Isolation, Selection, and Cultivation of Antagonistic Lactic Acid Bacteria Against Postharvest Pathogenic Fungi in Grapes
Main Article Content
Abstract
Incorporation of probiotics with edible coatings to preserve and improve the shelf life of fresh fruits has gained more popularity in the food industry. This study aimed to isolate, select lactic acid bacteria (LAB) that are antagonistic to postharvest pathogenic fungi in grapes (Vitis vinifera L.), and determine the optimal conditions for their biomass production. Fungi were isolated from decaying grapes on Potato Dextrose Agar medium (PDA) supplemented with 1% Chloramphenicol at 25 ºC for five to seven days. LAB were isolated on MRS agar medium at 37 ºC for two to three days. Isolates with antifungal capability were selected using the overlay technique. LAB were cultured in MRS broth at pH 4-8, 25-40 ºC for two to five days. Effects of pH and temperature on the antifungal activity of cell-free supernatant (CFS) were investigated at respectively pHs of 4.5 to 6.5 and temperatures of 60, 80 and 100 ºC for 2 hours. We identified and selected the fungal strain Nn4 with high infection density (1.5×105 CFU/g) to be the grape postharvest pathogenic fungus of interest. For antagonistic bacteria, we selected the lactic acid bacterial strain Ln9 with the highest antifungal activity against strain Nn4 (37.9 mm in diameter). The suitable cultivation conditions for biomass production of strain Ln9 were at pH 5.0, 30 ºC for 2 days. The antifungal activity of CFS was drastically reduced when the medium pH was adjusted to 6.5 but not reduced after heat treatment. It is evidential that the antifungal properties of probiotics can be related to the production of organic acids.
Keywords:
Antifungal activity, probiotics, postharvest pathogenic fungi, grapes, lactic acid bacteria.
References
[1] K. Ghafoor, J. Park, Y. Choi, Optimization of Supercritical Fluid Extraction of Bioactive Compounds from Grape (Vitis labrusca B.) Peel by using Response Surface Methodology, Innovative Food Science and Emerging Technologies,
Vol. 11, No. 3, 2010, pp. 485-490, https://doi.org/10.1016/j.ifset.2010.01.013.
[2] H. S. Mengal, M. A. Abro, G. H. Jatoi, L. Nawab, G. B. Poussio, N. Ahmed, A. Q. Zehri, A. Ali, Efficacy of Different Fungicides, Botanical Extracts and Bio-Control Agents Against Cladosporium Cladosporioides, the Causal Agent of Cladosporium Rot in Grapes, Acta Ecologica Sinica, Vol. 40, No. 4, 2020, pp. 300-305, https://doi.org/10.1016/j.chnaes.2019.08.002.
[3] H. Aloui, K. Khwaldia, L. S. Gonzalez,
L. Muneret, C. Jeandel, M. Hamdi, S. Desobry, Alginate Coatings Containing Grapefruit Essential Oil or Grapefruit Seed Extract or Grapes Preservation, International Journal of Food Science and Technology, Vol. 49, No. 4, 2014, pp. 952-959, https://doi.org/10.1111/ijfs.12387.
[4] A. F. Stocco, M. E. Diaz, M. C. R. Romera, L. A. Mercado, M. L. Rivero, M. L. Ponsone, Biocontrol of Postharvest Alternaria Decay in Table Grapes from Mendoza Province, Biological Control,
Vol. 134, 2019, pp. 114-122, https://doi.org/10.1016/j.biocontrol.2019.03.019.
[5] L. Palou, M. Serrano, D. M. Romero, D. Valero, New Approaches for Postharvest Quality Retention of Table Grapes, Fresh Produce, Vol. 4, 2010,
pp. 103-110.
[6] B. Yousuf, O. S. Qadri, A. K. Srivastava, Recent Developments in Shelf-Life Extension of Fresh-Cut Fruits and Vegetables by Application of Different Edible Coatings: A Review, LWT- Food Science and Technology, Vol. 89, 2018, pp. 198-209, https://doi.org/10.1016/j.lwt.2017.10.051.
[7] F. Pavli, C. Tassou, G. J. E. Nychas,
N. Chorianopoulos, Probiotic Incorporation in Edible Films and Coatings: Bioactive Solution for Functional Foods, International Journal of Molecular Sciences, Vol. 19, No. 1, 2018, pp. 150, https://doi.org/10.3390/ijms19010150.
[8] R. Massoud, D. Khodaeii, Z. H. Esfahani,
K. Khosravi-Darani, The Effect of Edible Probiotic Coating on Quality of Fresh Fruits and Vegetables: Fresh Strawberries as a Case Study, Biomass Conversion and Biorefinery, Vol. 4, 2021,
pp. 2517-2526, https://doi.org/10.1007/s13399-021-01332-0.
[9] A. Guimarães, A. Venancio, L. Abrunhosa, Antifungal Effect of Organic Acids from Lactic Acid Bacteria on Penicillium nordicum, Food Additives & Contaminants: Part A: Chemistry, Analysis, Control, Exposure & Risk Assessment, Vol. 35, No. 9, 2018, pp. 1803-1818, https://doi.org/10.1080/19440049.2018.1500718.
[10] A. Marín, A. Plotto, L. Atarés, A. Chiralt, Lactic Acid Bacteria Incorporated into Edible Coatings to Control Fungal Growth and Maintain Postharvest Quality of Grapes, HortScience, Vol. 54, No. 2, 2019, pp. 337-343, https://doi.org/10.21273/HORTSCI13661-18.
[11] DARD, The Situation of Grape Development in Ninh Thuan Province, 4/2023 (in Vietnamese).
[12] MOST, TCVN 9017: 2011-Vietnamese Standard on Fresh Fruits: Sampling Method on the Field
(in Vietnamese).
[13] MOST, TCVN 82751: 2010 (ISO 21527-2: 2008)- Vietnamese Standard on Microbiology of Food and Animal Feeding Stuffs: Horizontal Method for The Enumeration of Yeasts and Moulds - Part 1: Colony Count Technique In Products With Water Activity Less Than or Equal to 0,95
(in Vietnamese).
[14] D. Solairaj, N. N. G. Legrand, Q. Yang, H. Zhang, Isolation of Pathogenic Fungi Causing Postharvest Decay in Table Grapes and In Vivo Biocontrol Activity of Selected Yeasts Against Them, Physiological and Molecular Plant Pathology,
Vol. 110, 2020, pp. 101478, https://doi.org/10.1016/j.pmpp.2020.101478.
[15] R. Koch, Investigations into Bacteria: V. The Etiology of Anthrax, Based on the Ontogenesis of Bacillus anthracis, Cohns Beitrage zur Biologie der Pflanzen (in German), Vol. 2, No. 2, 1876,
pp. 277-310.
[16] E. B. Breitschwerdt, K. L. Linder, M. J. Day, R. G. Maggi, B. B. Chomel, V. A. J. Kempf, Koch's Postulates and the Pathogenesis of Comparative Infectious Disease Causation Associated with Bartonella Species, Journal of Comparative Pathology, Vol. 148, No. 2, 2013, pp. 115-125, https://doi.org/10.1016/j.jcpa.2012.12.003.
[17] R. Trias, L. Baneras, E. Montesinos, E. Badosa, Lactic Acid Bacteria From Fresh Fruit and Vegetables As Biocontrol Agents of Phytopathogenic Bacteria and Fungi, International Microbiology, Vol. 11, No. 4, 2008, pp. 231-236, https://doi.org/10.2436/20.1501.01.66.
[18] American Society for Microbiology, Catalase Test Protocol, https://asm.org/Protocols/Catalase-Test-Protocol, 2010 (accessed on: August 1st, 2023).
[19] American Society for Microbiology, Oxidase test protocol, https://asm.org/Protocols/Oxidase-Test-Protocol, 2010 (accessed on: August 1st, 2023).
[20] B. Taroub, L. Salma, Z. Manel, H. I. Ouzari,
Z. Hamdi, H. Moktar, Isolation of Lactic Acid Bacteria From Grape Fruit: Antifungal Activities, Probiotic Properties, and In Vitro Detoxification of Ochratoxin A, Annals of Microbiology, Vol. 69, No. 3, 2019, pp. 17-27, http://dx.doi.org/10.1007/s13213-018-1359-6.
[21] J. Sambrook, D. Russell, Molecular Cloning: A Laboratory Manual, 3rd edn, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.
[22] M. Lorenzini, B. Simonato, F. Favati, P. Bernardi, A. Sbarbati, G. Zapparoli, Filamentous Fungi Associated with Natural Infection of Noble Rot on Withered Grapes, International Journal of Food Microbiology, Vol. 272, 2018, pp. 83-86, https://doi.org/10.1016/j.ijfoodmicro.2018.03.004.
[23] U. Schillinger, F. K. Lüke, Identification of Lactobacilli From Meat and Meat Products, Food Microbiology, Vol. 4, No. 3, 1987, pp. 199-208, https://doi.org/10.1016/0740-0020(87)90002-5.
[24] S. Zhao, X. Hao, F. Yang, Y. Wang, X. Fan,
Y. Wang, Antifungal Activity of Lactobacillus plantarum ZZUA493 and Its Application to Extend The Shelf Life of Chinese Steamed Buns, Foods, Vol. 11, No. 2, 2022, pp. 195, https://doi.org/10.3390/foods11020195.
[25] F. A. Sadiq, B. Yan, F. Tian, J. Zhao, H. Zhang,
W. Chen, Lactic Acid Bacteria as Antifungal
and Anti-Mycotoxigenic Agents: a Comprehensive Review, Comprehensive Reviews in Food
Science and Food Safety, Vol. 18, No. 5, 2019,
pp. 1403-1436,
https://doi.org/10.1111/1541-4337.12481.
Vol. 11, No. 3, 2010, pp. 485-490, https://doi.org/10.1016/j.ifset.2010.01.013.
[2] H. S. Mengal, M. A. Abro, G. H. Jatoi, L. Nawab, G. B. Poussio, N. Ahmed, A. Q. Zehri, A. Ali, Efficacy of Different Fungicides, Botanical Extracts and Bio-Control Agents Against Cladosporium Cladosporioides, the Causal Agent of Cladosporium Rot in Grapes, Acta Ecologica Sinica, Vol. 40, No. 4, 2020, pp. 300-305, https://doi.org/10.1016/j.chnaes.2019.08.002.
[3] H. Aloui, K. Khwaldia, L. S. Gonzalez,
L. Muneret, C. Jeandel, M. Hamdi, S. Desobry, Alginate Coatings Containing Grapefruit Essential Oil or Grapefruit Seed Extract or Grapes Preservation, International Journal of Food Science and Technology, Vol. 49, No. 4, 2014, pp. 952-959, https://doi.org/10.1111/ijfs.12387.
[4] A. F. Stocco, M. E. Diaz, M. C. R. Romera, L. A. Mercado, M. L. Rivero, M. L. Ponsone, Biocontrol of Postharvest Alternaria Decay in Table Grapes from Mendoza Province, Biological Control,
Vol. 134, 2019, pp. 114-122, https://doi.org/10.1016/j.biocontrol.2019.03.019.
[5] L. Palou, M. Serrano, D. M. Romero, D. Valero, New Approaches for Postharvest Quality Retention of Table Grapes, Fresh Produce, Vol. 4, 2010,
pp. 103-110.
[6] B. Yousuf, O. S. Qadri, A. K. Srivastava, Recent Developments in Shelf-Life Extension of Fresh-Cut Fruits and Vegetables by Application of Different Edible Coatings: A Review, LWT- Food Science and Technology, Vol. 89, 2018, pp. 198-209, https://doi.org/10.1016/j.lwt.2017.10.051.
[7] F. Pavli, C. Tassou, G. J. E. Nychas,
N. Chorianopoulos, Probiotic Incorporation in Edible Films and Coatings: Bioactive Solution for Functional Foods, International Journal of Molecular Sciences, Vol. 19, No. 1, 2018, pp. 150, https://doi.org/10.3390/ijms19010150.
[8] R. Massoud, D. Khodaeii, Z. H. Esfahani,
K. Khosravi-Darani, The Effect of Edible Probiotic Coating on Quality of Fresh Fruits and Vegetables: Fresh Strawberries as a Case Study, Biomass Conversion and Biorefinery, Vol. 4, 2021,
pp. 2517-2526, https://doi.org/10.1007/s13399-021-01332-0.
[9] A. Guimarães, A. Venancio, L. Abrunhosa, Antifungal Effect of Organic Acids from Lactic Acid Bacteria on Penicillium nordicum, Food Additives & Contaminants: Part A: Chemistry, Analysis, Control, Exposure & Risk Assessment, Vol. 35, No. 9, 2018, pp. 1803-1818, https://doi.org/10.1080/19440049.2018.1500718.
[10] A. Marín, A. Plotto, L. Atarés, A. Chiralt, Lactic Acid Bacteria Incorporated into Edible Coatings to Control Fungal Growth and Maintain Postharvest Quality of Grapes, HortScience, Vol. 54, No. 2, 2019, pp. 337-343, https://doi.org/10.21273/HORTSCI13661-18.
[11] DARD, The Situation of Grape Development in Ninh Thuan Province, 4/2023 (in Vietnamese).
[12] MOST, TCVN 9017: 2011-Vietnamese Standard on Fresh Fruits: Sampling Method on the Field
(in Vietnamese).
[13] MOST, TCVN 82751: 2010 (ISO 21527-2: 2008)- Vietnamese Standard on Microbiology of Food and Animal Feeding Stuffs: Horizontal Method for The Enumeration of Yeasts and Moulds - Part 1: Colony Count Technique In Products With Water Activity Less Than or Equal to 0,95
(in Vietnamese).
[14] D. Solairaj, N. N. G. Legrand, Q. Yang, H. Zhang, Isolation of Pathogenic Fungi Causing Postharvest Decay in Table Grapes and In Vivo Biocontrol Activity of Selected Yeasts Against Them, Physiological and Molecular Plant Pathology,
Vol. 110, 2020, pp. 101478, https://doi.org/10.1016/j.pmpp.2020.101478.
[15] R. Koch, Investigations into Bacteria: V. The Etiology of Anthrax, Based on the Ontogenesis of Bacillus anthracis, Cohns Beitrage zur Biologie der Pflanzen (in German), Vol. 2, No. 2, 1876,
pp. 277-310.
[16] E. B. Breitschwerdt, K. L. Linder, M. J. Day, R. G. Maggi, B. B. Chomel, V. A. J. Kempf, Koch's Postulates and the Pathogenesis of Comparative Infectious Disease Causation Associated with Bartonella Species, Journal of Comparative Pathology, Vol. 148, No. 2, 2013, pp. 115-125, https://doi.org/10.1016/j.jcpa.2012.12.003.
[17] R. Trias, L. Baneras, E. Montesinos, E. Badosa, Lactic Acid Bacteria From Fresh Fruit and Vegetables As Biocontrol Agents of Phytopathogenic Bacteria and Fungi, International Microbiology, Vol. 11, No. 4, 2008, pp. 231-236, https://doi.org/10.2436/20.1501.01.66.
[18] American Society for Microbiology, Catalase Test Protocol, https://asm.org/Protocols/Catalase-Test-Protocol, 2010 (accessed on: August 1st, 2023).
[19] American Society for Microbiology, Oxidase test protocol, https://asm.org/Protocols/Oxidase-Test-Protocol, 2010 (accessed on: August 1st, 2023).
[20] B. Taroub, L. Salma, Z. Manel, H. I. Ouzari,
Z. Hamdi, H. Moktar, Isolation of Lactic Acid Bacteria From Grape Fruit: Antifungal Activities, Probiotic Properties, and In Vitro Detoxification of Ochratoxin A, Annals of Microbiology, Vol. 69, No. 3, 2019, pp. 17-27, http://dx.doi.org/10.1007/s13213-018-1359-6.
[21] J. Sambrook, D. Russell, Molecular Cloning: A Laboratory Manual, 3rd edn, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2001.
[22] M. Lorenzini, B. Simonato, F. Favati, P. Bernardi, A. Sbarbati, G. Zapparoli, Filamentous Fungi Associated with Natural Infection of Noble Rot on Withered Grapes, International Journal of Food Microbiology, Vol. 272, 2018, pp. 83-86, https://doi.org/10.1016/j.ijfoodmicro.2018.03.004.
[23] U. Schillinger, F. K. Lüke, Identification of Lactobacilli From Meat and Meat Products, Food Microbiology, Vol. 4, No. 3, 1987, pp. 199-208, https://doi.org/10.1016/0740-0020(87)90002-5.
[24] S. Zhao, X. Hao, F. Yang, Y. Wang, X. Fan,
Y. Wang, Antifungal Activity of Lactobacillus plantarum ZZUA493 and Its Application to Extend The Shelf Life of Chinese Steamed Buns, Foods, Vol. 11, No. 2, 2022, pp. 195, https://doi.org/10.3390/foods11020195.
[25] F. A. Sadiq, B. Yan, F. Tian, J. Zhao, H. Zhang,
W. Chen, Lactic Acid Bacteria as Antifungal
and Anti-Mycotoxigenic Agents: a Comprehensive Review, Comprehensive Reviews in Food
Science and Food Safety, Vol. 18, No. 5, 2019,
pp. 1403-1436,
https://doi.org/10.1111/1541-4337.12481.