Project title: Isolation, characterisation and exploitation of natural anti-yeast agents and their application as consumer-friendly preservatives in food and beverages.

 

Department of Agriculture, Food and the Marine (Food Institutionl Research Measure), Project Reference 15/F/731

 

This project ran from 2017 until 2021 and had significant scientific output. It originally set out to characterise both lactic acid bacteria and defensins/plant extracts for anti-yeast activity. The work was performed at both MTU with UCC partnering. A panel of typical spoilage yeasts from the food, feed and beverage sectors were chosen for testing including Kluyveromyces lactis, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Saccharomyces spp. and Debaryomyces hansenii. Typical food products spoiled by yeast include salad dressings (Debaryomyces), soft drinks (Zygosaccharomyces), dairy products (Kluyveromyces), and alcoholic beverages (Saccharomyces). In order to prevent the growth of such spoilage yeast, several methods of preservation have been adopted by food industries, ranging from chemical to physical preservation techniques. However, spoilage by yeast still occurs, hence this investigation.

 

Objectives

Briefly, the four technical objectives in the project were (a) to characterise bacterial or plant extracts with anti-yeast activity, (b) to identify, isolate and characterise the molecules involved (c) to apply them in food and beverage systems and (d) to synthesise the responsible molecules artificially and perform toxicity assays.

 

Results

While lactic acid bacteria did not show significant anti-yeast activity, a number of plant extracts showed considerable success. These included extracts of barley endosperm, potato tuber, radish seeds and white mustard seeds. Though other plant materials were also tested to perfect methodologies, the food-grade status of those below were considered an advantage. The extracts were isolated, purified and characterised in detail. Their characteristics are shown in table 1.

 

TABLE 1. Anti-yeast extracts and derived peptides
Plant source	Characteristics of peptide extracted	Solubility
Barley endosperm	47 amino acid sulphur rich defensin peptide (D-lp1)	Water soluble
Potato tuber	63-amino acid residue peptide with structural similarity to thionins (SN-1)	Water soluble
Radish seeds	Two similar 51-amino acid proteins, highly basic and rich in cysteine residues (Rs-AFP1 and Rs-AFP2)	Water soluble
White mustard seeds	Albumin protein, composed of 145 amino acid residues (with 8 cysteines) WMS-pep1 and WMS-pep3	Water soluble

 

Synthetic versions of all peptides were made and used in various detailed testing, which included evaluating stability of the peptides to proteolytic digestion, heat degradation, susceptibility to pH variation and different salts concentrations and detailed data is shown in the published papers. The designations of the peptides are shown in Table 2. Selected analytical experimental results are shown in the figures as explained in the respective figure legends, with all the in-depth information described in the scientific papers listed below. Potential for cytotoxicity against mammalian cells was also evaluated (as described in the various publications listed below) and no cytotoxic capacity was identified. The results indicated the peptides’ suitability for testing as potential preservatives peptide in different beverages and food systems as shown in Table 3.

 

TABLE 2. Synthetic peptides based on plant molecules investigated and their activity against food and beverage spoilage yeasts.
Name of the synthetic peptide	Derivation plant on which it is based	MICs range µg/ml against spoilage yeasts (microgram/ml)
		Z. bailli	Z. rouxii	K. lactis	D. hansenii
Rs-AFP1	Radish	25-50	-	-	-
Rs-AFP2	Radish	25-50	50-100	-	50-100
D-lp1	Barley	50-100	200-400	-	50-100
SN-1	Potatoes	100-200	-	-	200-400
KK-14	From sequences of different peptides with previously demonstrated activity against fungi	25-50	-	250-100	25-50
Dip KK-14		12.5-25	100-200	12.5-25	25-50
dKK-14		25-50	-	50-100	12.5-25
KK-14 (R10)		25-50	-	100-200	50-100
Pn-AMP1	Morning Glory	250-500	-	250-500	-
Pn-AMP2	Morning Glory	1000	-	-	-
WMS-pep1	White Mustard	800	-	-	800
WMS-pep3	White Mustard	100	-	-	100

 

 

 

 

TABLE 3. Food/beverage trials conducted giving data for the most susceptible target yeast
Food material	Peptides employed	Smaller concentration effective as preservative	Yeasts tested
Soft drink: Orange soda (fizzy)	KK-14	12.5 µg/ml	Z. bailii Sa1403
	Dip KK-14	12.5 µg/ml	Z. bailii Sa1403
	dKK-14	12.5 µg/ml	Z. bailii Sa1403
	KK-14 (R10)	12.5 µg/ml	Z. bailii Sa1403
	Rs-AFP1	50 µg/ml	Z. bailii Sa1403
	Rs-AFP2	50 µg/ml	Z. bailii Sa1403
	SN-1	100 µg/ml	Z. bailii Sa1403
	D-1p1	200 µg/ml	Z. bailii Sa1403
	Sin a 1	100 µg/ml	Z. bailii Sa1403
	Pn-AMP1	250 µg/ml	Z. bailii Sa1403
Soft drink: Cranberry juice	Rs-AFP1	50 µg/ml	Z. bailii Sa1403
	Rs-AFP2	50 µg/ml	Z. bailii Sa1403
	SN-1	100 µg/ml	Z. bailii Sa1403
	Sin a 1	50 µg/ml	Z. bailii Sa1403
	WMS-pep3	12.5 µg/ml	Z. bailii Sa1403
Soft drink: Apple juice	Rs-AFP1	200 µg/ml	Z. bailii Sa1403
	Rs-AFP2	200 µg/ml	Z. bailii Sa1403
	SN-1	200 µg/ml	Z. bailii Sa1403
	D-1p1	100 µg/ml	Z. bailii Sa1403
	Sin a 1	100 µg/ml	Z. bailii Sa1403
Viscous matrix: Salad dressing	Rs-AFP1	50 µg/ml	Z. bailii Sa1403
	Rs-AFP2	50 µg/ml	Z. bailii Sa1403
	Sin a 1	1000 µg/ml	Z. bailii Sa1403
Alcoholic beverage: White wine	D-1p1	400 µg/ml (partial inhibition)	Z. bailii Sa1403
	Sin a 1	1000 µg/ml	Z. bailii Sa1403
Dairy products: UHT milk	Pn-AMP1	1000 µg/ml	K. lactis ATCC 56498

 

 

Dissemination of project findings

 

The results of the project were disseminated at various meetings as follows:

• 35th European Peptide Symposium. Dublin, Ireland. August 26-31, 2018.
• 8th International Meeting on Antimicrobial Peptides. Edinburgh. September 2-5, 2018.
• 47th Annual Food Science and Technology Conference. Cork. Dec 6-7, 2018.
• FSAI Science Conference, Convention Centre, Dublin, August 21-22, 2019.
• 48th Annual Food Science and Technology Conference December 16, 2019, Limerick.
• Online end of project presentation via the APC Forum, June 1, 2021. Cork.

 

Papers published:

Shwaiki, L. N. Arendt, E. K. and Lynch, K. M. and Thery, T.L.C. (2019) ‘Inhibitory effect of four novel synthetic peptides on food spoilage yeasts’, International Journal of Food Microbiology. Elsevier, 300, pp. 43–52. doi: 10.1016/J.IJFOODMICRO.2019.04.005.

Shwaiki, L. N., Arendt, E. K. and Lynch, K. M. (2020) ‘Anti-yeast activity and characterisation of synthetic radish peptides Rs-AFP1 and Rs-AFP2 against food spoilage yeast’, Food Control, 113, p. 107178. doi: 10.1016/j.foodcont.2020.107178.

Shwaiki, L. N., Arendt, E. K. and Lynch, K. M. (2020) ‘Study on the characterisation and application of synthetic peptide Snakin-1 derived from potato tubers – Action against food spoilage yeast’, Food Control. Elsevier Ltd, 118. doi: 10.1016/j.foodcont.2020.107362.

Shwaiki, L. N., Sahin, A. W. and Arendt, E. K. (2020) ‘Study on the Inhibitory Activity of a Synthetic Defensin Derived from Barley Endosperm against Common Food Spoilage Yeast’, Molecules. MDPI AG, 26(1), p. 165. doi: 10.3390/molecules26010165.

Shwaiki, L. N., Arendt, E. K. and Lynch, K. M. (2021) ‘Plant compounds for the potential reduction of food waste–a focus on antimicrobial peptides’, Critical Reviews in Food Science and Nutrition. Bellwether Publishing, Ltd. doi: 10.1080/10408398.2021.1873733.

Shwaiki, L. N., Lynch, K. M. and Arendt, E. K. (2021) ‘Future of antimicrobial peptides derived from plants in food application – a focus on synthetic peptides’. Trends in Food Science and Technology. Elsevier. doi: 10.1016/j.tifs.2021.04.010.

Mignone, G. Shwaiki, L.N. Arendt, E.K. & Coffey, A. (2022). Isolation and characterisation of the antifungal activity of the mustard Napin protein Allergen Sin a 1. Biochemistry and Biophysics Reports (Elsevier). In Press. Reference BBREP-D-21-00478R1

 

 

CONFERENCE PRESENTATIONS and WORKSHOPS:

Shwaiki, L.N., Arendt, E.K., Lynch, K.M., Thery, T.L.C. (2018) Inhibitory Effect of Four Novel Synthetic Peptides on Food Spoilage Yeasts. Proceedings of 35th European Peptide Symposium. Dublin, Ireland. August 26-31, 2018 (Poster).

Shwaiki, L.N., Arendt, E.K., Lynch, K.M., Thery, T.L.C. (2018) Inhibitory Effect of Four Novel Synthetic Peptides on Food Spoilage Yeasts. Proceedings of 8th International Meeting on Antimicrobial Peptides. Edingburgh, UK. September 2-5, 2018 (Poster).

Mignone, G., Thery, T., Arendt, E.K., Coffey, A. (2018). Activity of synthetic peptide Pn-AMP1 against yeasts responsible of food spoilage. Proceedings of the 47th Annual Food Science and Technology Conference. Cork, Ireland. Dec 6-7, 2018 (Poster).

Shwaiki, L.N., Arendt, E.K., Lynch, K.M., Thery, T.L.C. (2018) Inhibitory Effect of Four Novel Synthetic Peptides on Food Spoilage Yeasts. Proceedings of the 47th Annual Food Science and Technology Conference. Cork, Ireland. Dec 6-7, 2018 (Oral Presentation).

Shwaiki, L.N., Lynch, K.M., Arendt, E.K.. (2019). Inhibitory effect of four novel synthetic peptides on food spoilage yeasts. FSAI Science Conference, Convention Centre Dublin, Ireland, August 21-22, 2019 (Poster)

Mignone, G., Arendt, E.K., Coffey, A. Inhibitory activity of two synthetic plant-based peptides against food and beverage spoilage yeasts. Proceedings of 48th Annual Food Science and Technology Conference. December 16, 2019, Limerick, Ireland (Poster)

Mignone G., Shwaiki L., Arendt, E.K., Coffey, A. Antifungal and antiyeast activity of an extract obtained from white mustard seeds and its potential use as preservative in food industry. Proceedings of the 49th Annual Food Science and Technology Conference. December 15, 2020, Online (Poster)

End of project presentation, APC Forum, Tuesday June 1st 2021. Presenter: Laila Nora Shwaiki. Work title: “Application of Synthetic Antimicrobial Peptides Derived from Plants for the Reduction of Yeast Spoilage in Food”. Chair - Silvia Melgar

 

• Figure 1. Scanning electron microscope (SEM) images of the yeast  Zygosaccharomyces bailii Sa1403 treated with the radish-derived peptides Rs-AFP1 and Rs-AFP2. A: untreated yeast cells; B & C: yeast cells exposed to the antifungal peptide Rs-AFP1 for 0 and 4 h, respectively. Rs-AFP1 has induced dramatic changes in the yeast cell morphology. Similarly with peptide Rs-AFP2 shown in images A (untreated) , D (0h)  and E (4h).

 

• Figure 2. Confocal laser scanning microscope (CLSM) images of Zygosaccharomyces bailii using the live-dead stain propidium iodide (PI). Red cells show dye penetration and staining of nucleic acid, indicating cell death in plate A, employing 400 μg/ml of SN-1. The assay thus shows that potato-derived peptide SN-1 permeabilises the membrane. Plates B and C show that lower concentrations of SN-1 (200 and 100 microgramme/ml respectively) results in fewer cells being killed. 

 

 

• Figure 3. Confocal laser scanning microscope (CLSM) images of Zygosaccharomyces bailii with an optical red filter (black background) and under brightfield illumination (grey background) for each sample. Red fluorescence by propidium iodide (PI) indicates the dye penetrating the damaged yeast cells staining the nucleic acid while the is dye excluded from live cells.

 

 

• Figure 4. Detection of overproduction of ROS (reactive oxygen species) in yeast in response to antiyeast peptides. The confocal laser scanning microscope (CLSM) images show Zygosaccharomyces bailii with an optical filter (black background) and under brightfield illumination (grey background) for each sample. The green fluorescence occurs when the dye Dihydrorhodamine penetrates yeast cells and becomes oxidised and the images show that the Barley-derived peptide D-lp1 induced overproduction of ROS, a classic antifungal mechanism: (A) exposure to 400 μg/ml, (B) 200 μg/ml, and (C) 100 μg/ml.

 

 

 

For further information

Contact

Aidan Coffey, Munster Technological University, Cork

Email: aidan.coffey@mtu.ie,

Phone: 021-4335486