149
Diversity of Bacteria and Yeasts on the Surface of
Table Grapes
Attila Kántor*, Miroslava Kačániová
Slovak University of Agriculture in Nitra, 949 76, Tr. A. Hlinku 2, Nitra
Abstract
Grapes are natural habitat for many different groups of microorganisms. The main groups of microorganisms that colonize the surface of the grapes are: yeasts, acetic acid bacteria and lactic acid bacteria. Grape berry surface is an unstable habitat that changes greatly according to the stage of grape ripening. The aim of this study was to isolate and identified wide range of different groups of microorganisms which colonize the grape berries. We used table grape cultivars Red Globe (red) and Sundance (white) in this study. Table grapes are grapes intended for consumption and different than grapes which use in winemaking. Bacterial and yeast species identified by MALDI-TOF MS Biotyper. We determine 12 yeasts and 30 species of bacteria. The dominant genera of microorganisms were
Bacillus, Candida, Lactobacillus, Staphylococcus and Aureobasidium. We also identified 4 different strains of Saccharomyces cerevisiae.
Keywords: microorganisms; table grapes; identification; MALDI-TOF
1. Introduction
All papers will be written in English. The journal The presence of yeasts and bacterial species on grape berry surfaces varies at the quantitative and qualitative levels as functions of several factors [1-5]. Grape berry surface is an unstable habitat that changes greatly according to the stage of grape ripening, which is itself dependent on several environmental factors, such as: rapid changes in temperature, humidity and UV radiation, nutritive limitations, and the application of agrochemical treatments [6-11]. The study of the microbial communities of grapes is usually addressed to berries, being well established that mature grapes harbour microbial populations at levels of 104–106 CFU.g-1 consisting mostly of yeasts and various species of lactic and acetic acid
* Corresponding author: Mgr. Attila Kántor, +421376415812, atic@centrum.sk
150 agents, stimulating plant growth and health [20-22]. Most research has concentrated on bacteria of oenological interest, like acetic and lactic acid bacteria [23-25], present in the microflora on grape berries. Little information is available about the effects of epiphytic bacteria on other plant parts, like leaves and bark. Despite their importance, the diversity of epiphytic bacteria on grape berries remains poorly described, as is the role of other plant parts and vineyard soil in bacterial colonization. Soil microorganisms are able to colonize parts of the plant above the ground, including leaves and fruit [26, 27]. The surface of grape berries represents a natural habitat for bacterial microbiota that has various impacts on the sanitary quality of grapes [28]. Aim of this study was to isolate bacteria and yeast species from the surface of table grapes and identify them with MALDI Biotyper.
2. Materials and Methods
Grape berries: We used two table grape cultivars
for this experiment. Table grapes was purchased in the local shop and aseptically inserted into the polybag. The first one was white seedless grape cultivar “Sundance” and the second was red seeded grape cultivar “Red globe”. Sundance has medium sized round shaped berries which are firm and crunchy. Red globe has very large round berries, and the ripe berries are mildly sweet and flavourful.
Preparation of berries: Grapes were removed
from bunches and separately inserted into the conical flask. The volume of conical flask was 1000 mL. Fifty berries placed into the conical flask from smaller white cultivar and 25 berries from larger red cultivar separately. After that, were poured physiological saline to the berries. We have added 250 mL to white berries and 500 mL to red berries. We placed the conical flasks into the laboratory shaker for 15 minutes at 200 rpm. Then we added 100 µL of not diluted sample on the surface of each cultivation media two times (first isolation). The conical flasks with berries in physiological saline were stored for a week at room temperature (25 ± 2°C) and after that we isolate the microorganisms, this was a second isolation.
Cultivation media: For detection of wide range of
bacterial and yeast species used six different cultivation media namely: MacConkey agar (Biolife, Italy), Nutrient agar (NA) (Biomark, India) MRS (Conda, Spain), Acetobacter agar with glucose (HiMedia, India), Malt extract agar with bromocresol green (MEA+BG) (Biomark, India) and Wild Yeast medium (HiMedia, India). Culture conditions were different for each group of bacteria or yeasts. Coliform bacteria cultivated on MacConkey agar at 37 °C for 24 h. Lactic acid bacteria cultivated on MRS at 37 °C for 72 h. Acetic acid bacteria and other common soil or plant bacteria cultivated on Acetobacter agar and nutrient agar at 30 °C for 48 h. Finally, yeast cultivated on malt extract agar with bromocresol green (0,020 g/L) and wild yeast medium for 3-5 days at 25 and 30 °C.
MALDI-TOF Mass Spectrometer: We used
151 with Realtime Classification software (RTC) (Bruker Daltonics, Germany).
3. Results and discussion
We identified 30 species of bacteria, mostly belonged to genus Bacillus, Staphylococcus, Lactobacillus and Microbacterium. We also identified 9 different genera of yeasts and 4 strains of Saccharomyces cerevisiae (total 12 species). Results from first isolation have been shown in table 1. As you seen we obtained total 97 isolates in first isolation. Every sample was created in duplicate and all isolated microorganisms was counted and recorded in table 1. In first isolation we not detected coliform bacteria on the grape berry surface. Also a few yeasts looking microorganisms detected at first isolation, just 7 colonies. We not detected lactic acid bacteria and wild yeast at first isolation. The highest number of detected bacteria was detected on nutrient and acetobacter agar, but after MALDI-TOF determination we not determine any acetic acid bacteria.
Table 1. Isolates number of bacteria and yeast 1st isolation Grape cultivar Cultivation media Sundance Red globe
MacConkey agar ND ND
Acetobacter agar 35 5
MRS 0 0
Nutrient agar 41 9
Malt Extract agar 4 3
Wild Yeast medium 0 0
ND: not detected
In first isolation from 97 isolates (80 isolates from Sundance and 17 isolates from Red globe) identified Bacillus species namely: Bacillus megaterium, Bacillus niacini and Bacillus cereus. We also identified many species of dermal staphylococci. The most often species was Staphylococcus warneri and Staphylococcus epidermidis. We identified Micrococcus luteus, Sphingomonas sp., Pseudomonas kilonensis and Microbacterium arborescens from nutrient agar. These species also grow on acetobacter agar. From yeast species we isolated just Kloeckera apiculata in first isolation. Overall we identified
10 species of microorganisms in first isolation. We discovered and identified more species after one week incubation of table grape berries at room temperature. The second isolation obtains more bacteria and yeast species than the first. Total we counted 1370 colonies of microorganisms in Sundance table grape and 523 colonies of microorganisms in Red globe table grape. We not detected bacteria on MacConkey and yeasts on Wild Yeast medium. Table 2 shows the results from second isolation after one week incubation of samples at room temperature.
Table 2. Isolates number of bacteria and yeast 2nd isolation Grape cultivar Cultivation media Sundance Red globe
MacConkey agar 12 ND
Acetobacter agar 340 31
MRS 91 264
Nutrient agar 451 196
Malt Extract agar 419 32
Wild Yeast medium 57 ND
*Results from count duplicate samples.
152
Table 3. All identified bacteria by MALDI-TOF
Bacterial species Gram stain Sundance Red globe
Acinetobacter junii G- ND D
Acinetobacter lwoffi G- ND D
Arthrobacter citreus G+ D ND
Arthrobacter tumbae G+ ND D
Bacillus cereus G+ D ND
Bacillus megaterium G+ D D
Bacillus mycoides G+ D ND
Bacillus niacini G+ D ND
Bacillus pumilus G+ ND D
Brevundimonas vesicularis G- ND D
Cellulosimicrobium cellulans G+ ND D
Lactobacillus acidophillus G+ D D
Lactobacillus bifermentans G+ D D
Lactobacillus nageli G+ ND D
Lactobacillus parabuchneri G+ ND D
Lactobacillus paracasei G+ D D
Lactobacillus plantarum G+ D D
Leuconostoc mesenteroides G+ D D
Microbacterium arborescens G+ D ND
Microbacterium oleivorans G+ D ND
Microbacterium oxydans G+ ND D
Micrococcus luteus G+ D D
Paenibacillus pabuli G+ ND D
Pantoea dispersa G- D ND
Pseudomonas kilonensis G- ND D
Sphingomonas sp. G- D D
Staphylococcus epidermidis G+ D D
Staphylococcus equorum G+ D ND
Staphylococcus hominis G+ D ND
Staphylococcus warneri G+ D D
153 As you can see in the table 3, we identified 30 species of bacteria belonged to 14 genera and mostly gram positive bacterial species (80%) than gram negative species (20%). From surface of the Sundance grape identified 19 species of bacteria and from Red globe surface 21 species. Some species were detected at both of grape varieties, for example Sphingomonas sp., Micrococcus luteus, Leuconostoc mesenteroides, Lactobacillus
paracasei, Staphylococcus warneri or Bacillus megaterium etc. Yeast species were detected at higher number in second isolation on the surface of Sundance grape. Mostly we have isolated Candida species, but also 4 strains of Saccharomyces cerevisiae. Table 4 shows the results from identified yeast species. We identified 5 Candida species, Candida valida, C. utilis, C. sorbosa, C. krusei and C. saitoana.
Table 4. All identified yeasts by MALDI-TOF
Anamorph [Teleomorph] Sundance Red globe
Candida valida [Pichia membranifaciens] D D Candida utilis [Pichia jandinii] ND D
Candida sorbosa [Pichia occidentalis] D ND
Candida krusei [Issatchenkia orientalis] D ND
Candida saitoana D ND
Kloeckera apiculata [Hanseniaspora uvarum] ND D
Cryptococcus diffluens ND D
Aureobasidium pullulans D D
Saccharomyces cerevisiae INVSc1 BRL ND D Saccharomyces cerevisiae BJ2168 BRL D ND
Saccharomyces cerevisiae DSM 1334 DSM D ND
Saccharomyces cerevisiae WS LLH ND D
D: detected, ND: not detected
Candida valida and Aureobasidium pullulans were identified at both of the grape varieties after one week incubation at room temperature (25 ± 2 °C). Other species identified just at one of the grape variety. On the surface of Sundance grape were identified 7 species and on the surface of Red globe also 7 species. Kloeckera apiculata was identified on the surface of Red globe grape. Kloeckera apiculata, Metschnikowia pulcherrima and Candida spp. belongs to the natural microflora of grape berries [12]. In this study not identified Metschnikowia species on the surface of table grapes. In fact table grapes are grapes intended for consumption and different than grapes which use in winemaking.
4. Conclusions
In this study we identified 30 species of bacteria belonging to 14 genera (5 Gram negative and 9 Gram positive) and 12 yeasts belonging to 5 genera. Total we identify 42 microorganism species by MALDI-TOF Mass Spectrometer after one week storage of grapes at room temperature.
Acknowledgements
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