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Exploring Microbial Peptidases for Cheese Production: A Viewpoint on the Current Conjecture Ronivaldo Rodrigues da Silva* Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, s/n Campus Universitário da USP, Ribeirão Preto, São Paulo 14040-903, Brazil (GRAS) microorganisms, such as Aspergillus niger var. awamori and Aspergillus oryzae, are conventional commercial coagulants employed in cheese making. Although the use of these milk coagulants constitutes the most common protocol to produce cheese, studies are being constantly undertaken in search of new enzymes with a high coagulant activity to produce good quality cheese at lower costs. Further, in this study, the economic value of this dairy product has been discussed to understand the global consumption of cheese and the economic benefits from this market. In the past few years, the global cheese production has significantly increased, which accounts for about 1/3 of the global consumption of cow milk. In 1980, the global cheese production was estimated to be 8.7 × 109 kg, while it reached 20 × 109 kg in 2011. According to global estimates, the cheese market is expected to continuously increase in the next few years.5 INTRODUCTION The increase in consumption of cheese is being driven by the Since long ago, enzymes have captured the interest of advancements in dairy technology, by which a variety of dairy investigators in food chemistry, particularly in dairy technology. products have been developed to target a wide spectrum of The increase in consumption of milk products, especially consumers, and this has led to enormous economic benefits. cheese, has led to the search for alternative milk coagulants that Therefore, numerous innovative technologies have been are cost-effective and are highly efficient for milk processing. In focused to identify prospective coagulants.2−4 this regard, microbial enzymes are important tools for the Subsequent to the evaluation of novel specific peptidases for preparation of cheese. milk coagulation, a variety of rennet enzymes have been In the past few years, increased scientific interest has been reported to be obtained from various fungal genera, such as devoted to microbial peptidases. Among these peptidases, few Mucor, Rhizomucor, Aspergillus, Endothia, Rhizopus, and of them are investigated to produce bioactive peptides, while Phanerochaete. Recently, the complete evaluation of the others are used for food processing.1 In particular, the latter catalytic specificity and milk coagulating properties of group of enzymes are highly applicable for dairy technology, extracellular aspartic peptidases obtained from Rhizomucor i.e., utilization of microbial peptidases as the substitutes for the miehei3 and Phanerochaete chrysosporium4 has been discussed. enzyme chymosin acquired from newborn ruminants. ChymoStudies on the specificity of peptidases are crucial to elucidate sin obtained from calves is associated with ethical issues. the catalytic properties of the enzymes. This information has Therefore, alternative enzymes have been investigated to been fundamental for their application in milk coagulation. The support the demand for cheese production.1,2 efficiency of a successful coagulant is assessed by the capacity of Recent reports1,3,4 have shown the production of fungal peptidases and their application for the coagulation of milk. peptidase to perform specific hydrolysis of the peptide bond These peptidases offer a promising alternative to reduce the between Phe105 and Met106 in the k-casein protein (Figure 1). cost of production for the preparation of cheese. However, These promising coagulants for cheese production need to further understanding is essential with regard to the effective exhibit higher coagulant activity than proteolytic activity.1,3,4 advances in the application of microbial peptidases in dairy A recent report4 regarding an extracellular aspartic peptidase technology and the future prospects of this sector with respect obtained from P. chrysosporium suggested that this enzyme to the current conjuncture. shows considerable essential characteristics required for cheese making. This enzyme exhibits properties to coagulate milk MICROBIAL PEPTIDASES AND THE CURRENT along with slight proteolytic activity. Upon neutral−alkaline CONJECTURE OF THE GLOBAL CHEESE MARKET
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To date, recombinant peptidases, e.g., calf chymosin and microbial rennet, expressed in “generally recognized as safe” © XXXX American Chemical Society
Received: January 2, 2018
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DOI: 10.1021/acs.jafc.8b00018 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
microbial diversity, and enzyme technology has led to costeffective production of good quality cheese.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ronivaldo Rodrigues da Silva: 0000-0002-6504-8406 Notes
The author declares no competing financial interest.
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REFERENCES
(1) da Silva, R. R. Bacterial and fungal proteolytic enzymes: Production, catalysis and potential applications. Appl. Biochem. Biotechnol. 2017, 183, 1−19. (2) Theron, L. W.; Divol, B. Microbial aspartic proteases: Current and potential applications in industry. Appl. Microbiol. Biotechnol. 2014, 98, 8853−8868. (3) da Silva, R. R.; Souto, T. B.; de Oliveira, T. B.; de Oliveira, L. C. G.; Karcher, D.; Juliano, M. A.; Juliano, L.; de Oliveira, A. H. C.; Rodrigues, A.; Rosa, J. C.; Cabral, H. Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei. J. Ind. Microbiol. Biotechnol. 2016, 43, 1059−1069. (4) da Silva, R. R.; de Oliveira, L. C. G.; Juliano, M. A.; Juliano, L.; de Oliveira, A. H. C.; Rosa, J. C.; Cabral, H. Biochemical and milkclotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium. Food Chem. 2017, 225, 45−54. (5) PM Food & Dairy Consulting. The World Cheese Market Report; PM Food & Dairy Consulting: Højbjerg, Denmark, 2014; pp 1−35. (6) Lemes, A. C.; Pavón, Y.; Lazzaroni, S.; Rozycki, S.; Brandelli, A.; Kalil, S. J. A new milk-clotting enzyme produced by Bacillus sp. P45 applied in cream cheese development. LWTFood Sci. Technol. 2016, 66, 217−224. (7) Ahmed, S. A.; Wehaidy, H. R.; Ibrahim, O. A.; Abd El Ghani, S.; El-Hofi, M. A. Novel milk-clotting enzyme from Bacillus stearothermophilus as a coagulant in UF-white soft cheese. Biocatal. Agric. Biotechnol. 2016, 7, 241−249. (8) Majumder, R.; Banik, S. P.; Khowala, S. Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091. Food Chem. 2015, 173, 441−448.
Figure 1. Specific hydrolysis of the peptide bond between Phe105 and Met106 in the k-casein protein.
conditions (pH > 6.5), it does not exhibit the proteolytic activity but can maintain the milk coagulation activity. Another recent study conducted by da Silva et al.3 described an aspartic peptidase secreted by R. miehei. The enzyme demonstrated greater coagulant activity than proteolytic activity. The enzyme was capable of performing specific hydrolysis in clotting milk sequence in k-casein (Phe105− Met106). Other recent studies have also investigated microbial peptidases for application in cheese production.6−8 In view of the studies conducted in the research for microbial coagulants, it is possible to defend the necessity for investments in this area of study. The enzyme technology has taken advantage of various microbial species with promising results that have been already mentioned in this viewpoint. In addition to these prospective microbial coagulant studies, the evaluation of substrate specificity using the fluorescence resonance energy transfer (FRET) substrates has been instrumental in elucidating the coagulation specificity of the enzymes.1,3,4
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REMARKS FOR FUTURE PROSPECTS The major challenges in using microbial rennet for cheese production are to improve the particular enzymatic properties, such as (i) higher coagulant activity than proteolytic activity, and (ii) low cost of enzyme production. Moreover, another important challenge for producing cheese of good quality (flavor and texture) is to reduce the bitterness derived from proteolysis that may release peptides containing aromatic amino acids. Importantly, exploration of microbial biodiversity for prospective coagulants is an interesting method for achieving advancements in cheese technology. The species diversity and biochemical variations contribute to consolidate the microorganisms as a valuable resource for the sustenance and development of this industrial sector. Currently, continuous investigations support better prospects for dairy technology. A practical example refers to the use of microbial rennet from R. miehei for cheese production and reinforces the potential to exploit microbial coagulants. Additionally, protein engineering techniques and heterologous expression of rennet enzymes are potential scientific tools to be exploited in this field of investigation. In conclusion, the global increase in the consumption of cheese encourages continual scientific research to improve the production technologies that result in appreciable products and economic benefits. Exploration of novel milk coagulants, B
DOI: 10.1021/acs.jafc.8b00018 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
