Skip to main content

No more cleaning up - Efficient lactic acid bacteria cell catalysts as a cost-efficient alternative to purified lactase enzymes

Abstract

β-galactosidases, commonly referred to as lactases, are used for producing lactose-free dairy products. Lactases are usually purified from microbial sources, which is a costly process. Here, we explored the potential that lies in using whole cells of a food-grade dairy lactic acid bacterium, Streptococcus thermophilus, as a substitute for purified lactase. We found that S. thermophilus cells, when treated with the antimicrobial peptide nisin, were able to hydrolyze lactose efficiently. The rate of hydrolysis increased with temperature; however, above 50 °C, stability was compromised. Different S. thermophilus strains were tested, and the best candidate was able to hydrolyze 80% of the lactose in a 50 g/L solution in 4 h at 50 °C, using only 0.1 g/L cells (dry weight basis). We demonstrated that it was possible to grow the cell catalyst on dairy waste, and furthermore, that a cell-free supernatant of a culture of a nisin-producing Lactococcus lactis strain could be used instead of purified nisin, which reduced cost of use significantly. Finally, we tested the cell catalysts in milk, where lactose also was efficiently hydrolyzed. The method presented is natural and low-cost, and allows for production of clean-label and lactose-free dairy products without using commercial enzymes from recombinant microorganisms.

Key points

• Nisin-permeabilized Streptococcus thermophilus cells can hydrolyze lactose efficiently.

• A low-cost and more sustainable alternative to purified lactase enzymes.

• Reduction of overall sugar content.

• Clean-label production of lactose-free dairy products.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Baltin KK, Akishev ZD, Abeldenov SK, Silayev DV, Khassenov BB (2017) Biochemical properties of recombinant Β-galactosidase from Streptococcus thermophilus. Eurasian J App Biotechnol 2:1-12

    Google Scholar 

  2. De Arauz LJ, Jozala AF, Mazzola PG, Vessoni Penna TC (2009) Nisin biotechnological production and application: a review. Trends Food Sci Technol 20:146-154

    Article  Google Scholar 

  3. De Vos WM, Mulders JWM, Siezen RJ, Hugenholtz J, Kuipers OP (1993) Properties of nisin Z and distribution of its gene, nisZ, in Lactococcus lactis. Appl Environ Microbiol 59:213-218

    Article  Google Scholar 

  4. Laridi R, Kheadr EE, Benech RO, Vuillemard JC, Lacroix C, Fliss I (2003) Liposome encapsulated nisin Z: optimization, stability and release during milk fermentation. Int Dairy J 13:325-336

    CAS  Article  Google Scholar 

  5. Paul Ross R, Morgan S, Hill C (2002) Preservation and fermentation: past, present and future. Int J Food Microbiol 79:3-16

    Article  Google Scholar 

  6. Saqib S, Akram A, Halim SA, Tassaduq R (2017) Sources of β-galactosidase and its applications in food industry. 3. Biotech 7:1-7

    Google Scholar 

  7. Chen J, Shen J, Ingvar Hellgren L, Jensen PR, Solem C (2015) Adaptation of Lactococcus lactis to high growth temperature leads to a dramatic increase in acidification rate. Sci Rep 5:1-15. https://doi.org/10.1038/srep14199

    CAS  Article  Google Scholar 

  8. De Carvalho CCCR (2017) Whole cell biocatalysts: essential workers from nature to the industry. Microb Biotechnol 10:250-263. https://doi.org/10.1111/1751-7915.12363

    Article  PubMed  Google Scholar 

  9. De Carvalho CCCR (2011) Enzymatic and whole cell catalysis: finding new strategies for old processes. Biotechnol Adv 29:75-83. https://doi.org/10.1016/j.biotechadv.2010.09.001

    CAS  Article  PubMed  Google Scholar 

  10. Dekker PJT, Koenders D, Bruins MJ (2019) Lactose-free dairy products: market developments, production, nutrition and health benefits. Nutrients 11:1-14. https://doi.org/10.3390/nu11030551

    CAS  Article  Google Scholar 

  11. Deng Y, Misselwitz B, Dai N, Fox M (2015) Lactose intolerance in adults: biological mechanism and dietary management. Nutrients 7:8020-8035. https://doi.org/10.3390/nu7095380

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Duetz WA, Van Beilen JB, Witholt B (2001) Using proteins in their natural environment: potential and limitations of microbial whole-cell hydroxylations in applied biocatalysis. Curr Opin Biotechnol 12:419-425. https://doi.org/10.1016/S0958-1669(00)00237-8

    CAS  Article  PubMed  Google Scholar 

  13. Greenberg NA, Mahoney RR (1982) Production and characterization of β-galactosidase from Streptococcus thermophilus. J Food Sci 47:1824-1835. https://doi.org/10.1111/j.1365-2621.1982.tb12891.x

    CAS  Article  Google Scholar 

  14. Hols P, Hancy F, Fontaine L, Grossiord B, Prozzi D, Leblond-Bourget N, Decaris B, Bolotin A, Delorme C, Ehrlich SD, Guédon E, Monnet V, Renault P, Kleerebezem M (2005) New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics. FEMS Microbiol Rev 29:435-463. https://doi.org/10.1016/j.femsre.2005.04.008

    CAS  Article  PubMed  Google Scholar 

  15. Hutkins RW, Morris HA (1987) Carbohydrate metabolism by Streptococcus thermophilus: a review. J Food Prot 50:876-884. https://doi.org/10.4315/0362-028x-50.10.876

    CAS  Article  PubMed  Google Scholar 

  16. Hutkins RW, Ponne C (1991) Lactose uptake driven by galactose efflux in Streptococcus thermophilus: evidence for a galactose-lactose antiporter. Appl Environ Microbiol 57:941-944. https://doi.org/10.1128/aem.57.4.941-944.1991

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Ishizaki A, Osajima K, Nakamura K, Kimura K, Hara T, Ezaki T (1990) Biochemical characterization of Lactococcus lactis IO-1 whose optimal temperature is as high as 37°C. J Gen Appl Microbiol 36:1-6. https://doi.org/10.2323/jgam.36.1

    CAS  Article  Google Scholar 

  18. Israelsen H, Madsen SM, Vrang A, Hansen EB, Johansen E (1995) Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80. Appl Environ Microbiol 61:2540-2547. https://doi.org/10.1128/aem.61.7.2540-2547.1995

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Lin B, Tao Y (2017) Whole-cell biocatalysts by design. Microb Cell Factories 16:1-12. https://doi.org/10.1186/s12934-017-0724-7

    CAS  Article  Google Scholar 

  20. Liu J, Chan SHJ, Chen J, Solem C, Jensen PR (2019) Systems biology - a guide for understanding and developing improved strains of lactic acid bacteria. Front Microbiol 10:1-19. https://doi.org/10.3389/fmicb.2019.00876

    Article  PubMed  PubMed Central  Google Scholar 

  21. Liu J, Dantoft SH, Würtz A, Jensen PR, Solem C (2016) A novel cell factory for efficient production of ethanol from dairy waste. Biotechnol Biofuels 9:33. https://doi.org/10.1186/s13068-016-0448-7

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory 466

  23. Misselwitz B, Butter M, Verbeke K, Fox MR (2019) Update on lactose malabsorption and intolerance: pathogenesis, diagnosis and clinical management. Gut 68:2080-2091. https://doi.org/10.1136/gutjnl-2019-318404

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Mulligan CN, Safi BF, Groleau D (1991) Continuous production of ammonium lactate by Streptococcus cremoris in a three-stage reactor. Biotechnol Bioeng 38:1173-1181

    CAS  Article  Google Scholar 

  25. Nickerson TA (1957) Lactose crystallization in ice cream. III. Mode of action of milk powder in preventing sandiness. J. Dairy Sci 40:309-313. https://doi.org/10.3168/jds.S0022-0302(57)94478-8

    CAS  Article  Google Scholar 

  26. Nijpels HH (1981) Lactases and their applications, in: Enzymes and food processing. https://doi.org/10.1007/978-94-011-6740-6_6

  27. Panesar PS, Panesar R, Singh RS, Kennedy JF, Kumar H (2006) Microbial production, immobilization and applications of β-D-galactosidase. J Chem Technol Biotechnol 81:530-543. https://doi.org/10.1002/jctb.1453

    CAS  Article  Google Scholar 

  28. Regenstein JM, Chaudry MM, Regenstein CE (2003) Halal food laws. Compr Rev Food Sci Food Saf 2:111-127. https://doi.org/10.1111/j.1541-4337.2003.tb00018.x

    Article  Google Scholar 

  29. Ribeiro Júnior JC, de Oliveira AM, de Silva F, G, Tamanini R, de Oliveira ALM, Beloti V (2018) The main spoilage-related psychrotrophic bacteria in refrigerated raw milk. J Dairy Sci 101:75-83. https://doi.org/10.3168/jds.2017-13069

  30. Shah NO, Nickerson TA (1978) Functional properties of hydrolyzed lactose: relative sweetness. J Food Sci 43:1575-1576. https://doi.org/10.1111/j.1365-2621.1978.tb02546.x

    CAS  Article  Google Scholar 

  31. Shen J, Chen J, Jensen PR, Solem C (2019) Sweet as sugar - efficient conversion of lactose into sweet sugars using a novel whole-cell catalyst. J Agric Food Chem 67:6257-6262. https://doi.org/10.1021/acs.jafc.9b01529

    CAS  Article  PubMed  Google Scholar 

  32. Shin KC, Sim DH, Seo MJ, Oh DK (2016) Increased production of food-grade d -tagatose from d -galactose by permeabilized and immobilized cells of Corynebacterium glutamicum, a GRAS host, Expressing d-Galactose Isomerase from Geobacillus thermodenitrificans. J Agric Food Chem 64:8146-8153. https://doi.org/10.1021/acs.jafc.6b03588

    CAS  Article  PubMed  Google Scholar 

  33. Somkuti GA, Dominiecki ME, Steinberg DH (1998) Permeabilization of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus with ethanol. Curr Microbiol 36:202-206. https://doi.org/10.1007/s002849900294

    CAS  Article  PubMed  Google Scholar 

  34. Somkuti GA, Steinberg DH (1994) Permeabilization of Streptococcus thermophilus and the expression of beta-galactosidase. Enzym Microb Technol 16:573-576. https://doi.org/10.1016/0141-0229(94)90121-X

    CAS  Article  Google Scholar 

  35. Virolainen N, Guglielmetti S, Arioli S, Karp M (2012) Bioluminescence-based identification of nisin producers - a rapid and simple screening method for nisinogenic bacteria in food samples. Int J Food Microbiol 158:126-132

    CAS  Article  Google Scholar 

  36. Zadow JG (1986) Lactose hydrolysed dairy products. Food Technology in Australia 38:460-462

    CAS  Google Scholar 

  37. Zendo T, Yoneyama F, Sonomoto K (2010) Lactococcal membrane-permeabilizing antimicrobial peptides. Appl Microbiol Biotechnol 88:1-9. https://doi.org/10.1007/s00253-010-2764-3

    CAS  Article  PubMed  Google Scholar 

  38. Zolnere K, Ciprovica I (2017) The comparison of commercially available ß-galactosidases for dairy industry: review. Res Rural Dev 1:215-222. https://doi.org/10.22616/rrd.23.2017.032

    Article  Google Scholar 

Download references

Acknowledgments

We acknowledge Sacco Srl., Italy, for providing strains.

Funding

This work was supported by the China Scholarship Council (Grant No. 201706300117), the DTU PoC Fund (“Sweet as Sugar” project), the Danish Dairy Research Foundation (Optimering af smagsdannelse i hårde oste), and Innovation Fund Denmark (Grant No. 6150-00036B).

Author information

Affiliations

Authors

Contributions

PRJ, CS, JL, and MF conceived and designed research. QW and HX conducted experiments. SKL and SMR contributed dairy waste materials. CS, JL, and QW analyzed data and wrote the manuscript. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Ming-Tao Fan, Christian Solem or Jian-Ming Liu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wang, Q., Lillevang, S.K., Rydtoft, S.M. et al. No more cleaning up - Efficient lactic acid bacteria cell catalysts as a cost-efficient alternative to purified lactase enzymes. Appl Microbiol Biotechnol 104, 6315–6323 (2020). https://doi.org/10.1007/s00253-020-10655-3

Download citation

Keywords

  • Lactase
  • Streptococcus thermophilus
  • Nisin
  • Permeabilization
  • Lactose-free