**1. Introduction**

The market for soft drinks and fruit juices is increasing annually with the release of new beverage products, which are gaining popularity among consumers. This market expansion has increased the challenge of addressing spoilage problems [1]. Yeasts are the most common group of microorganisms responsible for spoilage of soft drinks and fruit juices due to their ability to withstand juice acidity and resist the action of weak-acid preservatives [1,2]. Beverage industries are focusing on the application of novel antimicrobial agents derived from plant sources as an alternative solution to address beverage spoilage caused by weak-acid resistant yeasts [3]. Tasmanian pepper leaf (*Tasmannia lanceolata*), lemon myrtle (*Backhousia citriodora*) and anise myrtle (*Syzygium anisatum*) are three Australian native herbs in commercial production and there is a growing interest in their bioactive properties and assessing their potential applications as functional ingredients in the beverage industry [4].

Tasmanian pepper leaf belongs to the Winteraceae family and found in forested regions in Tasmania, Victoria [5]. It is used in food as a seasoning, flavoring, coloring and preservative and it is

incorporated into personal health care products [4,6,7]. Polygodial is the major bioactive compound in Tasmanian pepper leaf responsible for its strong pungen<sup>t</sup> flavor and reported to be the main contributor to the antibacterial and antifungal activities [4,5,8,9]. A chemical profiling of the essential oil of Tasmanian pepper leaf shows the following sesquiterpene compounds: polygodial (36.74%), guaiol (4.46%), calamenene (3.42%), spathulenol (1.94), drimenol (1.91%), cadina-1,4-diene (1.58%), 5-hydroxycalamenene (1.47%) bicyclogermacrene (1.15%), α-cubebene (0.88%), β-caryophyllene (0.87%), α-copaene (0.48%), cadalene (0.44%), d-cadinol (0.40%), elemol (0.39%), T-muurolol (0.39%) and germacrene-D (0.33%) [10]. Some cultivated Tasmanian pepper leaf clones are found to contain polygodial as high as 64% [10]. Phenolic compounds have been identified as the major class of antioxidant compounds in Tasmanian pepper leaf solvent and polyphenol rich extracts these include coumaric acid, cyanidin-3-glucoside, chlorogenic acid, quercetin, quercetin 3-rutinoside, cyanidin 3-rutinoside [7,11,12].

Lemon myrtle is a member of the Myrtaceae family and mainly grows in the subtropical rainforests of southeast regions of Queensland, Australia [13]. Lemon myrtle contains citral as a predominant compound with antimicrobial and insect repellent properties and is used as a cure for skin diseases [14–16]. It has a pleasant lemon flavor with mild sweet notes, it is currently one of the most cultivated and commercialized native plant species utilized in cosmetics, health products, herbal teas and flavoring agents in food and beverage systems [4,17–19]. According to reported literature, lemon myrtle essential oil contains citral (82–91%), 5-hepten-2-one,6-methyl (1.54–13.82%), 2,3-dehydro-1,8-cineole (3.52%), nerol (2.66%), germacrene B (0.2–2.18%), geraniol (0.8–1.26%), linalool (0.5–5.85%), myrcene (0.4–4.39%) and citronellal (0.25–2.19%) [15,16,20]. Phenolic compounds detected in solvent and polyphenol rich extracts of lemon myrtle contributing to antioxidant activity were myricetin, hesperetin rhamnoside hesperetin hexoside, quercetin, ellagic acid, ellagitannins and ellagic acid glycosides [11,12].

Anise myrtle also belongs to the Myrtaceae family and grows in subtropical rainforests of Bellingen and Nambucca Valleys of northeast New South Wales and some regions of Queensland [21,22]. Anise myrtle essential oil contains anethole (94.97%), methyl chavicol (4.43%), α-pinene (0.09%), 1,8-cineole (0.02%) and α-farnesene (0.07%) [23]. Anethole possesses antibacterial and antifungal activities and contributes to the intense licorice and aniseed aroma in anise myrtle leaves currently being utilized in cosmetics, savory cuisines, tea blends, body and mouth care products, alcoholic drinks and pharmaceutical industries [22,24–26]. Catechin, quercetin, hesperetin, myricetin, ellagic acid, ellagitannins and ellagic acid glycosides were the main antioxidant phenolic compounds in polyphenol rich and solvent extracts of anise myrtle leaves [11,12].

Essential oils of lemon and anise myrtle with citral and anethole as the major volatile compound have shown broad spectrum antimicrobial activity against bacteria, yeas<sup>t</sup> and fungi. The minimum inhibitory concentration (MIC) ranged from 0.16 to >1.25 (% *v*/*v*) for lemon myrtle and 0.63 to >1.25 (% *v*/*v*) for anise myrtle against the following bacteria: *Staphylococcus aureus*, *Escherichia coli*, *Bacillus cereus*, *Proteus vulgaris*, *Pseudomonas aeruginosa*, *Enterobacter aerogenes*, *Acinetobacter baumannii*, *Shewanella putrefaciens* and *Listeria monocytogenes* [27]. For yeas<sup>t</sup> (*Saccharomyces cerevisiae*) and fungi (*Geotricum candidum*) the MIC ranged from 0.04 to 0.08 (% *v*/*v*) for lemon myrtle and 0.16 to 0.08 (% *v*/*v*) anise myrtle respectively [27]. Hexane extracts of lemon myrtle leaves have shown anti-yeast activity against *Candida albicans*, *Candida colliculosa*, *Candida lipolytica*, *Hanseniaspora uvarum*, *Pichia anomala*, *Pichia membranifaciens*, *Rhodotorula mucilaginosa*, *Schizosaccharomyces octosporus*, however, the anise myrtle leaf hexane extracts did not show any activity against these yeasts [28]. Polygodial a major compound in Tasmanian pepper leaf extracts has shown anti yeas<sup>t</sup> activity against *Zygosaccharomyces bailii* and *Saccharomyces cerevisiae*, antifungal activity against *Sclerotinia libertiana*, *Mucor mucedo*, *Rhizopus chinensis*, *Aspergillus niger*, *Penicillium crustosum* and antibacterial activity against *Salmonella choleraesuis* [6,8]. Hexane and methanol extracts of Tasmanian pepper leaf have shown anti-yeast activity against *Candida albicans*, *Candida colliculosa*, *Candida lipolytica*, *Candida stellata*, *Hanseniaspora uvarum*, *Pichia anomala*, *Pichia membranifaciens*, *Rhodotorula mucilaginosa*, *Schizosaccharomyces octosporus* [28]. In the study by Zhao and Agboola [28] water extract had the least antimicrobial activity and hexane was the most potent.

Herb extracts could serve as functional ingredients in soft drinks and fruit beverages due to their antioxidant and antimicrobial properties. In recent years, much attention has been given to the application of natural compounds as an alternative solution to tackle beverage spoilage problems caused by weak-acid resistant yeasts [29–31]. The aim of this study is to measure the amounts of major compounds present in different solvent extracts of Tasmanian pepper leaf, lemon myrtle, and anise myrtle and assess the extracts bioactive properties and mechanism of action against weak-acid resistant yeasts and bacteria. This is the first study to assess the antimicrobial properties of different extracts of these herbs against a range of weak-acid resistant spoilage yeasts of importance to the beverage industry.

#### **2. Materials and Methods**

### *2.1. Plant Material*

Lemon myrtle and anise myrtle were supplied by Australian Rainforest Products Pty Ltd. (Lismore, NSW, Australia), and Tasmanian pepper leaf supplied by Diemen Pepper (Birchs Bay, Tasmania, Australia). Herbs received as dried whole leaves and stored at −20 ◦C until further use.
