Antimicrobial Properties of Chestnut Shell Extract as an Ecofriendly Approach for Food Preservation ^†

Abstract

The chestnut industry generates large quantities of by-products, including the chestnut shell, which is a source of phenolic compounds. In this study, the MIC (minimum inhibitory concentration) of chestnut shell extract was determined using the disk diffusion method. The chestnut shell was freeze-dried and milled. The extract was obtained using an ultrasound-assisted technique using water 70%: ethanol 30% (v/v) and subsequently lyophilized. Muller–Hinton plates were inoculated with ~10⁵ CFU/mL of microorganisms. Sterile paper discs (6 mm) were placed on the inoculated culture medium and impregnated with 10 µL of each extract. Seven concentrations of extract between 0.3% and 2.1% were tested. The plates were incubated for 24 h at 37 °C. The antibacterial efficacy of the extracts was indicated by a halo formed around the paper disk. This work was carried out in triplicate. Halos were found at 1.5%, 1.8%, and 2.1% on Listeria monocytogenes ATCC 7973 (8.32 ± 0.06 mm for 2.1%), Enterococcus faecalis 19433 (8.94 ± 0.41 mm for 2.1%), and Staphylococcus aureus ATCC (10.26 ± 0.19 mm at 2.1%). For the remaining microorganisms, no halos were observed. The tested extract showed antimicrobial activity, demonstrating its potential for the control of pathogens in the food industry.

1. Introduction

In Europe, the cultivation of chestnut trees (Castanea sativa, Mill.) has been increasing, as has the production of chestnuts [1]. Portugal is one of the European Union countries contributing most to the increase in production, with the Longal and Judia varieties being the most commonly cultivated [2]. Most chestnuts are eaten fresh, but their processing, whether to be sold frozen or in purees, for example, has been increasing [3].

The chestnut industry generates large quantities of by-products, including the chestnut shell, which is a source of phenolic compounds of great interest to the food and pharmaceutical industries [4,5]. The concept of the circular economy is increasingly being suggested and referred to, and the use of these by-products with added value can make a significant contribution to this concept and to the valorization of these products [6,7].

Phenolic compounds play a fundamental role in plants, both in their reproduction and growth, as well as for their adaptation and survival under stressful situations, such as attacks by pathogens [8]. Phenolic compounds also contribute to the organoleptic properties of foods. Of all the classes of phenolic compounds, flavonoids are the most abundant. They are also essential for food production and preservation, as they play a fundamental role in oxidation processes [9]. These compounds have been studied for their antimicrobial, antioxidant, anti-inflammatory, antiviral, and anti-hepatotoxic effects, among others [10].

The consumption of nuts has been increasingly recommended, with studies reporting that they help reduce cholesterol levels and are thus associated with a lower incidence of cardiovascular diseases, with these factors being associated with the antioxidant activity of the compounds present [11,12].

The search for natural sources of phenolic compounds with antimicrobial and antioxidant properties is therefore a current interest. This study is important due to the variability of the plant’s origins, varieties, and the variations of the technologies used for extraction that may influence the quantity of phenolic compounds and its activity. Also, the techniques used are constantly changing, and there may be alterations when compared to previous studies. The aim of this study was to extract phenolic compounds from chestnut shells and evaluate their antimicrobial activity.

2. Materials and Methods

2.1. Samples of Chestnut Shells

Chestnuts shells of the Judia variety were obtained from an industry in the north of Portugal. The samples were dried at 60 °C, vacuum-packed, and refrigerated (2 °C) until extraction.

2.2. Extraction

The extract was obtained using an ultrasound-assisted technique under a controlled temperature (40 °C) and time (30 min). The solvent used was water 70%: ethanol 30%, (v/v). For this stage, a ratio of 1 g of sample to 10 mL of solvent was used. The extract obtained was then centrifuged. The solvent was removed using a rotary evaporator at 38 °C under vacuum and lyophilized. For the analysis, the extract was reconstituted with water and filtered using a 0.20-μm syringe filter.

2.3. Antimicrobial Activity

According to the method outlined by Garcia et al. [13], we carried out the disk diffusion method. Eleven microorganisms were tested in this study to determine the antimicrobial properties of the chestnut shell extract under different concentrations. The microorganisms that were used are shown in Table 1. In order to prepare the inoculums, the microorganisms were cultivated in their respective enrichment medium (Table 1). Each isolate was then prepared in 0.1% tryptone salt, and the concentration of the inoculum was obtained using the McFarland method to a standard of 0.5 (approximately 10⁸ colony-forming units (CFUs) per mL). Each preparation was inoculated (0.1 mL) onto Mueller–Hinton agar. The inoculum was allowed to dry, and then the sterilized discs (6 mm) were placed with 10 µL of extract added to each one. Seven concentrations of extract were tested. The plates were then incubated for 37 °C/24 h for their subsequent visual analysis. The plates were checked for whether an inhibitory halo had formed, and when it did, it was measured (mm), and the values were recorded.

Table 1. Microorganisms that are likely to occur in meat products and their culture conditions.

Microorganism	Gram	Liquid Culture Medium, Temperature, and Incubation Time	Selective Culture Medium, Temperature, and Incubation Time
Escherichia coli O157:H7 9001	−	BHI, 37 °C, and 24 h/48 h (610495)	TBX (Tryptone Bile X-Glucuronide-84637), 44 °C, and 24 h
Yersinia enterocolitica ATCC 9018	−	BHI, 37 °C, and 24 h/48 h (610495)	Yersinia-selective agar base (VWR), 30 °C, and 24 h
Staphylococcus aureus ATCC	+	BHI, 37 °C, and 24 h/48 h (610495)	BP (Baird Parker (VWR)), 37 °C, and 24 h
Salmonella ATCC	−	BHI, 37 °C, and 24 h/48 h (610495)	Chromagar Salmonella, 37 °C, and 24 h
Salmonella typhimurium 14028	−	BHI, 37 °C, and 24 h/48 h (610495)	Chromagar Salmonella, 37 °C, and 24 h
Enterococcus faecalis 19433	+	BHI, 37 °C, and 24 h/48 h (610495)	VRBG (Violet Red Bile Glucose (VWR)), 30 °C, and 24 h/48 h
Enterococcus faecium 20477	+	BHI, 37 °C, and 24 h/48 h (610495)	VRBG (Violet Red Bile Glucose-VWR), 30 °C, and 24 h/48 h
Listeria monocytogenes ATCC 7973	+	BHI, 37 °C, and 24 h/48 h (610495)	Chromagar Listeria, 37 °C, and 24 h
Listeria ATCC 7644	+	BHI, 37 °C, and 24 h/48 h (610495)	Chromagar Listeria, 37 °C, and 24 h
Pseudomonas JI-Me-LM03	−	BHI, 37 °C, and 24 h/48 h (610495)	CFC (Pseudomonas-selective agar), 30 °C, and 48 h
Escherichia coli ATCC 1175	−	BHI, 37 °C, and 24 h/48 h (610495)	TBX (Tryptone Bile X-Glucuronide-84637), 44 °C, and 24 h

3. Results

The minimum inhibitory concentrations of the chestnut shell extract are shown in Table 2.

Table 2. Antimicrobial susceptibility—diameter (mean ± standard deviation) of inhibition halos (mm).

Microorganism	1.2%	1.5%	1.8%	2.1%
	DDA	DDA	DDA	DDA
Escherichia coli O157:H7 9001	-	-	-	-
Yersinia enterocolitica ATCC 9018	-	-	-	-
Staphylococcus aureus ATCC	-	-	-	10.26 ± 0.19
Salmonella ATCC	-	-	-	-
Salmonella typhimurium 14028	-	-	-	-
Enterococcus faecalis 19433	-	7 ± 0.25	7.74 ± 0.25	8.94 ± 0.41
Enterococcus faecium 20477	-	-	-	-
Listeria monocytogenes ATCC 7973	-	6.22 ± 0.18	6.99 ± 0.11	8.32 ± 0.06
Listeria ATCC 7644	-	-	-	-
Pseudomonas JI-Me-LM03	-	-	-	-
Escherichia coli ATCC 1175	-	-	-	-

Note: -, not determined.

The chestnut shell extract showed antimicrobial capacity against 3/11 of the microorganisms studied (27.27%) at the three highest concentrations that were studied (1.5%, 1.8%, and 2.1%). The microorganisms where an inhibitory halo was found were Gram positive, including Staphylococcus aureus ATCC (7.00 mm–9.47 mm), Enterococcus faecalis 19433 (6.65 mm–8.52 mm), and Listeria monocytogenes ATCC 7973 (6.04 mm–7.37 mm). These results are in agreement with other studies. J.-Y. An et al. [14] presented different extraction conditions that allowed for specific phenolic compounds to be isolated; however, they found an inhibitory halo for S. aureus and E. Faecium, as in our study. Silva et al. [2], used a higher concentration of extract than this study, which resulted in slightly larger halos; however, the microorganisms that were inhibited were similar. For Staphylococcus aureus and Enterococcus faecalis, they obtained inhibitory halos of 12 mm and 11 mm, respectively.

4. Conclusions

The results that were obtained demonstrate the interesting antimicrobial potential of chestnut by-products, such as the shell. The extract obtained is rich in phenolic compounds, and could be interesting to use as an antimicrobial and antioxidant additive. It also offers a strong possibility of adding economic value to the region and the chestnut industry. It is necessary to complement this study with others, such as the identification of phenolic compounds present in the shell and their antioxidant capacity.