Antimicrobial Compounds from Plant and Agrowaste

Substantial amount of reusable substances, such as soluble sugars, proteins, phenols, and fibers, are present in the agro-industrial wastes which can be very valuable for the production of value-added antimicrobial products. Normally, one or two parts of plants, vegetables, fruits, cereals, etc., are used for some useful product and the rest of the parts such as peels, pomace, seed, leaves, hull, bark, and root are dumped as waste. A number of research groups are working to explore various possibilities of utilizing the plant and agrowaste as value-added products (Table 14.1). Some of the important works on exploration of these natural wastes for production of antimicrobial compounds are summed up below.

Antimicrobial Compounds from Fruit Waste

Huge quantities of fruits are used worldwide but their peel, seed, and pomace remain mostly unutilized, thus generating huge amount of waste. These wastes, though highly perishable and seasonal, are a serious challenge for the processing industries and pollution monitoring agencies. Suitable methods can be adopted to utilize this waste into value-added products, which can improve the overall economics of processing units and can help to reduce environmental pollution. A lot of developments have taken place in the field of utilizing fruit waste and by-products for extraction of antimicrobial compounds. Some of the important studies on utilization of fruit wastes for production of antimicrobial agents are briefed here. Antimicrobial and Antioxidant Potential of Juice Pressing Waste

Anthocyanins, tannins, starches, saponins, polypeptides, and lectins were found in the water extract, and polyphenols, lactones, flavones, and phenons were additional

Table 14.1 Antimicrobial compounds extracted from plant and agrowaste

phytochemicals traced in the extracts of the pomace (peels, seeds, flesh) remaining after pressing the juice of Fragaria ananassa (strawberry), Prunus cerasus (sour cherry), Ribes nigrum (black currant), Ribes rubrum (red currant), Rubus fruticosus (blackberry), and Rubus idaeus (raspberry). Antimicrobial and antioxidant potential of water and methanol extracts was investigated on Bacillus cereus,

B. subtilis, Campylobacter jejuni, E. coli, Salmonella typhimurium and Serratia marcescens, C. albicans, C. krusei, C. glabrata, C. pulcherrima, and C. parapsilosis by broth dilution method (Krisch et al. 2009). Both aqueous and methanol extracts inhibited growth of almost all bacteria. Methanol extracts had stronger inhibitory effect than water extracts.

Pomace of beetroot is also disposed as low-value feed or manure, although it is rich in phenols. During juice pressing, most of the secondary plant metabolites and dietary fiber compounds of beetroot are not transferred into the liquid phase and remain in the pomace (Will et al. 2000). Beetroot (Beta vulgaris L. ssp. vulgaris) has very high total phenolic content, i.e., in the range of 50–60 µmol/g dry weight and very good antioxidant property (Vinson et al. 1998; Kahkonen et al. 1999). Beetroot peels are reported to contain l-tryptophan, p-coumaric, and ferulic acids as well as cyclodopa glucoside derivatives (Kujala et al. 2001).

Phenolic content in beetroot is mainly present in the peel (50 %), crown (37 %), and flesh (13 %) (Canadanovic et al. 2011). Canadanovic et al. (2011) reported phenolic content (376.4 mg/g of dry beetroot pomace extract), flavonoid content (269.70 mg/g), and betalain (41.85 mg/g) in the ethanolic extract of the beetroot. They evaluated antibacterial activity of ethanol extract of beetroot pomace by disk diffusion and microdilution method against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa. Gram-positive bacteria Staphylococcus aureus and Bacillus cereus demonstrated higher susceptibility than Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa against beetroot extract. Antimicrobial Activity of Citrus Fruit Peels

Citrus is one of the major fruit commercially grown all over the world. Huge quantity of wastes such as peels are generated every year, as juice yield of citrus is not even half of the total fruit mass (Manthey and Grohmann 2001). Citrus peels are rich in nutrients and contain many phytochemicals like flavanones and polymethoxylated flavones (Ahmad et al. 2006), thus can be explored for antimicrobial compounds. Therapeutic value of citrus oil as an antidiabetic, antimicrobial, antifungal, hypotensive agent, antioxidant, antibacterial, and antiviral agent has been studied by many research groups (Kumamoto et al. 1986; Caccioni et al. 1998; Hamendra and Anand 2007; Kanaze et al. 2008).

Phytochemical analysis and antimicrobial activities of peel of Citrus limon (lemon) and Citrus sinensis (sweet orange) were studied by Ashok et al. (2011). They used five different solvent extracts (ethyl acetate, acetone, ethanol, petroleum ether, and water) for both types of peels and screened their extract against five

pathogenic bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, and Salmonella typhi. They carried out phytochemical analysis of powdered plant parts and reported presence of flavonoids, saponins, tannins, alkaloids, and terpenoids in the peel extracts. Solvent extracts of Citrus sinensis peel and Citrus limon showed significant activities and presence of different phytochemicals. Acetone peel extract of Citrus sinensis showed highest antibacterial activity followed by the ethyl acetate peel extract of Citrus limon.

Chanthaphon et al. (2008) studied ethyl acetate extracts and hydrodistillated essential oils of Citrus spp. and kaffir lime peels (Citrus hystrix DC.) for antimicrobial activities against food-related microorganisms. Ethyl acetate extract from kaffir lime contained limonene (31.64 %), citronellal (25.96 %), and β-pinene (6.83 %). Essential oil obtained from hydrodistillation contained β-pinene (30.48 %), sabinene (22.75 %), and citronellal (15.66 %). They evaluated antimicrobial activities of ethyl acetate extracts from fresh peels and dried peels of tropical citrus fruits (lime), kaffir lime, and pomelo peels against pathogenic E. coli. Extracts from both fresh and dried limes, kaffir lime, and pomelo peels showed antibacterial activity against S. aureus, but the ones from fresh peels showed higher activity. Similarly, the extracts from fresh lime and kaffir lime peels showed activity against E. coli but no activity was observed with dried peels. They also observed that ethyl acetate extracts from all citrus peels showed better antimicrobial activities than their essential oils. The ethyl acetate extract of peels inhibited Gram-positive bacteria, yeast, and molds: Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Saccharomyces cerevisiae var. sake, and Aspergillus fumigatus. Antioxidant and Antimicrobial Activity of Pomegranate Peel and Seed

Pomegranate peel extract (PE) also shows excellent antioxidant and antimicrobial activity. Kanatt et al. (2010) studied the antimicrobial and antioxidant properties of pomegranate peel and seed extracts. Antimicrobial activity of pomegranate peel extract was tested against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and S. typhimurium. It inhibited growth of Staphylococcus aureus and Bacillus cereus but Escherichia coli and S. typhimurium were resistant to it. Pomegranate peel extract (PE) was found very effective in scavenging hydroxyl and superoxide anion radicals. Investigators used pomegranate peel extract to enhance shelf life of chicken meat products successfully by 2–3 weeks during chilled storage.

Tehranifara et al. (2011) also investigated the antioxidant properties of peel, seed, and leaf of pomegranate (Punica granatum L.). They used aqueous and methanolic extraction with different concentrations (0, 500, 1,000, and 1,500 ppm) on three fungus Penicillium italicum, Rhizopus stolonifer, and Botrytis cinerea. Methanolic extract showed the highest inhibitory effect on the mycelia growth and spore germination. Peel and seed extracts showed more inhibitory effect than leaf extract. Antioxidant capacities of peel, seed, and leaf extracts of pomegranate were

55.3 %, 35.7 %, and 16.4 %, respectively. The phenolic content was 2.8 times higher

in peel extract than leaf extract, which may be the reason for better antimicrobial activity of peel extract. These studies propose pomegranate peel and seed as source of powerful antioxidant and antifungal compounds. Antimicrobial Effect of Apple Skins

Apples are high on phenolic compounds (Mangas et al. 1999; Podsedek et al. 2000; Shoji et al. 2003). Apple pulp and skins are reported to contain catechin, procyanidin, caffeic acid, and chlorogenic acid among other compounds. Skin of apple also contains flavonoids such as quercetin glycosides and cyanidin glycosides which are not present in pulp (Escarpa and Gonzalez 1998; Vander et al. 2001). Alberto et al. (2006) examined the antimicrobial activity of phenolic compounds extracted from the skin of two apple varieties Royal Gala and Granny Smith using the agar diffusion method. The phenolic compounds were extracted in acetone, methanol, and ethanol solvents. Total phenolic and flavonoid content was obtained with Folin–Ciocalteu reagent (Singleton and Rossi 1965). Granny Smith variety skin was found containing more polyphenols and flavonoid than Royal Gala, whereas maximum phenolics and flavonoids were obtained in acetone extract for both varieties of apples. The highest inhibitory effect of both apple varieties corresponded to extract which contained high phenolic content. Antimicrobial activities of different extracts were evaluated against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, and Listeria monocytogenes. Extracts of both the apple skins were reported to inhibit these microorganisms; however, extracts of Granny Smith were found more effective demonstrating a direct relationship between the phenolic content of the extracts and the antimicrobial effect. This study established the usefulness of apple skin for its antibacterial properties, thereby adding value to the already known benefits of apple to the human health. Fruit and Vegetable Peels

Chanda et al. (2011) evaluated seven fruit and vegetable peels for their antimicrobial properties: Mangifera indica L. (Anacardiaceae), Lagenaria siceraria (Molina) Standl. (Cucurbitaceae), Solanum tuberosum L. (Solanaceae), Ananas comosus (Linnaeus) Merr. (Bromeliaceae), Luffa acutangula (L.) Roxb. (Cucurbitaceae), Momordica charantia L. (Cucurbitaceae), and Moringa oleifera Lam. (Moringaceae). Antimicrobial activities of hexane, chloroform, acetone, and methanol extracts of these samples were evaluated by agar well diffusion method against Staphylococcus aureus, Staphylococcus subflava, Corynebacterium rubrum, Salmonella typhimurium, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, Cryptococcus luteolus, Candida albicans, Candida tropicalis, and Candida glabrata. Mangifera indica peel showed best and promising antimicrobial activity. Polar solvents (acetone and methanol) were found more effective than nonpolar solvents (hexane and chloroform). Activities shown by acetone extracts were the best, followed by methanol extracts. The extracts showed better antifungal activity than antibacterial activity. C. glabrata and K. pneumoniae were the most susceptible organisms. Report suggested that broad spectrum of antibacterial activity by M. indica peels may help to discover new chemical classes of antibiotic substances. Antibacterial Effect of Grape Seeds

Grape seeds, which are normally a waste of winery or juice pressing, can be effectively utilized for production of antioxidants and antimicrobial compounds. Jayaprakasha et al. (2003) have reported antimicrobial activity of acetone and methanol extracts of grape seed against Bacillus cereus, Bacillus coagulans, Bacillus subtilis, S. aureus, E. coli, and P. aeruginosa. The authors reported that the extracts were free radical inhibitors and primary antioxidants that react with free radicals.

In another report on antimicrobial activity of grape seed extract, Baydar et al. (2004) revealed that the grape seed extracts in acetone/water/acetic acid and ethyl acetate/methanol/water solvents inhibited various test organisms: Aeromonas hydrophila, B. brevis, B. cereus, B. megaterium, B. subtilis, E. faecalis, E. coli, Klebsiella pneumoniae, L. monocytogenes, Mycobacterium smegmatis, Proteus vulgaris, P. aeruginosa, and S. aureus. The grape seed extracts were found to contain high total phenolics, which should be the reason for their strong antimicrobial activity.

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