Development of a novel selective medium for the isolation and enumeration of acetic acid bacteria from various foods

Development of a novel selective medium for the isolation and enumeration of acetic acid bacteria from various foods

Food Control 106 (2019) 106717 Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont Short comm...

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Food Control 106 (2019) 106717

Contents lists available at ScienceDirect

Food Control journal homepage: www.elsevier.com/locate/foodcont

Short communication

Development of a novel selective medium for the isolation and enumeration of acetic acid bacteria from various foods

T

Dong-Hyeon Kima, Jung-Whan Chona, Hyunsook Kimb, Kun-Ho Seoa,∗ a b

Center for One Health, College of Veterinary Medicine, Konkuk University, Seoul, 05029, South Korea Department of Food & Nutrition, College of Human Ecology, Hanyang University, Wangsimni-ro, Seongdong-gu, Seoul, South Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Acetic acid bacteria Selective media Culture Food Isolation

Acetic acid bacteria (AAB) are gram-negative, obligate aerobic bacteria that produce acetic acid from alcohols or sugars. AAB are ubiquitous and found in many food products, including vinegar and wine. Owing to the lack of a suitable selective culture medium and the fastidious nature of AAB in food samples, ecological studies have been fundamentally limited for decades. Here, we developed a novel selective medium (AAB-selective agar; ABS) for AAB from food samples and evaluated its performance in comparison with glucose-yeast extract-calcium carbonate agar (GYC). In inclusivity tests, 16 species of AAB showed good growth on both ABS and GYC. Remarkably, the growth of 21 non-AAB strains was inhibited on ABS but not on GYC, suggesting the high exclusivity of the novel selective medium (p < 0.05). In artificial AAB-positive food samples, including wine, vinegar, yogurt, fruit, and kimchi, ABS provided better visual differentiation of AAB colonies, showing green colonies with a yellow halo surrounded by no competing colonies. Moreover, in ABS, a significantly greater number of AAB colonies were recovered from yogurts, fruits, and kimchi (Acetobacter aceti ATCC 15973 in yogurt, 4.24 ± 0.17 vs. 3.80 ± 0.14; Gluconobacter oxydans ATCC 15163 in fruit, 4.87 ± 0.07 vs. 4.53 ± 0.15; Gluconacetobacter hansenii ATCC 35959 in kimchi, 4.25 ± 0.14 vs. 3.85 ± 0.12; p < 0.05). Finally, a total of five novel AAB strains were isolated from 10 vinegar and 10 kimchi samples using ABS, while unsuccessful using GYC, demonstrating the superior performance of ABS in isolating new AAB strains or detecting AAB contamination.

1. Introduction Acetic acid bacteria (AAB) are a large group of gram-negative, obligate aerobic bacteria belonging to the family Acetobacteriaceae and the class Alphaproteobacteria (Sievers & Swings, 2015). They possess the ability to incompletely oxidize alcohols or sugars to corresponding organic compounds, especially acetic acid, as end products (Cleenwerck & De Vos, 2008). This microbial group includes 19 genera and 46 species, of which the major genera are Acetobacter, Gluconobacter, and Gluconacetobacter (Cleenwerck & De Vos, 2008; Matsutani, Hirakawa, Yakushi, & Matsushita, 2011; Malimas et al., 2017). Although they form the large microbial group and have distinct metabolic and biochemical activities among food microorganisms, culture media for selectively detecting or isolating AAB have not been developed for several decades, although some modifications have been tried in some studies (De Vero & Giudici, 2013). AAB play a pivotal role in the fermented food industry, and the

consumption of AAB-fermented foods such as vinegar and kefir is increasingly getting popular worldwide (Raspor & Goranovič, 2008; Mamlouk & Gullo, 2013; Kim et al., 2015). In contrast, they are well recognized as spoilage bacteria, which deteriorate the organoleptic quality of fermented foods such as wine, yogurt, fruits, and kimchi (Bartowsky & Henschke, 2008; Mamlouk & Gullo, 2013; Soni & Dey, 2014). Therefore, an understanding of the dynamics and activities of AAB in these foods can be useful for further processing optimization and microbial quality control, positively impacting production (Gullo, Verzelloni, & Canonico, 2014). Given that the majority of commercial fermented foods harbor multiple strains of bacteria, culture media should be able to selectively recover AAB. To the best of our knowledge, however, no selective culture methods are available for AAB isolation, detection, and enumeration from these foods, which is the major hurdle in investigating the dynamics and physiology of AAB population and isolating novel high-functional AAB strains (Vegas et al., 2010). Although several alternative molecular biology methods to screen

Abbreviations: AAB, acetic acid bacteria; ABS, acetic acid bacteria-selective agar; GYC, glucose-yeast-calcium carbonate agar ∗ Corresponding author. E-mail address: [email protected] (K.-H. Seo). https://doi.org/10.1016/j.foodcont.2019.106717 Received 26 March 2019; Received in revised form 13 June 2019; Accepted 15 June 2019 Available online 17 June 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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Gluconobacter frateurii IFO3251, Gluconobacter morbifer JCM 15512, Gluconobacter oxydans ATCC 15163, and Gluconobacter oxydans IFO 3293. Non-AAB strains included Bacillus subtilis KU18601, Bacillus siamensis KU18602, Torulaspora delbrueckii KU18603, Weissella hellenica KU18701, Leuconostoc mesenteroides KU18702, Saccharomyces servazzii KU18703, Bacillus luti KU18704, Bacillus megaterium KU18705, Leuconostoc citreum KU18706, Lactobacillus acidophilus 07012, Lactobacillus kefiranofaciens 07188, Lactobacillus kefiri 07088, Lactobacillus bulgaricus KU18101, Lactobacillus plantarum KU18102, Lactobacillus rhamnosus KU18103, Lactococcus lactis subsp. lactis KU18104, Lactococcus lactis subsp. cremoris KU18105, Bifidobacterium longum 720, Bifidobacterium animalis subsp. lactis KU18106, Saccharomyces unisporus 2601, and Enterococcus durans KU16801. The non-AAB strains were selected because they were found and isolated from major AAB habitats including fruits, fermented vegetables and dairy products, which suggests that they may form competing colonies in the culture media for AAB. The strains were cultured on GYC for two passages. A single colony of each strain removed from GYC was then streaked onto ABS and GYC plates and incubated at 30 °C for 72 h. On ABS, a green colony with a yellow halo formed by typical AAB growth was regarded as positive (Supplementary data 1). For GYC, a white colony with a clear halo was regarded as positive.

the presence of AAB have been developed, including denaturing gradient gel electrophoresis, temporal temperature gradient gel electrophoresis, and TaqMan probe-based real-time PCR (Lopez et al., 2003; De Vero et al., 2006; Torija, Mateo, Guillamón, & Mas, 2010; Valera, Torija, Mas, & Mateo, 2013), culture methods easily isolating AAB from foods remain insufficient (Vegas et al., 2010). Several non-selective culture media have been used to maintain and preserve AAB strains, including glucose-yeast extract-calcium carbonate agar (GYC; the medium most commonly used to culture and isolate AAB strains from sugar-rich samples), acetic acid-ethanol (AE) and reinforced acetic acidethanol (RAE) media (containing acetic acid and ethanol; used in isolating high acid-producing AAB strains from spirit vinegar), as well as yeast extract-peptone-mannitol (YPM) and malt extract-yeast extract (MYA) agar (containing mannitol or malt extract; used to recover some AAB strains) (Giudici, Gullo, & De Vero, 2017). None of these media are selective; therefore, some modifications for enhancing their selectivity have been applied without clear concensus, including acidification and the addition of antimicrobial agents to inhibit the growth of competing yeast and lactic acid bacteria (Giudici et al., 2017). In addition, no clearly defined formula has been documented to prepare AAB selective media, nor has such a medium been validated for its performance with food samples. Here, to overcome this drawback, a novel culture medium was developed to selectively isolate and enumerate AAB from food samples that are not sterilized and have complex microbiota. The novel medium, i.e., acetic acid bacteria-selective agar (ABS), was tested for inclusivity and exclusivity using AAB and non-AAB strains and validated with artificial AAB-positive food samples (wine, yogurt, fruits, vinegar, and kimchi) in comparison with GYC. GYC was chosen for comparison because it is the most widely used medium for AAB, and previous studies have shown that it enables several AAB strains to be recovered from food samples (Vegas et al., 2010). In addition, food samples were chosen based on their positive and negative association in food quality. Finally, the novel medium was used to detect and isolate AAB strains from field food samples.

2.3. Performance of the novel medium for enumeration of AAB For experimental spiking, Acetobacter aceti ATCC 15973, Gluconobacter oxydans ATCC 15163, and Gluconacetobacter hansenii ATCC 35959 grown on GYC were serially diluted in autoclaved 0.9% saline. Five types of foods, i.e., wine, yogurt, vinegar, fruit, and kimchi, were purchased from a retail market in Seoul, Korea. AAB was only artificially inoculated into AAB-negative food samples, which was confirmed by real-time PCR assays targeting AAB group, to exclude naturally contaminated positives arising from natural contamination (Kim, Lim, Kim, & Seo, 2019). In addition, mesophilic aerobic bacteria were counted according to the previous study (Kang, Kim, Chon, & Seo, 2018). AAB cells were spiked into each food sample in a sterilized stomacher bag (JS-UniTech, Seoul, Korea). The AAB inoculation levels were 4–5 log CFU/g. The inoculated liquid food samples (wine, yogurt, and vinegar) were diluted at a 1:10 ratio in phosphate-buffered saline (PBS, pH 7.4; Sigma), and inoculated solid food samples (kimchi and fruits) were rinsed with an equal volume of PBS. Serial dilutions were performed to generate 30–300 colonies on the medium, and 100 μL of diluted or rinsed PBS was spread-plated on GYC and ABS, respectively. The plates were observed and enumerated after incubation at 30 °C for 72 h.

2. Materials and methods 2.1. Culture media ABS, the novel culture medium, was formulated after preliminary optimization study (patent pending) as follows. D-(+) Glucose 50 g (Sigma, St. Louis, MO, USA), yeast extract 10 g (Sigma), bromophenol blue 20 mg (Sigma), and bacteriological agar 20 g (Oxoid, Basingstoke, Hampshire, UK) were dissolved in 1 L of distilled water and autoclaved at 121 °C for 15 min. After cooling in a water bath at 50 °C, 1 mL of glacial acetic acid (Junsei, Tokyo, Japan), 50 mL of pure ethanol (Merck, Darmstadt, Germany), and 5000 U of penicillin dissolved in distilled water were added to the medium. After thorough mixing, 20 mL of the medium was poured into each Petri dish. The GYC medium included 50 g/L glucose (Sigma), 10 g/L yeast extract (Sigma), 5 g/L CaCO3 (Sigma), and 20 g/L agar (Oxoid). After autoclaving, 20 mL of medium was poured into each Petri dish. The pH of the ABS plate was 4.8 ± 0.1 at 25 °C. All plates were stored at 4 °C and used within 14 days.

2.4. Performance of the novel medium for isolating new AAB from acidic food samples In total, 10 vinegar and 10 kimchi samples were purchased in Korea for AAB isolation. The vinegar samples were diluted 1:10 in PBS (Sigma), and 100 μL was spread-plated on ABS and GYC. The plates were observed after incubation at 30 °C for 72 h. Colonies obtained on ABS and GYC were identified based on 16S rRNA sequence identity using BLAST according to our previous study (Kim et al., 2017).

2.2. Microbial strains and inclusivity/exclusivity test

2.5. Statistical analysis

A total of 16 AAB and 21 non-AAB strains were used in this study. AAB strains included Acetobacter aceti ATCC 15973, Acetobacter aceti IFO 3281, Acetobacter estunensis ATCC 23753, Acetobacter liquefaciens ATCC 14835, Acetobacter orleanensis ATCC 12876, Acetobacter pasteurianus ATCC 12875, Acetobacter pasteurianus ATCC 7839, Acetobacter persicus A396, Gluconacetobacter hansenii ATCC 35959, Gluconacetobacter medellinensis IFO 3288, Gluconacetobacter xylinus subsp. xylinus ATCC 11142, Gluconobacter cerinus ATCC 49206,

The inclusivity and exclusivity of ABS and GYC were calculated as follow: inclusivity (%) = (the number of AAB strains recovered on each plate/total number of AAB strains tested) × 100; exclusivity (%) = (the number of non-AAB strains inhibited on each plate/total number of non-AAB strains tested) × 100. The inclusivity and exclusivity of both media were compared by Fisher's exact tests using GraphPad Instat 3 (GraphPad Instat Software, Inc., La Jolla, CA, USA). Colony counts in food samples were converted to log CFU/g and analyzed by two-tailed 2

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AAB are ubiquitous microorganisms found in many types of food (Kommanee et al., 2012), where they act as fermentation starters (e.g., in natural fermented vinegar and fermented milk products) or as contaminants (e.g., in alcoholic beverages, such as wine and beer) (Torija et al., 2010). In addition, AAB demonstrate probiotic qualities, such as acid and bile resistance, activity against microbial pathogens, and anticancer properties (Haghshenas et al., 2015; Aghazadeh, Pouralibaba, & Khosroushahi, 2017). Therefore, it is important to assess the presence of AAB in food samples using reliable culture methods. Although several molecular assays have been developed, culture methods are still essential to identify and characterize AAB populations (Vegas et al., 2010). Some culture media have been recommended including YPM (0.5% yeast extract, 0.3% peptone, 2.5% mannitol, 1.5% agar), GYC (1% yeast 3

3.85 ± 0.12B 4.36 ± 0.18B 3.73 ± 0.11B 4.25 ± 0.14A 4.81 ± 0.09A 4.08 ± 0.14A 4.02 ± 0.14A 4.53 ± 0.15B 3.82 ± 0.20A 4.13 ± 0.13A 4.87 ± 0.07A 4.02 ± 0.11A 3.80 ± 0.14B 4.68 ± 0.15A 3.79 ± 0.08B 4.24 ± 0.17A 4.79 ± 0.20A 4.04 ± 0.07A 4.36 ± 0.17A 4.95 ± 0.08A 4.15 ± 0.13A 4.37 ± 0.11A 4.92 ± 0.09A 4.14 ± 0.20A 4.31 ± 0.15A 4.89 ± 0.17A 4.13 ± 0.25A 4.30 ± 0.22A 4.94 ± 0.10A 4.18 ± 0.18A

GYC ABS GYC

ABS

GYC

ABS

GYC

Kimchi Fruit

4.49 ± 0.12 5.07 ± 0.09 4.32 ± 0.12

4. Discussion

Acetobacter aceti ATCC 15973 Gluconobacter oxydans ATCC 15163 Gluconacetobacter hansenii ATCC 35959

From 10 vinegar and 10 kimchi samples, a total of five AAB strains were isolated, as shown in Table 2. The isolates showed a uniform colony morphology but differed in the time needed to form a visible colony (Table 2). The isolates were confirmed by 16S rRNA gene sequencing, and the sequences have been deposited in the GenBank database (www.ncbi.nlm.nih.gov/Genbank/; Table 2).

ABS

3.3. Performance of the novel medium for isolating new AAB from acidic food samples

GYC

The enumeration of AAB from artificial AAB-positive samples using ABS and GYC is summarized in Table 1, and the observations for the respective culture plates are represented in Fig. 1. In food samples with no competing microbiota (mesophilic aerobic bacterial count < 2 log CFU/ml of samples), such as wine and vinegar, all three AAB species grew well on both media, and there were no significant differences in recovered AAB cells between ABS and GYC (Table 1). In contrast, in food samples with competing microbiota, such as yogurt and kimchi (mesophilic aerobic bacterial count, 7.24 ± 0.37 and 6.33 ± 0.54 log CFU/ml or g of samples, respectively), and fruit (mesophilic aerobic bacterial count, 2.28 ± 0.42 log CFU/g of samples), ABS recovered a significantly higher number of AAB colonies than GYC (p < 0.05, Table 1), and it was difficult to differentiate and isolate AAB cells on GYC (Fig. 1). Remarkably, AAB colonies could be easily differentiated and were not covered by competing microbiota on ABS, facilitating the enumeration and isolation of colonies (Fig. 1). Especially, the same results were observed, regardless of the genus and species of inoculated AAB.

ABS

3.2. Performance of the novel medium for enumeration of AAB

Yogurt

All 16 standard AAB strains exhibited growth on both ABS and GYC agar. Non-AAB strains did not grow on ABS, whereas all 12 strains including B. subtilis KU18601, B. siamensis KU18602, T. delbrueckii KU18603, W. hellenica KU18701, L. mesenteroides KU18702, S. servazzii KU18703, B. luti KU18704, B. megaterium KU18705, L. citreum KU18706, L. lactis subsp. lactis KU18104, L. lactis subsp. cremoris KU18105, and E. durans KU16801 grew on GYC. Therefore, ABS showed the same inclusivity (ABS, 16 out of 16 strains, 100% vs. GYC, 16 out of 16 strains, 100%) and better exclusivity than GYC (ABS, 0 out of 21 strains, 100% vs. GYC, 9 out of 21 strains, 42.85%; p < 0.05, Fisher's exact tests).

Vinegar

3.1. Inclusivity and exclusivity

Wine

3. Results

Inoculum

Student's t-tests using SPSS (version 18.0, SPSS Inc., Chicago, IL, USA). A p-value of less than 0.05 was considered statistically significant.

Strain

Table 1 Enumeration of acetic acid bacteria (AAB) from artificial AAB-positive food samples using acetic acid bacteria-selective media (ABS) and glucose-yeast extract-calcium carbonate agar (GYC). Each food sample was artificially inoculated with Acetobacter aceti ATCC 15973, Gluconobacter oxydans ATCC 15163, and Gluconacetobacter hansenii ATCC 35959, and the diluents or rinsates were spread-plated on both media to enumerate AAB colonies. The plates were observed after 72 h of incubation at 30 °C, aerobically. Counts are expressed as log CFU per milliliter or gram of samples. The experiments were performed in quadruplicate.

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Fig. 1. Recovery of acetic acid bacteria (AAB) from artificial AAB-positive food samples using acetic acid bacteria-selective media (ABS) and glucose-yeast extractcalcium carbonate agar (GYC). Each food sample was artificially inoculated with Acetobacter aceti ATCC 15973, Gluconobacter oxydans ATCC 15163, and Gluconacetobacter hansenii ATCC 35959, and the diluents or rinsates were spread-plated on both media to enumerate AAB colonies. The plates were observed after 72 h of incubation at 30 °C. AAB and non-AAB colonies are indicated by black and white arrows, respectively.

functional AAB strains (Vegas et al., 2010). To overcome this lack of exclusivity and recoverability, we developed a novel medium named as ABS for the isolation and enumeration of AAB from various food samples. The novel medium showed an excellent inclusivity and exclusivity, supporting the growth of all 16 AAB species and inhibiting 21 non-AAB species. In addition, ABS provided better visual differentiation and recoverability in AAB-spiked food samples, especially yogurt, fruit, and kimchi, harboring high levels of competing microbiota, which were failed in GYC. ABS enabled the formation of a green colony with a yellow halo, which is specific to AAB growth, surrounded by a navy-colored background of uncultured zone, which also helps researchers and lab workers find or isolate AAB readily. Bromophenol blue, a pH indicator, plays a pivotal role in the development of this green-to-yellow colony color by acid-producing microorganisms (Flores, 1978). In food samples, AAB usually co-exist with other microorganisms such as lactic acid bacteria, Bacillus spp., yeast, and other environmental microbes (Soni & Dey, 2014). To isolate and enumerate AAB selectively, it is essential to hinder the growth of these unwanted microorganisms. The strategy for AAB selection is to employ multiple selective agents when formulating the medium. In ABS, glacial acetic acid is a selective agent that lowers the pH of the medium to 4.8, at which a wide spectrum of food microorganisms including bacteria and yeast are unable to grow and form colonies (Jay, Loessner, & Golden,

extract, 10% glucose, 2% CaCO3, 1.5% agar), RAE agar (1% yeast extract, 4% glucose, 1% peptone, 0.338% Na2HPO4·2H2O, 0.15% citric acid × H2O, 2% (w/v) acetic acid, 1% (w/v) ethanol; bottom: 1% agar, top: 2% agar), AE media (0.5% glucose, 0.3% yeast extract, 0.4% peptone, 3% absolute ethanol, 3% glacial acetic acid), and AG media (0.1% glucose, 0.5% yeast extract, 0.5% peptone, 0.7% CaCO3, 0.15% glycerol, 0.2% malt extract, 1.5% agar), but no selective media are specifically available for AAB strains (Andres-Barrao, Weber, Chappuis, Theiler, & Barja, 2011; De Vero & Giudici, 2013). Taking a closer look at the formulation, it is evident that these media consist of common ingredients at different proportions (De Vero & Giudici, 2013). In addition, these agar media are not selective, which limits their application to only maintaining and storing AAB strains (De Vero & Giudici, 2013). Cirigliano (1982) developed dextrose sorbitol mannitol (DSM) agar (1% proteose peptone; 0.3% yeast extract; 1.5% calcium lactate; 0.1% dextrose; 0.1% d-sorbitol; 0.2% d-mannitol; 0.1% monopotassium phosphate; 0.002% manganese sulfate monohydrate; 0.003% bromocresol purple; 0.0004% cycloheximide; 0.0l% sodium desoxycholate or 0.00295% brilliant green; 1.5% agar) for the selective isolation of AAB and differentiation between Gluconobacter spp. and Acetobacter spp., but its performance in selective enumeration of AAB has not been addressed in food samples. Thus, the existing culture media cannot be used extensively for various types of food products to investigate the dynamics of AAB population, detect AAB contamination, and isolate novel

Table 2 Isolation of acetic acid bacteria (AAB) strains from vinegar and kimchi using acetic acid bacteria-selective media (ABS). No.

Species (%)

Sample

Colony morphology

1 2 3 4 5

Acetobacter pasteurianus Gluconacetobacter europaeus Gluconacetobacter kakiaceti Gluconacetobacter europaeus Gluconobacter frateurii

Makgeoli vinegar Blueberry vinegar Rice grain vinegar Persimmon vinegar Kimchi

Round, Round, Round, Round, Round,

convex, convex, convex, convex, convex,

4

green green green green green

colony colony colony colony colony

with with with with with

yellow yellow yellow yellow yellow

halo halo halo halo halo

Colony formation within

Accession No.

24–48 h 48–72 h 48–72 h 48–72 h 24–48 h

MH845625.1 MH845623.1 MH845617.1 MH845618.1 MN080427.1

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Appendix A. Supplementary data

2005). In contrast, a previous study reported that the optimal pH for AAB growth ranged from 3.0 to 5.0 (Sharafi, Rasooli, & Beheshti-Maal, 2010). In addition, some genera of AAB such as Acetobacter, Gluconoacetobacter, and Acidomonas are known to utilize acetic acid as an energy source (Cleenwerck & De Vos, 2008). Likewise, ethanol generally acts as an antimicrobial molecule against many food microorganisms, whereas AAB oxidize ethanol to acetic acid to generate energy (Bartowsky & Henschke, 2008). Penicillin is a selective agent for inhibiting the growth of gram-positive bacteria, such as Bacillus and lactic acid bacteria (Rice, 2006). In addition, aerobic culture conditions provide the preferred growth environment for AAB compared to lactic acid bacteria, as the former are obligate aerobes and the latter are facultative anaerobes (Cleenwerck & De Vos, 2008; Salvetti, Torriani, & Felis, 2012). Finally, the ABS plate was incubated up to 72 h to prevent the delayed growth of yeasts and molds (Jay et al., 2005). In addition to this formula, the selectivity of ABS could be further enhanced by the supplementation of 5 mg/L of cycloheximide to inhibit the growth of yeasts and molds in foods, especially samples with high levels of yeasts and molds (data not shown). Together, these components of ABS selectively support AAB growth, resulting in the formation of a green colony with a yellow halo, which differentiates them from other competing microorganisms. A large fraction of microorganisms in both natural and industrial environments are hard to culture, and their viability is also difficult to recover under standard laboratory conditions (Gullo et al., 2014). Similarly, AAB adapt to extreme food matrices such as wine and vinegar, thereby making it difficult to be selectively recovered on culture plates (Vegas et al., 2010). Accordingly, the detection or enumeration of AAB has been conducted by direct microscopic observation, direct epifluorescence methods (Andres-Barrao et al., 2011), and PCR-based molecular assays (González, Hierro, Poblet, Mas, & Guillamón, 2006). Although wines are contaminated with bacteria that are easily visible under a microscope, this is not the case for fermented milk or vegetables which naturally contain other fermentative microorganisms, making the differentiation of AAB impossible (Vegas et al., 2010). Notably, we successfully and selectively isolated novel AAB strains from vinegar and kimchi samples, indicating that the novel medium could be used in the food and biotechnological industry. Although there is heterogeneity among these isolates in the time needed to form colonies, which could be attributed to the difference in metabolic activities of these species (Bartowsky & Henschke, 2008), it is noteworthy that the colonies of all five isolates appeared alike, suggesting that ABS is able to provide a uniform morphology over various AAB compared to non-AAB microorganisms. In conclusion, we successfully developed a novel selective medium by formulating multiple carbon sources, selective and visualizing agents, and its superior performance over GYC for the detection, enumeration, and isolation of AAB were confirmed using artificially-contaminated and field food samples. This medium can provide an accurate and reliable method for AAB detection and quantification in fermented food production, and can facilitate the effective isolation of novel functional AAB strains from various sources.

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Conflicts of interest None. Acknowledgements This research was supported by the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (as part of the (multi-ministerial) Genome Technology to Business Translation Program) (Grant number 918015-2). In addition, this paper was supported by the KU Research Professor Program of Konkuk University. 5