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Recombinant production and characterization of Aspergillus niger prolyl endopeptidase enzyme for gluten-free food production

Yıl 2021, , 287 - 293, 15.09.2021
https://doi.org/10.31015/jaefs.2021.3.5

Öz

Gluten is a protein group found in wheat, barley, rye, and oats, known as cereals. When this vegetable protein is introduced into the body, celiac disease can occurs. The use of bacterial and fungal oligopeptidase to ensure the cleavage of gluten into non-toxic fragments are considered a promising alternative for celiac disease. In this study, the Aspergillus niger Prolyl EndoPeptidase (AN-PEP) enzyme was cloned into pET22b vector and recombinantly produced in BL21 (DE3) pLysE cells. PEP enzyme expressed as inclusion body and was recovered by refolding. And N-terminal His-tagged recombinant protein was purified by nickel affinity chromatography. 280 mg AN-PEP enzyme from 1L bacterial culture was purified at very high yield, and this protein was 90% purity. As a result; It has been determined that the recombinantly produced PEP enzyme can digest gluten. This study shows that recombinantly produced AN-PEP (rAN-PEP) has great potential to use in the production processes of gluten-free foods.

Destekleyen Kurum

Tokat Gaziosmanpaşa University Research Fund under

Proje Numarası

2018/32.

Teşekkür

This work was supported by the Tokat Gaziosmanpaşa University Research Fund under Grant number:2018/32.

Kaynakça

  • Babaeipour, V., Abbas, M. P., Sahebnazar, Z., & Alizadeh, R. (2010). Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation. Bioprocess Biosyst Eng, 33(5), 591-598. Doi: https://doi.org/10.1007/s00449-009-0380-3
  • Ciacci, C., Ciclitira, P., Hadjivassiliou, M., Kaukinen, K., Ludvigsson, J. F., McGough, N., Sanders, D. S., Woodward, J., Leonard, J. N., & Swift, G. L. (2015). The gluten-free diet and its current application in coeliac disease and dermatitis herpetiformis. United European Gastroenterol J, 3(2), 121-135.Doi: https://doi.org/10.1177/2050640614559263
  • Comino, I., Fernandez-Banares, F., Esteve, M., Ortigosa, L., Castillejo, G., Fambuena, B., Ribes-Koninckx, C., Sierra, C., Rodriguez-Herrera, A., Salazar, J. C., Caunedo, A., Marugan-Miguelsanz, J. M., Garrote, J. A., Vivas, S., Lo Iacono, O., Nunez, A., Vaquero, L., Vegas, A. M., Crespo, L., Fernandez-Salazar, L., Arranz, E., Jimenez-Garcia, V. A., Antonio Montes-Cano, M., Espin, B., Galera, A., Valverde, J., Giron, F. J., Bolonio, M., Millan, A., Cerezo, F. M. (2016). Fecal Gluten Peptides Reveal Limitations of Serological Tests and Food Questionnaires for Monitoring Gluten-Free Diet in Celiac Disease Patients. Am J Gastroenterol, 111(10), 1456-1465. Doi: https://doi.org/10.1038/ajg.2016.439
  • Di Sabatino, A., & Corazza, G. R. (2009). Coeliac disease. Lancet, 373(9673), 1480-1493. Doi: https://doi.org/10.1016/S0140-6736(09)60254-3
  • Edens, L., Dekker, P., van der Hoeven, R., Deen, F., de Roos, A., & Floris, R. (2005). Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates. J Agric Food Chem, 53(20), 7950-7957. Doi: https://doi.org/10.1021/jf050652c
  • Ehren, J., Moron, B., Martin, E., Bethune, M. T., Gray, G. M., & Khosla, C. (2009). A food-grade enzyme preparation with modest gluten detoxification properties. PLoS One, 4(7), e6313. Doi: https://doi.org/10.1371/journal.pone.0006313
  • Elli, L., Branchi, F., Tomba, C., Villalta, D., Norsa, L., Ferretti, F., Roncoroni, L., & Bardella, M. T. (2015). Diagnosis of gluten related disorders: Celiac disease, wheat allergy and non-celiac gluten sensitivity. World J Gastroenterol, 21(23), 7110-7119. Doi: https://doi.org/10.3748/wjg.v21.i23.7110
  • Gass, J., Bethune, M. T., Siegel, M., Spencer, A., & Khosla, C. (2007). Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue. Gastroenterology, 133(2), 472-480. Doi: https://doi.org/10.1053/j.gastro.2007.05.028
  • Gessendorfer, B., Hartmann, G., Wieser, H., Koehler, P. (2011). Determination of celiac disease-specific peptidase activity of germinated cereals. European Food Research and Technology, 232, 205–209. Doi: https://doi.org/10.1007/s00217-010-1375-7
  • Helmerhorst, E. J., Zamakhchari, M., Schuppan, D., & Oppenheim, F. G. (2010, Oct 11). Discovery of a novel and rich source of gluten-degrading microbial enzymes in the oral cavity. PLoS One, 5(10), e13264. Doi: https://doi.org/10.1371/journal.pone.0013264
  • Jiang, B., Wang, M., Wang, X., Wu, S., Li, D., Liu, C., Feng, Z., & Li, J. (2021). Effective separation of prolyl endopeptidase from Aspergillus Niger by aqueous two phase system and its characterization and application. Int J Biol Macromol, 169, 384-395. Doi: https://doi.org/10.1016/j.ijbiomac.2020.12.120
  • Kaplan, Ö., İmamoğlu, R., Şahingöz, İ., Gökçe, İ. (2021). Recombinant production of Thermus aquaticus single-strand binding protein for usage as PCR enhancer. Int. Adv. Res. Eng. J., 5(1), 42-46. Doi: https://doi.org/10.35860/iarej.766741
  • Krishnareddy, S., Stier, K., Recanati, M., Lebwohl, B., & Green, P. H. (2017). Commercially available glutenases: a potential hazard in coeliac disease. Therap Adv Gastroenterol, 10(6), 473-481. Doi: https://doi.org/10.1177/1756283X17690991
  • Kuduğ, H., Ataman, B., İmamoğlu, R., Düzgün, D., Gökçe, İ. (2019). Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization. Int. Adv. Res. Eng. J., 3(1), 20-25. Retrieved from https://dergipark.org.tr/en/pub/iarej/issue/44303/429547
  • Lopez, M., & Edens, L. (2005). Effective prevention of chill-haze in beer using an acid proline-specific endoprotease from Aspergillus niger. J Agric Food Chem, 53(20), 7944-7949. Doi: https://doi.org/10.1021/jf0506535
  • Loponen, J., Kanerva, P., Zhang, C., Sontag-Strohm, T., Salovaara, H., & Ganzle, M. G. (2009). Prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs determined by chromatographic and immunological methods. J Agric Food Chem, 57(2), 746-753. Doi: https://doi.org/10.1021/jf803243w
  • Melissis, S. C., Papageorgiou, A. C., Labrou, N. E., & Clonis, Y. D. (2010). Purification of M-MLVH- RT on a 9-aminoethyladenine-(1,6-diamine-hexane)-triazine selected from a combinatorial library of dNTP-mimetic ligands. J Chromatogr Sci, 48(6), 496-502. Doi: https://doi.org/10.1093/chromsci/48.6.496
  • Moreno, M. L., Cebolla, A., Munoz-Suano, A., Carrillo-Carrion, C., Comino, I., Pizarro, A., Leon, F., Rodriguez-Herrera, A., & Sousa, C. (2017). Detection of gluten immunogenic peptides in the urine of patients with coeliac disease reveals transgressions in the gluten-free diet and incomplete mucosal healing. Gut, 66(2), 250-257. Doi: https://doi.org/10.1136/gutjnl-2015-310148
  • Ortiz, C., Valenzuela, R., & Lucero, A. Y. (2017). Celiac disease, non celiac gluten sensitivity and wheat allergy: comparison of 3 different diseases triggered by the same food. Rev Chil Pediatr, 88(3), 417-423. Doi: https://doi.org/10.4067/S0370-41062017000300017
  • Palmer, I., & Wingfield, P. T. (2012). Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Curr Protoc Protein Sci, Chapter 6, Unit6 3. Doi: https://doi.org/10.1002/0471140864.ps0603s70
  • Rodrigues, M., Yonamine, G. H., & Fernandes Satiro, C. A. (2018). Rate and determinants of non-adherence to a gluten-free diet and nutritional status assessment in children and adolescents with celiac disease in a tertiary Brazilian referral center: a cross-sectional and retrospective study. BMC Gastroenterol, 18(1), 15. Doi: https://doi.org/10.1186/s12876-018-0740-z
  • Schwalb, T., Wieser, H., Koehler, P. (2012). Studies on the gluten-specificpeptidase activity of germinated grains from different cereal species and cultivars. European Food Research and Technology, 235,1161–1170.
  • Sebela, M., Rehulka, P., Kabrt, J., Rehulkova, H., Ozdian, T., Raus, M., Franc, V., & Chmelik, J. (2009). Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics. J Mass Spectrom, 44(11), 1587-1595. Doi: https://doi.org/10.1002/jms.1667
  • Shan, L., Marti, T., Sollid, L. M., Gray, G. M., & Khosla, C. (2004). Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue. Biochem J, 383(Pt 2), 311-318. Doi: https://doi.org/10.1042/BJ20040907
  • Siegel, M., Bethune, M. T., Gass, J., Ehren, J., Xia, J., Johannsen, A., Stuge, T. B., Gray, G. M., Lee, P. P., & Khosla, C. (2006). Rational design of combination enzyme therapy for celiac sprue. Chem Biol, 13(6), 649-658. Doi: https://doi.org/10.1016/j.chembiol.2006.04.009
  • Stepniak, D., Spaenij-Dekking, L., Mitea, C., Moester, M., de Ru, A., Baak-Pablo, R., van Veelen, P., Edens, L., & Koning, F. (2006). Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease. Am J Physiol Gastrointest Liver Physiol, 291(4), G621-629. Doi: https://doi.org/10.1152/ajpgi.00034.2006
  • Tye-Din, J. A., Anderson, R. P., Ffrench, R. A., Brown, G. J., Hodsman, P., Siegel, M., Botwick, W., & Shreeniwas, R. (2010). The effects of ALV003 pre-digestion of gluten on immune response and symptoms in celiac disease in vivo. Clin Immunol, 134(3), 289-295. Doi: https://doi.org/10.1016/j.clim.2009.11.001
  • Vallejo, L. F., & Rinas, U. (2004). Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Fact, 3(1), 11. Doi: https://doi.org/10.1186/1475-2859-3-11
  • Walter, T., Wieser, H., Koehler, P. (2015). Degradation of gluten in rye sourdough products by means of a proline-specific peptidase. . European Food Research and Technology, 240, 517–524. Doi: https://doi.org/10.1007/s00217-014-2350-5
  • Wei, G., Helmerhorst, E. J., Darwish, G., Blumenkranz, G., & Schuppan, D. (2020). Gluten Degrading Enzymes for Treatment of Celiac Disease. Nutrients, 12(7). Doi: https://doi.org/10.3390/nu12072095
  • Wei, G., Tian, N., Valery, A. C., Zhong, Y., Schuppan, D., & Helmerhorst, E. J. (2015). Identification of Pseudolysin (lasB) as an Aciduric Gluten-Degrading Enzyme with High Therapeutic Potential for Celiac Disease. Am J Gastroenterol, 110(6), 899-908. Doi: https://doi.org/10.1038/ajg.2015.97
  • Zamakhchari, M., Wei, G., Dewhirst, F., Lee, J., Schuppan, D., Oppenheim, F. G., & Helmerhorst, E. J. (2011). Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract. PLoS One, 6(9), e24455. Doi: https://doi.org/10.1371/journal.pone.0024455
Yıl 2021, , 287 - 293, 15.09.2021
https://doi.org/10.31015/jaefs.2021.3.5

Öz

Proje Numarası

2018/32.

Kaynakça

  • Babaeipour, V., Abbas, M. P., Sahebnazar, Z., & Alizadeh, R. (2010). Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation. Bioprocess Biosyst Eng, 33(5), 591-598. Doi: https://doi.org/10.1007/s00449-009-0380-3
  • Ciacci, C., Ciclitira, P., Hadjivassiliou, M., Kaukinen, K., Ludvigsson, J. F., McGough, N., Sanders, D. S., Woodward, J., Leonard, J. N., & Swift, G. L. (2015). The gluten-free diet and its current application in coeliac disease and dermatitis herpetiformis. United European Gastroenterol J, 3(2), 121-135.Doi: https://doi.org/10.1177/2050640614559263
  • Comino, I., Fernandez-Banares, F., Esteve, M., Ortigosa, L., Castillejo, G., Fambuena, B., Ribes-Koninckx, C., Sierra, C., Rodriguez-Herrera, A., Salazar, J. C., Caunedo, A., Marugan-Miguelsanz, J. M., Garrote, J. A., Vivas, S., Lo Iacono, O., Nunez, A., Vaquero, L., Vegas, A. M., Crespo, L., Fernandez-Salazar, L., Arranz, E., Jimenez-Garcia, V. A., Antonio Montes-Cano, M., Espin, B., Galera, A., Valverde, J., Giron, F. J., Bolonio, M., Millan, A., Cerezo, F. M. (2016). Fecal Gluten Peptides Reveal Limitations of Serological Tests and Food Questionnaires for Monitoring Gluten-Free Diet in Celiac Disease Patients. Am J Gastroenterol, 111(10), 1456-1465. Doi: https://doi.org/10.1038/ajg.2016.439
  • Di Sabatino, A., & Corazza, G. R. (2009). Coeliac disease. Lancet, 373(9673), 1480-1493. Doi: https://doi.org/10.1016/S0140-6736(09)60254-3
  • Edens, L., Dekker, P., van der Hoeven, R., Deen, F., de Roos, A., & Floris, R. (2005). Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates. J Agric Food Chem, 53(20), 7950-7957. Doi: https://doi.org/10.1021/jf050652c
  • Ehren, J., Moron, B., Martin, E., Bethune, M. T., Gray, G. M., & Khosla, C. (2009). A food-grade enzyme preparation with modest gluten detoxification properties. PLoS One, 4(7), e6313. Doi: https://doi.org/10.1371/journal.pone.0006313
  • Elli, L., Branchi, F., Tomba, C., Villalta, D., Norsa, L., Ferretti, F., Roncoroni, L., & Bardella, M. T. (2015). Diagnosis of gluten related disorders: Celiac disease, wheat allergy and non-celiac gluten sensitivity. World J Gastroenterol, 21(23), 7110-7119. Doi: https://doi.org/10.3748/wjg.v21.i23.7110
  • Gass, J., Bethune, M. T., Siegel, M., Spencer, A., & Khosla, C. (2007). Combination enzyme therapy for gastric digestion of dietary gluten in patients with celiac sprue. Gastroenterology, 133(2), 472-480. Doi: https://doi.org/10.1053/j.gastro.2007.05.028
  • Gessendorfer, B., Hartmann, G., Wieser, H., Koehler, P. (2011). Determination of celiac disease-specific peptidase activity of germinated cereals. European Food Research and Technology, 232, 205–209. Doi: https://doi.org/10.1007/s00217-010-1375-7
  • Helmerhorst, E. J., Zamakhchari, M., Schuppan, D., & Oppenheim, F. G. (2010, Oct 11). Discovery of a novel and rich source of gluten-degrading microbial enzymes in the oral cavity. PLoS One, 5(10), e13264. Doi: https://doi.org/10.1371/journal.pone.0013264
  • Jiang, B., Wang, M., Wang, X., Wu, S., Li, D., Liu, C., Feng, Z., & Li, J. (2021). Effective separation of prolyl endopeptidase from Aspergillus Niger by aqueous two phase system and its characterization and application. Int J Biol Macromol, 169, 384-395. Doi: https://doi.org/10.1016/j.ijbiomac.2020.12.120
  • Kaplan, Ö., İmamoğlu, R., Şahingöz, İ., Gökçe, İ. (2021). Recombinant production of Thermus aquaticus single-strand binding protein for usage as PCR enhancer. Int. Adv. Res. Eng. J., 5(1), 42-46. Doi: https://doi.org/10.35860/iarej.766741
  • Krishnareddy, S., Stier, K., Recanati, M., Lebwohl, B., & Green, P. H. (2017). Commercially available glutenases: a potential hazard in coeliac disease. Therap Adv Gastroenterol, 10(6), 473-481. Doi: https://doi.org/10.1177/1756283X17690991
  • Kuduğ, H., Ataman, B., İmamoğlu, R., Düzgün, D., Gökçe, İ. (2019). Production of red fluorescent protein (mCherry) in an inducible E. coli expression system in a bioreactor, purification and characterization. Int. Adv. Res. Eng. J., 3(1), 20-25. Retrieved from https://dergipark.org.tr/en/pub/iarej/issue/44303/429547
  • Lopez, M., & Edens, L. (2005). Effective prevention of chill-haze in beer using an acid proline-specific endoprotease from Aspergillus niger. J Agric Food Chem, 53(20), 7944-7949. Doi: https://doi.org/10.1021/jf0506535
  • Loponen, J., Kanerva, P., Zhang, C., Sontag-Strohm, T., Salovaara, H., & Ganzle, M. G. (2009). Prolamin hydrolysis and pentosan solubilization in germinated-rye sourdoughs determined by chromatographic and immunological methods. J Agric Food Chem, 57(2), 746-753. Doi: https://doi.org/10.1021/jf803243w
  • Melissis, S. C., Papageorgiou, A. C., Labrou, N. E., & Clonis, Y. D. (2010). Purification of M-MLVH- RT on a 9-aminoethyladenine-(1,6-diamine-hexane)-triazine selected from a combinatorial library of dNTP-mimetic ligands. J Chromatogr Sci, 48(6), 496-502. Doi: https://doi.org/10.1093/chromsci/48.6.496
  • Moreno, M. L., Cebolla, A., Munoz-Suano, A., Carrillo-Carrion, C., Comino, I., Pizarro, A., Leon, F., Rodriguez-Herrera, A., & Sousa, C. (2017). Detection of gluten immunogenic peptides in the urine of patients with coeliac disease reveals transgressions in the gluten-free diet and incomplete mucosal healing. Gut, 66(2), 250-257. Doi: https://doi.org/10.1136/gutjnl-2015-310148
  • Ortiz, C., Valenzuela, R., & Lucero, A. Y. (2017). Celiac disease, non celiac gluten sensitivity and wheat allergy: comparison of 3 different diseases triggered by the same food. Rev Chil Pediatr, 88(3), 417-423. Doi: https://doi.org/10.4067/S0370-41062017000300017
  • Palmer, I., & Wingfield, P. T. (2012). Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Curr Protoc Protein Sci, Chapter 6, Unit6 3. Doi: https://doi.org/10.1002/0471140864.ps0603s70
  • Rodrigues, M., Yonamine, G. H., & Fernandes Satiro, C. A. (2018). Rate and determinants of non-adherence to a gluten-free diet and nutritional status assessment in children and adolescents with celiac disease in a tertiary Brazilian referral center: a cross-sectional and retrospective study. BMC Gastroenterol, 18(1), 15. Doi: https://doi.org/10.1186/s12876-018-0740-z
  • Schwalb, T., Wieser, H., Koehler, P. (2012). Studies on the gluten-specificpeptidase activity of germinated grains from different cereal species and cultivars. European Food Research and Technology, 235,1161–1170.
  • Sebela, M., Rehulka, P., Kabrt, J., Rehulkova, H., Ozdian, T., Raus, M., Franc, V., & Chmelik, J. (2009). Identification of N-glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics. J Mass Spectrom, 44(11), 1587-1595. Doi: https://doi.org/10.1002/jms.1667
  • Shan, L., Marti, T., Sollid, L. M., Gray, G. M., & Khosla, C. (2004). Comparative biochemical analysis of three bacterial prolyl endopeptidases: implications for coeliac sprue. Biochem J, 383(Pt 2), 311-318. Doi: https://doi.org/10.1042/BJ20040907
  • Siegel, M., Bethune, M. T., Gass, J., Ehren, J., Xia, J., Johannsen, A., Stuge, T. B., Gray, G. M., Lee, P. P., & Khosla, C. (2006). Rational design of combination enzyme therapy for celiac sprue. Chem Biol, 13(6), 649-658. Doi: https://doi.org/10.1016/j.chembiol.2006.04.009
  • Stepniak, D., Spaenij-Dekking, L., Mitea, C., Moester, M., de Ru, A., Baak-Pablo, R., van Veelen, P., Edens, L., & Koning, F. (2006). Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease. Am J Physiol Gastrointest Liver Physiol, 291(4), G621-629. Doi: https://doi.org/10.1152/ajpgi.00034.2006
  • Tye-Din, J. A., Anderson, R. P., Ffrench, R. A., Brown, G. J., Hodsman, P., Siegel, M., Botwick, W., & Shreeniwas, R. (2010). The effects of ALV003 pre-digestion of gluten on immune response and symptoms in celiac disease in vivo. Clin Immunol, 134(3), 289-295. Doi: https://doi.org/10.1016/j.clim.2009.11.001
  • Vallejo, L. F., & Rinas, U. (2004). Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Fact, 3(1), 11. Doi: https://doi.org/10.1186/1475-2859-3-11
  • Walter, T., Wieser, H., Koehler, P. (2015). Degradation of gluten in rye sourdough products by means of a proline-specific peptidase. . European Food Research and Technology, 240, 517–524. Doi: https://doi.org/10.1007/s00217-014-2350-5
  • Wei, G., Helmerhorst, E. J., Darwish, G., Blumenkranz, G., & Schuppan, D. (2020). Gluten Degrading Enzymes for Treatment of Celiac Disease. Nutrients, 12(7). Doi: https://doi.org/10.3390/nu12072095
  • Wei, G., Tian, N., Valery, A. C., Zhong, Y., Schuppan, D., & Helmerhorst, E. J. (2015). Identification of Pseudolysin (lasB) as an Aciduric Gluten-Degrading Enzyme with High Therapeutic Potential for Celiac Disease. Am J Gastroenterol, 110(6), 899-908. Doi: https://doi.org/10.1038/ajg.2015.97
  • Zamakhchari, M., Wei, G., Dewhirst, F., Lee, J., Schuppan, D., Oppenheim, F. G., & Helmerhorst, E. J. (2011). Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract. PLoS One, 6(9), e24455. Doi: https://doi.org/10.1371/journal.pone.0024455
Toplam 32 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Gıda Mühendisliği
Bölüm Makaleler
Yazarlar

Belma Şenol Bu kişi benim 0000-0003-3021-0087

Özlem Kaplan 0000-0002-3052-4556

Rizvan İmamoğlu 0000-0002-6306-4760

İsa Gökçe 0000-0002-5023-9947

Proje Numarası 2018/32.
Yayımlanma Tarihi 15 Eylül 2021
Gönderilme Tarihi 10 Şubat 2021
Kabul Tarihi 4 Haziran 2021
Yayımlandığı Sayı Yıl 2021

Kaynak Göster

APA Şenol, B., Kaplan, Ö., İmamoğlu, R., Gökçe, İ. (2021). Recombinant production and characterization of Aspergillus niger prolyl endopeptidase enzyme for gluten-free food production. International Journal of Agriculture Environment and Food Sciences, 5(3), 287-293. https://doi.org/10.31015/jaefs.2021.3.5

by-nc.png

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