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Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated Bacillus licheniformis BL312

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Abstract

Purpose

This study was conducted to find a microbial milk-clotting enzyme (MCE) with a high and stable milk-clotting activity (MCA) to proteolytic activity (PA) ratio suitable for the cheese industry.

Methods

Microbial strains were isolated from soil suspensions cultured in solid casein medium. 16S rDNA of representative isolates were sequenced to identify the microbial species. Nutrition and fermentation conditions were systematically examined to optimize MCA of the selected MCE. Protease inhibitors were used to identify the type of MCE. The casein hydrolysis was analyzed through reversed-phase HPLC (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Results

The Bacillus licheniformis BL312 was identified from 50 bacterial strains. BL312 MCE achieved a maximal MCA (460 ± 15 SU/mL) at 48 h that was 2.7-fold higher than the control, and the MCA/PA ratio (9.0) and pH (6.6) remained stable throughout the fermentation process. Medium containing 30 g/L wheat bran shorts, 5 g/L glucose, and 3 g/L corn steep liquor was sufficient for optimal BL312 MCE production. Fermentation conditions of an inoculum size of 7.0% (v/v), fermentation temperature of 37 °C, agitation speed of 210 rpm, and initial pH 6.6 were required to achieve maximal MCA. BL312 MCE was inhibited by phenylmethanesulfonyl fluoride (PMSF) and high concentrations of ethylenediaminetetraacetic acid (EDTA) (5–25 mM). The αs-casein (αs-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE and calf rennet were different.

Conclusions

BL312 MCE is a serine/alkaline protease that exhibits high MCA and various hydrolysis for caseins in comparison with calf rennet.

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References

  • Ageitos JM, Vallejo JA, Sestelo ABF, Poza M, Villa TG (2007) Purification and characterization of a milk-clotting protease from Bacillus licheniformis strain USC13. J Appl Microbiol 103(6):2205–2213

    CAS  PubMed  Google Scholar 

  • Ahmed SA, Helmy WA (2012) Comparative evaluation of Bacillus licheniformis 5A5 and Aloe variegata milk-clotting enzymes. Braz J Chem Eng 29(1):69–76

    CAS  Google Scholar 

  • An ZG, He XL, Gao WD, Zhao W, Zhang WB (2014) Characteristics of miniature cheddar-type cheese made by microbial rennet from Bacillus amyloliquefaciens: a comparison with commercial calf rennet. J Food Sci 79:M214–M221

    CAS  PubMed  Google Scholar 

  • Arima K, Yu J, Iwasaki S, Tamura G (1968) Milk-clotting enzyme from microorganisms: V. purification and crystallization of Mucor rennin from Mucor pusillus var. Lindt Appl Microbiol 16(11):1727–1733

    CAS  PubMed  Google Scholar 

  • Ben AA, Makhlouf I, Flaviu PR, Francis F, Bauwens J, Attia H, Besbes S, Blecker C (2017) Effect of extraction pH on techno-functional properties of crude extracts from wild cardoon (Cynara cardunculus L.) flowers. Food Chem 225:258–266

    Google Scholar 

  • Cai QH, Yue XY, Niu TG, Ji C (2004) The screening of culture condition and properties of xylanase by white-rot fungus Pleurotus ostreatus. Process Biochem 39(11):1561–1566

    CAS  Google Scholar 

  • Cavalcanti MTH, Martinez CR, Furtado VC, Neto BB, Teixeira MF, Lima Filho JL, Porto ALF (2005) Milk-clotting protease production by Nocardiopsis sp. in an inexpensive medium. World J Microbiol Biotechnol 21(2):151–154

    CAS  Google Scholar 

  • Celebi M, Topuzogullari M, Kuzu H (2016) Thermal destabilization of Rhizomucor miehei rennet with aldehyde dextran sulfate: purification, bioconjugation and milk-clotting activities. Appl Biochem Biotechnol 180(2):261–273

    CAS  PubMed  Google Scholar 

  • Chwen-Jen S, Lan APT, Ing LS (2009) Milk-clotting enzymes produced by culture of Bacillus subtilis natto. Biochem Eng 43(1):85–91

    Google Scholar 

  • Ding ZY, Liu SP, Gu ZH, Zhang L, Zhang KC, Shi GY (2011) Production of milk-clotting enzyme by Bacillus subtilis B1 from wheat bran. Afr J Biotechnol 10(46):9370–9378

    CAS  Google Scholar 

  • Ding ZY, Ai LZ, Ouyang A, Ding ML, Wang WF, Wang BD, Liu SP, Gu ZH, Zhang L, Shi GY (2012) A two-stage oxygen supply control strategy for enhancing milk-clotting enzyme production by Bacillus amyloliquefaciens. Eur Food Res Technol 34(6):1043–1048

    Google Scholar 

  • Fazouane F, Mechakra A, Abdellaoui R, Nouani A (2010) Characterization and cheese-making properties of rennet-like enzyme produced by a local Algerian isolate of Aspergillus niger. Food Biotechnol 24(3):258–269

    Google Scholar 

  • Gagaoua M, Ziane F, Nait RS, Boucherba N, Ait KEEA, Bouanane-Darenfed A, Hafid K (2017) Three phase partitioning, a scalable method for the purification and recovery of cucumisin, a milk-clotting enzyme, from the juice of Cucumis melo var. reticulatus. Int J Biol Macromol 102:515–525

    CAS  PubMed  Google Scholar 

  • Ghareib MH, Hamdy HS, Khalil AA (2001) Production of intracellular milk-clotting enzyme in submerged cultures of Fusarium subglutinans. Acta Microbiol Pol 50(2):139–147

    CAS  PubMed  Google Scholar 

  • Hang F, Liu PY, Wang QB, Han J, Wu ZJ, Gao CX, Liu ZM, Zhang H, Chen W (2016) High milk-clotting activity expressed by the newly isolated Paenibacillus spp. strain BD3526. Molecules 21(1):73

    PubMed  PubMed Central  Google Scholar 

  • Hashem AM (1999) Optimization of milk-clotting enzyme productivity by Penicillium oxalicum. Bioresour Technol 70(2):203–207

    CAS  Google Scholar 

  • Hayaloglu AA, Karatekin B, Gurkan H (2014) Thermal stability of chymosin or microbial coagulant in the manufacture of Malatya, a halloumi type cheese: proteolysis, microstructure and functional properties. Int Dairy J 38(2):136–144

    CAS  Google Scholar 

  • He XL, Zhang WB, Ren FZ, Gan BZ, Guo HY (2012) Screening fermentation parameters of the milk-clotting enzyme produced by newly isolated Bacillus amyloliquefaciens D4 from the Tibetan Plateau in China. Ann Microbiol 62(1):357–365

    CAS  Google Scholar 

  • Lee KD, Warthesen JJ (2010) Mobile phases in reverse-phase HPLC for the determination of bitter peptides in cheese. J Food Sci 61:291–294

    Google Scholar 

  • Li Y, Liang S, Zhi DJ, Chen P, Su F, Li HY (2012) Purification and characterization of Bacillus subtilis milk-clotting enzyme from Tibet Plateau and its potential use in yak dairy industry. Eur Food Res Technol 234(4):733–741

    CAS  Google Scholar 

  • Li L, Zheng Z, Zhao X, Wu FY, Zhang J, Yang ZN (2019) Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1. Food Sci Biotechnol. https://doi.org/10.1007/s10068-018-0539-2

    CAS  PubMed  Google Scholar 

  • Liburdi K, Emiliani SS, Benucci I, Lombardelli C, Esti M (2018) A preliminary study of continuous milk coagulation using Cynara cardunculus flower extract and calf rennet immobilized on magnetic particles. Food Chem 239:157–164

    CAS  PubMed  Google Scholar 

  • Luo J, Xiao C, Zhang H, Ren FZ, Lei XG, Yang ZB, Yu ZQ (2018) Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases. J Dairy Sci 101(4):2842–2850

    CAS  PubMed  Google Scholar 

  • Majumder R, Banik SP, Khowala S (2015) Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091. Food Chem 173:441–448

    CAS  PubMed  Google Scholar 

  • Marcial J, Pérez De Los Santos AI, Fernández FJ, Díaz-Godínez G, Montiel-González AM, Tomasini A (2011) Characterization of an aspartic protease produced by Amylomyces rouxii. Rev Mex Cienc Geol 10(1):9–16

    CAS  Google Scholar 

  • Meng FQ, Chen R, Zhu XY, Lu YJ, Nie T, Lu FX, Lu ZX (2018) A newly effective milk-clotting enzyme from Bacillus subtilis and its application in cheese-making. J Agric Food Chem 66(24):6162–6169

    CAS  PubMed  Google Scholar 

  • Merheb-Dini C, Gomes E, Boscolo M, Silva RD (2010) Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31 (milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31). Food Chem 120(1):87–93

    CAS  Google Scholar 

  • Moon SH, Parulekar SJ (2010) A parametric study ot protease production in batch and fed-batch cultures of Bacillus firmus. Biotechnol Bioeng 37(5):467–483

    Google Scholar 

  • Patel R, Dodia M, Singh SP (2005) Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: production and optimization. Process Biochem 40(11):3569–3575

    CAS  Google Scholar 

  • Preetha S, Boopathy R (1997) Purification and characterization of a milk clotting protease from Rhizomucor miehei. World J Microbiol Biotechnol 13(5):573–578

    CAS  Google Scholar 

  • Qiu WW, Curtin D, Johnstone P, Beare M (2016) Small-scale spatial variability of plant nutrients and soil organic matter: an arable cropping case study. Commun Soil Sci Plan 47(19):2189–2199

    CAS  Google Scholar 

  • Salehi M, Aghamaali MR, Sajedi RH, Asghari SM, Jorjani E (2017) Purification and characterization of a milk-clotting aspartic protease from Withania coagulans fruit. Int J Biol Macromol 98:847–854

    CAS  PubMed  Google Scholar 

  • Sato S, Tokuda H, Koizumi T, Nakanishi K (2007) Purification and characterization of fan extracellular proteinase having milk-clotting activity from Enterococcus faecalis TUA2495L. Food Sci Technol Res 10(1):44–50

    Google Scholar 

  • Sen S, Veeranki VD, Mandal B (2009) Effect of physical parameters, carbon and nitrogen sources on the production of alkaline protease from a newly isolated Bacillus pseudofirmus SVB1. Ann Microbiol 59(3):531–538

    CAS  Google Scholar 

  • Shata HMA (2005) Extraction of milk-clotting enzyme produced by solid state fermentation of Aspergillus oryzae. Pol J Microbiol 54(3):241–247

    CAS  PubMed  Google Scholar 

  • Sun Q, Wang XP, Yan QJ, Chen W, Jiang ZQ (2014) Purification and characterization of a chymosin from Rhizopus microsporus var. rhizopodiformis. Appl Biochem Biotechnol 174(1):174–185

    CAS  PubMed  Google Scholar 

  • Tang XM, Shen W, Lakay FM, Shao WL, Wang ZX, Prior BA, Zhuge J (2004) Cloning and over-expression of an alkaline protease from Bacillus licheniformis. Biotechnol Lett 26(12):975–979

    CAS  PubMed  Google Scholar 

  • Thakur M, Karanth NG, Nand K (1990) Production of fungal rennet by Mucor miehei using solid state fermentation. Appl Microbiol Biotechnol 32(4):409–413

    CAS  Google Scholar 

  • Uda K, Abe K, Dehara Y, Mizobata K, Edashige Y, Nishimura R, Radkov AD, Moe LA (2017) Triple serine loop region regulates the aspartate racemase activity of the serine/aspartate racemase family. Amino Acids 49(10):1743–1754

    CAS  PubMed  Google Scholar 

  • Vallejo JA, Ageitos JM, Poza M, Villa TG (2012) Short communication: a comparative analysis of recombinant chymosins. J Dairy Sci 95(2):609–613

    CAS  PubMed  Google Scholar 

  • Vishwanatha K, Rao AA, Singh SA (2010) Acid protease production by solid-state fermentation using Aspergillus oryzae MTCC 5341: optimization of process parameters. J Ind Microbiol Biotechnol 37(2):129–138

    CAS  PubMed  Google Scholar 

  • Wang J, Li C, Yang H, Mushegian A, Jin S (1998) A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice. J Bacteriol 180(24):6764–6768

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wasko A, Kieliszek M, Targonski Z (2012) Purification and characterization of a proteinase from the probiotic Lactobacillus rhamnosus OXY. Prep Biochem Biotechnol 42:476–488

    CAS  PubMed  Google Scholar 

  • Wu JJ, Chen HB, Chen WP (2008) Fermentation parameter and partial biochemical characterisation of milk clotting enzyme from Chinese distiller’s yeast. Ann Microbiol 58(4):717–722

    CAS  Google Scholar 

  • Yegin S, Goksungur Y, Fernandez-Lahore M (2012) Purification, structural characterization, and technological properties of an aspartyl proteinase from submerged cultures of Mucor mucedo DSM 809. Food Chem 133(4):1312–1319

    CAS  Google Scholar 

  • Yu PJ, Chou CC (2005) Factors affecting the growth and production of milk-clotting enzymes by Amylomyces rouxii in rice liquid medium. Food Technol Biotechnol 43(3):283–288

    CAS  Google Scholar 

  • Zhou J, Wang RQ, Guo WH, Zhou GJ, Wang Q, Wang W, Han XM, Pang XG, Zhan JC, Dai JR (2011) Soil microbial community diversity and its relationships with geochemical elements under different farmlands in Shouguang, China. Commun Soil Sci Plan 42(9):1008–1026

    CAS  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the National Key R&D Program of China (grant no. 2018YFD0502306) and Shanghai Engineering Research Center of Food Microbiology (grant no. 19DZ2281100) for the granted fellowships.

Funding

This work was supported by the National Key R&D Program of China (grant no. 2018YFD0502306) and Shanghai Engineering Research Center of Food Microbiology (grant no. 19DZ2281100).

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Correspondence to Lianzhong Ai.

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Zhang, Y., Xia, Y., Lai, P.FH. et al. Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated Bacillus licheniformis BL312. Ann Microbiol 69, 1289–1300 (2019). https://doi.org/10.1007/s13213-019-01513-3

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