Wine Microbial Consortium: Seasonal Sources and Vectors Linking Vineyard and Winery Environments
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sampling and Microbial Recovery
2.1.1. Samples Collection
- (a)
- Soil: five soil samples (30 g) were taken, four from the vineyard areas and one from a forest area far from the vineyard areas (Figure 1A). Sampling occurred during the four annual periods mentioned above.
- (b)
- Tree and vine bark: five samples were collected from five trees (one sample per tree) close to the vineyard area indicated in Figure 1B, including a wild olive (Olea europaea var. sylvestris Mill.), a cork oak (Quercus suber L.), a cypress (Cupressus sempervirens L.), a Japanese cheese wood (Pittosporum tobira Ait.), and from a cypress far from the vineyard area (Figure 1B). Tree samples were taken during the four annual periods mentioned above. The bark samples of two vines (one sample per vine) located in the two different plots were collected during the spring, véraison, and harvest seasons.
- (c)
- Insects: five yellow adhesive chromotropic traps (20 cm × 25 cm, Biosani, Portugal) were set for six days on the places indicated in Figure 1C, including four vines (Vitis vinifera L.) and on a cypress far from the vineyard area. Insects (five specimens in each trap) were randomly collected during the four periods mentioned above and were subjected to a simplified identification at Order level [24], considering the Orders Heteroptera and Homoptera as Hemiptera.
- (d)
- Vine leaves: five vine leaves were collected from the places indicated in Figure 1D during the spring, véraison, and harvest seasons.
- (e)
- Grapes: five healthy berries were collected in sterile plastic bags from the locations indicated in Figure 1D at véraison and harvest seasons. Five damaged berries were only collected at harvest time.
- (f)
- Winery: the winery and its equipment were analyzed during véraison and harvest. Five samples from the walls, vats, press, crusher, and cold-stabilizer (one sample each) were collected with cotton swabs.
- (g)
- Grape juices: one sample each of 250 mL red and white grape musts in the winery before starter addition were obtained during the harvest season and analyzed after processing.
2.1.2. Sample Suspension
2.1.3. Selective and Differential Media
2.2. Microbial Screening and Identification
2.2.1. Isolate Purification, Maintenance and Phenotypic Tests
2.2.2. DNA Extraction and PCR Amplification
Yeasts
Bacteria
3. Results and Discussion
3.1. Seasonal Isolation of Yeasts and Bacteria
3.2. Microbial Identification
3.2.1. Yeast Species
3.2.2. Acetic Acid Bacterial Species
3.2.3. Lactic Acid Bacterial Species
3.3. Origin and Dissemination of the WMC throughout the Year
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Season | Reservoir | Samples | Isolates | Bacteria | Yeasts | Presumptive Lactic Acid Bacteria | Presumptive Acetic Acid Bacteria | Ascomycetous Yeasts |
---|---|---|---|---|---|---|---|---|
Winter | Soil | 5 | 91 | 82 | 9 | 0 | 21 | 0 |
Bark | 5 | 82 | 44 | 38 | 0 | 6 | 5 | |
Insects | 25 | 67 | 48 | 19 | 0 | 6 | 5 | |
Total | 35 | 240 | 174 | 66 | 0 | 33 | 10 | |
Total/sample | - | 6.9 | 5.0 | 1.9 | 0 | 0.9 | 0.3 | |
Spring | Soil | 5 | 60 | 60 | 0 | 1 | 0 | 0 |
Tree and vine bark | 7 | 82 | 60 | 22 | 3 | 0 | 0 | |
Insects | 25 | 133 | 67 | 66 | 0 | 0 | 0 | |
Vine leaves | 5 | 41 | 25 | 16 | 4 | 0 | 12 | |
Total | 42 | 316 | 212 | 104 | 8 | 0 | 12 | |
Total/sample | - | 7.5 | 5.0 | 2.5 | 0.2 | 0 | 0.3 | |
Véraison | Soil | 5 | 75 | 54 | 21 | 2 | 9 | 0 |
Tree and vine bark | 7 | 71 | 46 | 25 | 2 | 10 | 3 | |
Insects | 25 | 130 | 84 | 46 | 13 | 2 | 10 | |
Vine leaves | 5 | 42 | 21 | 21 | 1 | 1 | 0 | |
Healthy grapes | 5 | 67 | 38 | 29 | 0 | 3 | 0 | |
Winery equipment | 5 | 37 | 37 | 0 | 0 | 0 | 0 | |
Total | 52 | 422 | 280 | 142 | 18 | 25 | 13 | |
Total/sample | - | 8.1 | 5.4 | 2.7 | 0.4 | 0.5 | 0.3 | |
Harvest | Soil | 5 | 45 | 45 | 0 | 4 | 5 | 0 |
Tree and vine bark | 7 | 76 | 58 | 18 | 3 | 4 | 1 | |
Insects | 25 | 84 | 71 | 13 | 25 | 6 | 4 | |
Vine leaves | 5 | 44 | 31 | 13 | 5 | 0 | 0 | |
Healthy grapes | 5 | 54 | 36 | 18 | 6 | 1 | 0 | |
Damaged grapes | 5 | 45 | 27 | 18 | 0 | 0 | 13 | |
Winery equipment | 5 | 11 | 9 | 2 | 0 | 0 | 2 | |
Must/enriched must | 5 | 74 | 18 | 58 | 9 | 5 | 58 | |
Fermented must | 1 | 13 | 0 | 11 | 0 | 0 | 11 | |
Total | 63 | 446 | 295 | 151 | 52 | 21 | 89 | |
Total/sample | - | 7.1 | 4.7 | 2.4 | 0.8 | 0.3 | 1.4 | |
Annual total | 192 | 1424 | 961 | 463 | 78 | 79 | 114 | |
Total/sample | - | 7.4 | 5 | 2.4 | 0.4 | 0.4 | 0.6 |
Season | Species | Insects Far from Vineyard | Insects in the Vineyard | Tree Bark Close to Vineyard | Vine Bark | Damaged White Grapes |
---|---|---|---|---|---|---|
Winter | Debaryomyces hansenii | 3 | ||||
D. robertsiae | 4 | 1 | ||||
Spring | Candida cellae | 4 | ||||
C. parapsilopsis | 1 | 3 | ||||
Metschnikowia spp. | 1 | |||||
M. chrysoperlae | 3 | |||||
Véraison | C. albicans | 2 | ||||
C. apicola | 1 | |||||
C. hawaiiana | 2 | |||||
D. fabryi | 1 | |||||
D. hansenii | 1 | |||||
Metschnikowia spp. | 1 | |||||
M. reukaufii | 1 | |||||
Harvest | C. hawaiiana | 1 | ||||
C. infanticola | 1 | |||||
Hanseniaspora optuntiae | 1 | |||||
H. uvarum | 1 | 2 | ||||
Pichia ciferrii | 1 | |||||
P. guilliermondii | 1 |
Season | Species | Non-Enriched Must | Enriched Must | Fermented White Juice | Winery Equipment |
---|---|---|---|---|---|
Harvest | Candida boidinii | 1 | 1 | ||
C. diversa | 2 | ||||
C. hellenica | 1 | ||||
C. zemplinina | 1 | 1 | |||
Hanseniaspora uvarum | 1 | 2 | |||
Issatchenkia terricola | 2 | 1 | |||
Lachancea thermotolerans | 1 | 1 | |||
Metschnikowia spp. | 1 | ||||
M. pulcherrima | 1 | 1 | 1 | ||
P. fermentans | 1 | 1 | |||
P. kluyveri | 1 | 1 | |||
P. manshurica | 2 | ||||
P. occidentalis | 1 | ||||
Saccharomyces cerevisiae | 1 | 1 | |||
Zygosaccharomyces bailii | 1 |
Season | Species | Vineyard Soil | Soil Far from Vinery | Tree Bark Close to Vineyard | Tree Far from Winery |
---|---|---|---|---|---|
Winter | Acetobacter tropicalis | 1 | |||
Acidisphaera rubrifaciens | 1 | ||||
Gluconobacter oxydans | 5 | 1 | 1 | ||
Gluconacetobacter asukensis | 5 | 1 | |||
Gl. liquefaciens | 1 | ||||
Granulibacter bethesdensis | 1 | ||||
Rhodopila globiformis | 1 | ||||
Roseomonas cervicalis | 1 | ||||
Harvest | Acidiphilium spp. | 1 | |||
G. cerinus | 1 | ||||
G. oxydans | 2 |
Season | Species | Soil Vineyard | Insects in the Vineyard | Insects Far from Vineyard | Tree Bark Close to Vineyard | Vine Leaf | Sound Berry | White Grape Must |
---|---|---|---|---|---|---|---|---|
Spring | Enterococcus sp. | 1 | ||||||
E. faecalis | 1 | |||||||
E. faecium | 1 | |||||||
Lactococcus lactis | 1 | 2 | ||||||
Véraison | E. casseliflavus | 1 | ||||||
E. faecalis | 1 | |||||||
E. gallinarum | 1 | |||||||
Lactobacillus kunkeei | 1 | |||||||
La. lactis | 2 | |||||||
Leuconostoc lactis | 1 | 1 | ||||||
Le. mesenteroides | 1 | |||||||
Vagococcus carniphilus | 1 | |||||||
V. teuberi | 1 | |||||||
Harvest | Enterococcus spp. | 1 | ||||||
E. absuriae | 1 | |||||||
E. faecalis | 5 | 1 | ||||||
E. faecium | 1 | 1 | 2 | 1 | ||||
E. gallinarum | 1 | 1 | ||||||
E. hirae | 1 | |||||||
E. lactis | 1 | 1 | 1 | |||||
E. mundtii | 1 | |||||||
E. pallens | 1 | |||||||
E. raffini | 1 | |||||||
La. lactis | 1 | 2 | 1 | 2 | ||||
Le. mesenteroides | 1 | |||||||
Le. pseudomesenteroides | 1 | |||||||
Fructobacillus tropaeoli | 1 |
Technological Significance | Species | Reservoir | Season |
---|---|---|---|
Innocuous yeasts | Candida parapsilopsis | Insects | Spring |
C. apicola | Insects | Véraison | |
C. diversa | Grape must | Harvest | |
C. zemplinina | Grape must | Harvest | |
Debaryomyces hansenii | Tree bark | Winter, véraison | |
Hanseniaspora uvarum | Insects, damaged grapes, grape must | Harvest | |
Issatchenkia terricola | Grape must | Harvest | |
Lachancea thermotolerans | Grape must | Harvest | |
Metschnikowia pulcherrima | Grape must, winery | Harvest | |
Pichia fermentans | Grape must | Harvest | |
P. guilliermondii | Damaged grapes | Harvest | |
P. kluyveri | Grape must | Harvest | |
P. occidentalis | Fermented must | Harvest | |
Fermenting yeasts | Saccharomyces cerevisiae | Fermented must, winery | Harvest |
Spoilage yeasts | Zygosaccharomyces bailii | Fermented must | Harvest |
Acetic acid bacteria | Acetobacter tropicalis | Soil | Winter |
Gluconobacter oxydans | Soil, tree bark | Winter, harvest | |
G. cerinus | Soil | Harvest | |
G. liquefaciens | Soil | Winter | |
Spoilage lactic acid bacteria | Lactobacillus kunkeei | Insects | Véraison |
Leuconostoc mesenteroides | Insects | Véraison, harvest |
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Camilo, S.; Chandra, M.; Branco, P.; Malfeito-Ferreira, M. Wine Microbial Consortium: Seasonal Sources and Vectors Linking Vineyard and Winery Environments. Fermentation 2022, 8, 324. https://doi.org/10.3390/fermentation8070324
Camilo S, Chandra M, Branco P, Malfeito-Ferreira M. Wine Microbial Consortium: Seasonal Sources and Vectors Linking Vineyard and Winery Environments. Fermentation. 2022; 8(7):324. https://doi.org/10.3390/fermentation8070324
Chicago/Turabian StyleCamilo, Sofia, Mahesh Chandra, Patrícia Branco, and Manuel Malfeito-Ferreira. 2022. "Wine Microbial Consortium: Seasonal Sources and Vectors Linking Vineyard and Winery Environments" Fermentation 8, no. 7: 324. https://doi.org/10.3390/fermentation8070324
APA StyleCamilo, S., Chandra, M., Branco, P., & Malfeito-Ferreira, M. (2022). Wine Microbial Consortium: Seasonal Sources and Vectors Linking Vineyard and Winery Environments. Fermentation, 8(7), 324. https://doi.org/10.3390/fermentation8070324