proteolysis which, MA and MA/PА ratio are increased. in turn, influences the ripening time, physical, chemical МА dependence on calcium ions content in the and organoleptic properties of cheese. All this substrate. In native milk, due to exposed carboxyl and determines the tactics for practical use of ME with phosphate groups, the surface of casein micelles is different thermal stability. negatively charged, which prevents them from binding and provides for aggregation stability of the colloidal system. During pasteurization process, calcium salts 100 present in milk partially convert into insoluble state. This prolongs the duration of rennet coagulation, and 80 results in formation of a flaccid milk clot. In order to 2+ compensate for the reduced Ca content after 60 pasteurization, CaCl2 in concentration of 0.2–0.5 g/l is introduced into the milk mixture prepared for 40 coagulation. Introduced CaCl2 produces a two-fold effect. First, in presence of Ca2+, negative charge from (%) time Clotting the surface of casein micelles is partially removed, and 20 K-caseins, located in the “hairy” layer become more available for attacks of milk-clotting enzymes. Second, 0 2+ Cа participates in formation of ion “bridges” between 0 1 2 3 4 5 micelles during their aggregation phase. As a result, Calcium chloride concentration (mM) milk clotting time decreases. Increased CaCl2 content does not only lead to a higher MA, but to a higher overall PA of the enzyme as well. Excessive addition of CaCl2 to the milk mixture Fig. 2. Dependence of clotting time on CaCl2 content.

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Thermolabile enzymes are preferably used in 110 production of hard and semi-hard cheese varieties, with 100 high second heating temperature (52–58○С), and lengthy ripening duration and extended storage time. 90 According to a widely applied cheesemaking practice, 80 cheese is produced in summer, in presence of a 70 thermolabile ME which is completely inactivated by 60 high temperature during the second heating. This leads 50 to a reduced proteolysis rate during cheese ripening 40 and storage. Products made in summer with the help of МА (%) a thermolabile ME are set for a lengthy storage and 30 distributed in autumn and in winter, when cheese 20 output drops. 10 Thermostable milk-clotting enzymes that remain 0 ○ proteolytically active at high temperatures (60–70 С) -10 are used for production of soft cheese varieties with 30 40 50 60 70 short ripening and storage time, or for cheese varieties Temperature (Celsius) that do not require ripening. In complex MEAs that consist of milk-clotting enzymes with high and low thermal stability, milk coagulation (at Т~32○С) occurs through the joint activity of participating enzymes, Fig. 3. Milk-clotting activity dependence on enzyme while after heat treatment (72–74○С), the required heating temperature (thermal stability). proteolysis level in ripening cheese is ensured by thermostable component only. According to the classification elaborated by “Chr. Information on polyporopepsin thermal stability Hansen” company, microfungal milk coagulants used presented on the BRENDA enzyme portal [- in cheesemaking are subdivided into several types: enzymes.org/enzyme.php?ecno=3.4.23.29] is contra- L type, natural (thermostable) enzymes; XL type, dictory: some data suggest that the coagulant is stable thermolabile coagulants obtained by oxidation; XP at 30○С (рН 4.6) and is completely inactivated at 45○С type and XLG type, these are chromatographically (рН 4.6), while according to other data, the enzyme can purified XL preparations, free from amylase/lipase/ resist heating for 15 minutes at 50○С (рН 4.5), and all cellulase admixtures [2]. In accordance with this activity stops altogether at 60○С (рН 4.5). classification, the I. lacteus coagulant should be E. Kikuchi et al. [16] conducted a comparative classified as XL type, albeit its thermal lability is a study of thermal stability in calf rennet (“Chr. natural property of this enzyme, and not a result of Hansen”), mucorpepsin R. pusillus (“Meito”), and chemical modification. I. lacteus ME. It was shown that the I. lacteus milk- Thus, as far as thermal lability is concerned, the clotting enzyme lost approx. 40% of the baseline MA I. lacteus milk-clotting enzyme is comparable to ICS at 45○С already, while at approx. 55○С it became CR, and, consequently, complies with the requirements completely inactive. Control enzymes, calf rennet and of the cheesemaking industry. mucorpepsin, were inactivated at approx. 60○С and MA dependence on рН level of the substrate. An approx. 70○С, respectively. Yu.P. Zagnitko [19] enzyme used for production of most varieties of cheese studied thermal stability of milk clotting preparations shall prove efficient in clotting milk within the рН obtained by SA sedimentation from cultural liquid of range of 6.4–6.7. I. lacteus (strain В-02), and noticed that the coagulant Variations of рН level of the milk produce certain retained its high МА upon incubation at 25–40○С, with changes in electrostatic and hydrophobic properties of maximum МА (230 RU/ml) at 30○С. However, at the milk casein micelle. As рН level grows and is heating temperatures above 45○С, inactivation of distanced from the casein рI, their cumulative negative enzyme was observed. In the range of 45–60○С the charges increase. As a result, forces of intermicellar inactivation degree of I. lacteus preparations (В-02 electrostatic repulsion are also increased. At the same strain) was directly proportional to the exposure. time, casein-casein hydrophobic interactions are Results of comparative thermal stability study diminished, which increases clot formation time during contrasting I. lacteus coagulant, ICS CR, and CP are rennet coagulation of the milk. Therefore, whenever presented in Fig. 3. At 40○С, all the test enzymes рН level of the milk mixture increases within retained their MA around 100%. I. lacteus coagulant the range of 5.0–7.0, rennet clotting time becomes and ICS CR were almost completely inactivated at slower. 60○С, residual MA amounting to 0.8% and 5.1% of the Yet another factor affecting the MA of an enzyme baseline value. However, the dynamics of inactivation is the optimum pH which depends on the substrate for these enzymes within the range of 40–60○С is nature. Polyporopepsin, chymosin, and pepsin are acid different. At 50○С, the I. lacteus coagulant was proteinases. That is why, as the substrate is alkalized inactivated by 55%, while ICS CR retained almost 90% and its pH is distanced from the pH optimum of the test of the baseline МА. The highest thermal stability was enzymes, clotting duration should increase. demonstrated by CP, as its activity was reduced by I. lacteus coagulant and ICS CR are similarly 96% only after heating at 70○С. dependant on pH changes in their MA (Fig. 4).

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Acylation by acid anhydrides significantly enhances MA of mucorpepsin produced by R. pusillus [24]. Maximum effect was observed during exposure of R. pusillus coagulant to maleic anhydride, however, that agent also inhibited МА in R. miehei mucorpepsin. T. Higashi et al. [26] treated R. pusillus coagulant with succinic anhydride, and achieved an increase in its specificity due to increased МА and МА/PА ratio. Oxidation by hydrogen peroxide run in presence of maleic anhydrid is used in order to reduced thermal stability and to prevent MA loss in R. pusillus mucorpepsin [27]. Methods for enzymic modification of microfungal coagulants have also been reported. Endoglycosidase H treatment for native mucorpepsin R. miehei ("Hannilase"), its partially oxydized version ("Modilase S") and R. miehei coagulant preparation ("Novoren XL"), derived from a heterologous producer (Aspergillus oryzae (Ahlb.) Cohn), allowed to obtain

deglycosylated proteases with enhanced cheesemaking Fig. 4. Dependence of clotting time on рН. properties. Milk-clotting activity of Hannilase and Modilase S would increase by over 30%, and in case of Both preparations demonstrate high clotting activity recombinant mucorpepsin R. miehei produced by within the pH range of 6.0–6.5, and gradually become A. oryzae ("Novoren XL"), increase in specific activity inactivated at рН 6.5–7.0. Such results seem coherent amounted to approx. 45%. Besides, overall PA would with the BRENDA [brenda-enzymes.org/enzyme. decrease by ~10%, specificity (МА/PА) would php?ecno=3.4.23.29] data, suggesting that the optimum increase, thermal stability would go down, pH range рН of polyporopepsin lies within the range of 2.5–4.0, for MA would be more extensive, and cohesion of and partially coincides with the chymosin optimum of coagulants with milk clot would improve [28, 29]. 3.7–4.9. The optimum of pepsin enzymatic activity lies Another potential method for influencing within a more acid range of рН 1.8–2.2. Consequently, technological properties of microfungal coagulants is CP preparation starts losing its activity earlier: at рН induced mutagenesis. Several focal substitutions can be 6.5, activity loss is considerable, while at рН 7.0 the singled out that might positively influence the enzyme is almost completely inactivated (at рН 7.0, technological properties of mucorpepsins. A101T or clotting duration is increased by 46 times). G186D replacement in a molecule of R. pusillus Thus, MA of I. lacteus coagulant dependence on mucorpesin and mutant expression in S. cerevisiae рН within the range of 5.5–7.0 is similar to that of the allows to enhance significantly thermal stability of the reference milk milk-clotting enzyme, - calf rennet. coagulant. Curiously, the same result is ensured by any Methods for correcting the technological amino acid substitution in G186 position. Mutant properties of microfungal milk-clotting enzymes. enzyme with two simultaneous substitutions, A101T There are several methods for correcting MA, and G186D, proved to be even less thermostable than specificity, and thermal stability of microfungal mucorpepsins with single replacements, and at the enzymes in cheesemaking. An attempt to influence one same time, did not show any decrease in МА/PА ratio. technological parameter usually results in modification Focal substitutions of E19V and Q266E in a of the others. Thus, although thermal stability of mucorpepsin derived from a wild strain of R. miehei I. lacteus coagulant does not require any adjustment, (CBS 182-67) result in a 4.6 times specificity increase, we shall consider main methods and examples of and a 1.51% growth in cheese output, which is a very changing the technological parameters of a microfungal significant achievement in cheesemaking [2]. Site- milk-clotting enzymes in a complex. targeted mutagenesis method allowed to obtain a Thermal stability of mucorpepsins is known to be mucorpepsin (the producer being M. pusillus) with two positively related to the degree of glycosylation. In simultaneous amino acids substitutions, G186D and order to reduce the glycolysation of coagulants E13D, which led to an increased enzymatic specificity belonging to the Rhizomucor genus, several methods of due to a higher МА [13]. chemical modification were suggested: oxidation, One of the above listed methods for reducing high nitrification, acylation, carbamylation, periodate- non-specific PA in mucorpepsin might lead to a higher induced and enzymatic deglycolysation [2]. D.A. specificity (МА/PА) of the I. lacteus coagulant, which Cornelius [24] introduced a method for reducing in future might allow for its application in thermal stability of R. miehei and R. pusillus cheesemaking. proteinases by oxidation run with hydrogen peroxide or Results. We carried out a complex study of photosensibilized oxidation in presence of a colorant. technological properties of a milk coagulant produced Oxidation does not lead to considerable loss of MA, by a higher basidial fungus, Irpex lacteus (I. lacteus), and is currently widely used in order to reduce thermal and evaluated each parameter in accordance with its stability of R. miehei coagulants [2]. suitability for cheesemaking industry application.

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I. lacteus coagulant is shown to be on par with natural strategies to be used in order to obtain preparations ME of animal origin used in dairy industry in a series with a high specific activity: selecting highly of technological parameters, such as thermal stability, productive strains, perfecting cultivation methods, MA dependence on pH level, and Ca2+ ions content. achieving optimal purification, and/or using methods of Practical application of the milk-clotting enzyme chemical, biochemical, and genetic modification. produced by I. lacteus is considerably challenged by its The data we obtained might be used in further work low МА and high overall PA. We need to achieve a aimed at studying and improving technological considerable increase in МА of the coagulant in order properties of a milk-clotting enzyme produced by the to remove this obstacle. There are a number of I. lacteus fungus.

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Please cite this article in press as: Lebedev L.R., Kosogova Т.А., Teplyakova Т.V., Kriger A.V., Elchaninov V.V., Belov А.N., and Коval' А.D. Study of technological properties of milk-clotting enzyme from Irpex lacteus (Irpex lacteus (Fr.) Fr.). Foods and Raw Materials, 2016, vol. 4, no. 2, pp. 58–65. DOI: 10.21179/2308-4057-2016-2-58-65.

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