Advertisement

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

  • Yao Zhang
  • Yongjun Xia
  • Phoency F.-H. Lai
  • Xiaofeng Liu
  • Zhiqiang Xiong
  • Jichao Liu
  • Lianzhong AiEmail author
Original Article
  • 98 Downloads

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.

Keywords

Isolation Fermentation Bacillus licheniformis Milk-clotting enzyme Serine/alkaline protease Casein hydrolysis 

Notes

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 information

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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

This manuscript is approved by all authors for publication.

References

  1. 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):2205–2213CrossRefGoogle Scholar
  2. Ahmed SA, Helmy WA (2012) Comparative evaluation of Bacillus licheniformis 5A5 and Aloe variegata milk-clotting enzymes. Braz J Chem Eng 29(1):69–76CrossRefGoogle Scholar
  3. 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–M221CrossRefGoogle Scholar
  4. 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):1727–1733Google Scholar
  5. 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 225:258–266CrossRefGoogle Scholar
  6. 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):1561–1566CrossRefGoogle Scholar
  7. 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):151–154CrossRefGoogle Scholar
  8. 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):261–273CrossRefGoogle Scholar
  9. Chwen-Jen S, Lan APT, Ing LS (2009) Milk-clotting enzymes produced by culture of Bacillus subtilis natto. Biochem Eng 43(1):85–91CrossRefGoogle Scholar
  10. 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):9370–9378CrossRefGoogle Scholar
  11. 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):1043–1048CrossRefGoogle Scholar
  12. 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):258–269CrossRefGoogle Scholar
  13. 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 102:515–525CrossRefGoogle Scholar
  14. Ghareib MH, Hamdy HS, Khalil AA (2001) Production of intracellular milk-clotting enzyme in submerged cultures of Fusarium subglutinans. Acta Microbiol Pol 50(2):139–147Google Scholar
  15. 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):73Google Scholar
  16. Hashem AM (1999) Optimization of milk-clotting enzyme productivity by Penicillium oxalicum. Bioresour Technol 70(2):203–207CrossRefGoogle Scholar
  17. 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):136–144CrossRefGoogle Scholar
  18. 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):357–365CrossRefGoogle Scholar
  19. Lee KD, Warthesen JJ (2010) Mobile phases in reverse-phase HPLC for the determination of bitter peptides in cheese. J Food Sci 61:291–294CrossRefGoogle Scholar
  20. 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):733–741CrossRefGoogle Scholar
  21. 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
  22. 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 239:157–164CrossRefGoogle Scholar
  23. 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):2842–2850CrossRefGoogle Scholar
  24. Majumder R, Banik SP, Khowala S (2015) Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091. Food Chem 173:441–448CrossRefGoogle Scholar
  25. 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):9–16Google Scholar
  26. 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):6162–6169CrossRefGoogle Scholar
  27. 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):87–93CrossRefGoogle Scholar
  28. Moon SH, Parulekar SJ (2010) A parametric study ot protease production in batch and fed-batch cultures of Bacillus firmus. Biotechnol Bioeng 37(5):467–483CrossRefGoogle Scholar
  29. Patel R, Dodia M, Singh SP (2005) Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: production and optimization. Process Biochem 40(11):3569–3575CrossRefGoogle Scholar
  30. Preetha S, Boopathy R (1997) Purification and characterization of a milk clotting protease from Rhizomucor miehei. World J Microbiol Biotechnol 13(5):573–578CrossRefGoogle Scholar
  31. 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):2189–2199CrossRefGoogle Scholar
  32. 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 98:847–854CrossRefGoogle Scholar
  33. 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):44–50CrossRefGoogle Scholar
  34. 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):531–538CrossRefGoogle Scholar
  35. Shata HMA (2005) Extraction of milk-clotting enzyme produced by solid state fermentation of Aspergillus oryzae. Pol J Microbiol 54(3):241–247Google Scholar
  36. 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):174–185CrossRefGoogle Scholar
  37. 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):975–979CrossRefGoogle Scholar
  38. Thakur M, Karanth NG, Nand K (1990) Production of fungal rennet by Mucor miehei using solid state fermentation. Appl Microbiol Biotechnol 32(4):409–413CrossRefGoogle Scholar
  39. 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):1743–1754CrossRefGoogle Scholar
  40. Vallejo JA, Ageitos JM, Poza M, Villa TG (2012) Short communication: a comparative analysis of recombinant chymosins. J Dairy Sci 95(2):609–613CrossRefGoogle Scholar
  41. 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):129–138CrossRefGoogle Scholar
  42. 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):6764–6768Google Scholar
  43. Wasko A, Kieliszek M, Targonski Z (2012) Purification and characterization of a proteinase from the probiotic Lactobacillus rhamnosus OXY. Prep Biochem Biotechnol 42:476–488CrossRefGoogle Scholar
  44. 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):717–722CrossRefGoogle Scholar
  45. 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):1312–1319CrossRefGoogle Scholar
  46. 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):283–288Google Scholar
  47. 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):1008–1026CrossRefGoogle Scholar

Copyright information

© Università degli studi di Milano 2019

Authors and Affiliations

  • Yao Zhang
    • 1
  • Yongjun Xia
    • 1
  • Phoency F.-H. Lai
    • 1
  • Xiaofeng Liu
    • 1
  • Zhiqiang Xiong
    • 1
  • Jichao Liu
    • 2
  • Lianzhong Ai
    • 1
    Email author
  1. 1.Shanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food EngineeringUniversity of Shanghai for Science and TechnologyShanghaiChina
  2. 2.Beijing Sanyuan Foods Co., LtdBeijingChina

Personalised recommendations