La microbiología a la disposición de la producción de vinagres
Caracterización microbiológica de la biomasa presente en los
acetificadores
Proyecto WINEGAR
F. BARJA
0
2
4
6
8
10
12
0 50 90 182 231 345 399
Tiempo (h)
°AcH
, °Et
OH
-1.00E+08
5.00E+07
2.00E+08
3.50E+08
5.00E+08
6.50E+08
8.00E+08
Bac
teria
s/m
l
(°AcH)(°EtOH)Biomasa
TRANSMISSION ELECTRON MICROCOPY ANALYIS OF AAB FROM INDUSTRIAL ACETATOR
Microscopia electrónica de barrido de muestras tomadas directamente del fermentador pilote
SCANNING ELECTRON MICROSCOPY OF ABB FROM SOLID MEDIA CULTURE AND ACETATOR
1 µm
1 µm1 µm
1 µm
A
C
B
TRANSMISSION ELECTRON MICROCOPY OF polysaccharides in acetic acid bacteria
24.85%21.71%50.14%50.71%17.14%63.14%100%
7.78 E+071.01 E+085.56 E+089.69 E+086.56 E+081.00 E+092.82 E+09
7.75 E+075.56 E+077.45 E+081.01 E+098.58 E+078.86 E+081.20 E+09
8.58 E+088.21 E+088.25 E+082.23 E+081.23 E+089.02 E+081.05 E+09
8.58 E+088.59 E+075.56 E+077.75 E+084.43 E+088.89 E+081.50 E+09
7.38 E+074.35 E+052.35 E+061.15 E+085.28 E+063.35 E+086.00 E+08
6.36 E+061.25 E+084.35 E+083.35 E+074.53 E+074.25 E+085.35 E+08
5.56 E+077.78 E+071.78 E+082.24 E+087.75 E+079.65 E+071.60 E+08
YPE 4-3%YPE 2-3%YPE 4%YPE 2%V-VM 10Cámara (tot)
ACETIC ACID BACTERIA INNOCULATED IN SOLIDE MEDIA (M10)ACETIC ACID BACTERIA INNOCULATED IN SOLIDE MEDIA (M10)
START OF PROCESSSTART OF PROCESS MIDDLE OF PROCESSMIDDLE OF PROCESS
END OF PROCESSEND OF PROCESS
Ratio of cell colonies during the cycle on industrial vinegar production
End of the cycle
white colonies
Yellow colonies
Red colonies
16% 8%
76%
Midle of the cycle
white colonies
Yellow colonies
Red colonies
28% 8%
64%
Start of the cycle
white colonies
Yellow colonies
Red colonies
11% 6%
83%
Muestras aisladas desde colonias blancas en distintos medios de cultivo
Muestras aisladas desde colonias amarillas en distintos medios de cultivo
Ratio of Taq I restriction patterns after amplification of 16S rDNA of Yellow, White and Red colonies
White colonies
A.aceti
A. pasteurianus
30%A. Pasteurianus
70%A. aceti
Yelow colonies
A.aceti
A. pasteurianus
N. D.
20%A. Pasteurianus
N.D. 10% 70%A. aceti
Red colonies
A. pasteurianus
100%A. Pasteurianus
Fermentation stage Alcool concentration (%) Acetic acid concentration (%) Total concentration (%) Microorganisms (%)
Initial 4 - 5 5 - 6 10 - 11 Acet. Aceti 62.3
Acet. Pasteurianus 29.4
n.d. 8.3
Mid-fermentation 2 - 3 8 - 9 10 - 11 Acet. Aceti 50.4
Acet. Pasteurianus 43.2
n.d. 6.4
End of fermentation 0.5 -1 9 - 10 10 - 11 Acet. Aceti 58.8
Acet. Pasteurianus 33.6
n.d. 7.6
Table 2 Evolution of acetic acid bacteria during red wine vinegar process cycle
24.85%21.71%50.14%50.71%17.14%63.14%100%
7.78 E+071.01 E+085.56 E+089.69 E+086.56 E+081.00 E+092.82 E+09
7.75 E+075.56 E+077.45 E+081.01 E+098.58 E+078.86 E+081.20 E+09
8.58 E+088.21 E+088.25 E+082.23 E+081.23 E+089.02 E+081.05 E+09
8.58 E+088.59 E+075.56 E+077.75 E+084.43 E+088.89 E+081.50 E+09
7.38 E+074.35 E+052.35 E+061.15 E+085.28 E+063.35 E+086.00 E+08
6.36 E+061.25 E+084.35 E+083.35 E+074.53 E+074.25 E+085.35 E+08
5.56 E+077.78 E+071.78 E+082.24 E+087.75 E+079.65 E+071.60 E+08
YPE 4-3%YPE 2-3%YPE 4%YPE 2%V-VM 10Cámara (tot)
Direct Epifluorescent Filter technique (DEFT)Direct Epifluorescent Filter technique (DEFT)
13 mm
Centrifugation
8000 g, 10 min
Buffer or water(1 ml)
Fluorochromes,Methods
B
D
A
C
Bacterial cells recovered from industrial acetators by membrane filtration, stained with different dyes and visualized by
epifluorescent microscopy. A) BacLight. B) BacLight, killed controls. C) DAPI. D) CTC.
Direct Epifluorescent Filter technique (DEFT)Direct Epifluorescent Filter technique (DEFT)
13 mm
Centrifugation
8000 g, 10 min
Buffer or water(1 ml)
Fluorochromes,Methods
• CTC/DAPI staining
• CTC was used in conjugation with DAPI to differentiate metabolically active cells from inactive cells
• The combined use of DAPI / CTC presented some complications.
Direct Epifluorescent Filter technique (DEFT)Direct Epifluorescent Filter technique (DEFT)
• Estimation of number cells:
DVAFCN ×
××=
N = Cells ml-1C = Number of cellsF = Filtration area (mm2)A = Observation field area (mm2)V = Volume of sample filteredD = Dilution factor
• The relationship between the direct countmethods was determined using regressionanalysis
4.00
6.00
8.00
10.00
12.00
4.00 6.00 8.00 10.00 12.00
DAPI total countslog cells/ml
BacL
ight
tota
l cou
nts
log
cells
/ml
r2=0.99
Relationship between enumeration of total bacteria using BacLight and DAPI (strain CECT 474)
4.00
5.00
6.00
7.00
4.00 5.00 6.00 7.00
CTC viable countslog cells/ml
BacL
ight
via
ble
coun
tslo
g ce
lls/m
l
r2=0.99
Relationship between enumeration of viable bacteria using BacLight and CTC (samples from industrial acetators).
• Determination of viable and non viable cells concentration with epifluorescence staining technique.
vinegarsample
direct counting afterincubation at roomtemperature in thedark for 15 minutes
dilution (sample/water)
(1 ml / 5 ml)
(sample/BacLightTM dyes)
(1 ml/1.5 µl SYTO9/1.5 µl Propid. iodide)
• LIVE/DEAD® BacLightTM Bacterial Viability Kits.
· SYTO® 9 green-fluorescent nucleic acid stain.· Propidium iodide red-fluorescent nucleic acid stain.
· Bacteria with intact cell membranes stain fluorescent green.
· Bacteria with damaged membranes stain fluorescent red.
· Background remains nonfluorescent.
5 cells in thismini square
4 viable cells
1 non viable cells
• Counting cells in each mini-square.• Green: viable
• Red: non-viable
Filter: Olympus MNB2 Filter: Olympus MNG2
• Estimation of number cells.
Concentration cells = x F(cells / ml) S x D
C
Where:
C = Counted cells (cells)
S = Counted squares surface (mm2)
D = Chamber depth (mm)
F = dilution factor
CONTAMINACIONES BIOLOGICAS POSIBLES EN VINAGRES
Threeday
growth
offungifromvinegar
M10
MA
LT
MF 4
MF 2
MF 3
MF 1
Fig. 3
A B
C D
Vinaigre de cidre biologique (A) (B)
A B
A B
Comparaison entre bactéries acétiques et bactéries lactiques
A1 µm 1 µm
B
A. Bactéries acétiques grossies 10’000 fois
B. Bactéries lactiques grossies 10’000 fois
Comparaison entre bactéries acétiques et bactéries lactiques
1-15(variable, dépend de l’espèce)
-+-bactéries lactiques
1-4+-+bactériesacétiques
Taille (µm)CatalaseGram +Gram -
Diminution de l'acidité lors du stockage du vinaigre
0
1
2
3
4
5
6
7
0 18Temps [mois]
Aci
dité
[%]
Acidité partie sup.Acidité partie moy.Acidité partie inf.
WIN
E
Bitterness Bacteria (threads) Pasteurization
Tourne Bacterium. tartarophthorum SO2
Oxidation of alcoholto CO2 and H2O
Yeasts (Mycoderma vini)on the tank surface
Addition of filtered vinegar to the mash,total acidity 1,5%,time of contact: 3 weeks
Vinegar eels(in wine vinegar also)
Turbatrix aceti Filtering cartridge in charging pipe,sterilizing with H2O2 + peracetic acid
VIN
EGA
R
diseases their causes means of prevention
Ferm
enta
tion
stop
page
s
Lactic acid bacteria
Examination of wine and vinegar under the microscope. Mash to be tested on Pilot Acetator, to ascertainthat it is not infected. If sodisinfection with H2O2.
Budding yeasts in spirit vinegar, molds:genus Moniliellagenus Sporotrichum
Hygiene, disinfection(see before)
Acetobacter peroxydans,too low total concentration, mutations. Overoxidation ofacetic acid in CO2 and H2O
Total concentration (TC) of 8-10, culti-vate selected strain on Pilot Acetator
Heavy metals(Cd, Hg, Pb, Zn, Cu)
Control of raw material
Phage: fermenter near generator (wood shavings)
Acetobacter xylinusCloudy vinegar(mothers of vinegar)
Ultra-violet rays in charging pipe,separation of vinegar produced on Acetator and generator
Sterile filtrationStrict hygiene
WIN
E
Bitterness Bacteria (threads) Pasteurization
Tourne Bacterium. tartarophthorum SO2
Oxidation of alcoholto CO2 and H2O
Yeasts (Mycoderma vini)on the tank surface
Addition of filtered vinegar to the mash,total acidity 1,5%,time of contact: 3 weeks
Vinegar eels(in wine vinegar also)
Turbatrix aceti Filtering cartridge in charging pipe,sterilizing with H2O2 + peracetic acid
VIN
EGA
R
diseases their causes means of prevention
Ferm
enta
tion
stop
page
s
Lactic acid bacteria
Examination of wine and vinegar under the microscope. Mash to be tested on Pilot Acetator, to ascertainthat it is not infected. If sodisinfection with H2O2.
Budding yeasts in spirit vinegar, molds:genus Moniliellagenus Sporotrichum
Hygiene, disinfection(see before)
Acetobacter peroxydans,too low total concentration, mutations. Overoxidation ofacetic acid in CO2 and H2O
Total concentration (TC) of 8-10, culti-vate selected strain on Pilot Acetator
Heavy metals(Cd, Hg, Pb, Zn, Cu)
Control of raw material
Phage: fermenter near generator (wood shavings)
Acetobacter xylinusCloudy vinegar(mothers of vinegar)
Ultra-violet rays in charging pipe,separation of vinegar produced on Acetator and generator
Sterile filtrationStrict hygiene
WIN
E
Bitterness Bacteria (threads) Pasteurization
Tourne Bacterium. tartarophthorum SO2
Oxidation of alcoholto CO2 and H2O
Yeasts (Mycoderma vini)on the tank surface
Addition of filtered vinegar to the mash,total acidity 1,5%,time of contact: 3 weeks
Vinegar eels(in wine vinegar also)
Turbatrix aceti Filtering cartridge in charging pipe,sterilizing with H2O2 + peracetic acid
VIN
EGA
R
diseases their causes means of prevention
Ferm
enta
tion
stop
page
s
Lactic acid bacteria
Examination of wine and vinegar under the microscope. Mash to be tested on Pilot Acetator, to ascertainthat it is not infected. If sodisinfection with H2O2.
Budding yeasts in spirit vinegar, molds:genus Moniliellagenus Sporotrichum
Hygiene, disinfection(see before)
Acetobacter peroxydans,too low total concentration, mutations. Overoxidation ofacetic acid in CO2 and H2O
Total concentration (TC) of 8-10, culti-vate selected strain on Pilot Acetator
Heavy metals(Cd, Hg, Pb, Zn, Cu)
Control of raw material
Phage: fermenter near generator (wood shavings)
Acetobacter xylinusCloudy vinegar(mothers of vinegar)
Ultra-violet rays in charging pipe,separation of vinegar produced on Acetator and generator
Sterile filtrationStrict hygiene
WIN
E
Bitterness Bacteria (threads) Pasteurization
Tourne Bacterium. tartarophthorum SO2
Oxidation of alcoholto CO2 and H2O
Yeasts (Mycoderma vini)on the tank surface
Addition of filtered vinegar to the mash,total acidity 1,5%,time of contact: 3 weeks
Vinegar eels(in wine vinegar also)
Turbatrix aceti Filtering cartridge in charging pipe,sterilizing with H2O2 + peracetic acid
VIN
EGA
R
diseases their causes means of prevention
Ferm
enta
tion
stop
page
s
Lactic acid bacteria
Examination of wine and vinegar under the microscope. Mash to be tested on Pilot Acetator, to ascertainthat it is not infected. If sodisinfection with H2O2.
Budding yeasts in spirit vinegar, molds:genus Moniliellagenus Sporotrichum
Hygiene, disinfection(see before)
Acetobacter peroxydans,too low total concentration, mutations. Overoxidation ofacetic acid in CO2 and H2O
Total concentration (TC) of 8-10, culti-vate selected strain on Pilot Acetator
Heavy metals(Cd, Hg, Pb, Zn, Cu)
Control of raw material
Phage: fermenter near generator (wood shavings)
Acetobacter xylinusCloudy vinegar(mothers of vinegar)
Ultra-violet rays in charging pipe,separation of vinegar produced on Acetator and generator
Sterile filtrationStrict hygiene
WIN
E
Bitterness Bacteria (threads) Pasteurization
Tourne Bacterium. tartarophthorum SO2
Oxidation of alcoholto CO2 and H2O
Yeasts (Mycoderma vini)on the tank surface
Addition of filtered vinegar to the mash,total acidity 1,5%,time of contact: 3 weeks
Vinegar eels(in wine vinegar also)
Turbatrix aceti Filtering cartridge in charging pipe,sterilizing with H2O2 + peracetic acid
VIN
EGA
R
diseases their causes means of prevention
Ferm
enta
tion
stop
page
s
Lactic acid bacteria
Examination of wine and vinegar under the microscope. Mash to be tested on Pilot Acetator, to ascertainthat it is not infected. If sodisinfection with H2O2.
Budding yeasts in spirit vinegar, molds:genus Moniliellagenus Sporotrichum
Hygiene, disinfection(see before)
Acetobacter peroxydans,too low total concentration, mutations. Overoxidation ofacetic acid in CO2 and H2O
Total concentration (TC) of 8-10, culti-vate selected strain on Pilot Acetator
Heavy metals(Cd, Hg, Pb, Zn, Cu)
Control of raw material
Phage: fermenter near generator (wood shavings)
Acetobacter xylinusCloudy vinegar(mothers of vinegar)
Ultra-violet rays in charging pipe,separation of vinegar produced on Acetator and generator
Sterile filtrationStrict hygiene
VIN
EGA
R
diseases their causes means of prevention
Ferm
enta
tion
stop
page
s
Lactic acid bacteria
Examination of wine and vinegar under the microscope. Mash to be tested on Pilot Acetator, to ascertainthat it is not infected. If sodisinfection with H2O2.
Budding yeasts in spirit vinegar, molds:genus Moniliellagenus Sporotrichum
Hygiene, disinfection(see before)
Acetobacter peroxydans,too low total concentration, mutations. Overoxidation ofacetic acid in CO2 and H2O
Total concentration (TC) of 8-10, culti-vate selected strain on Pilot Acetator
Heavy metals(Cd, Hg, Pb, Zn, Cu)
Control of raw material
Phage: fermenter near generator (wood shavings)
Acetobacter xylinusCloudy vinegar(mothers of vinegar)
Ultra-violet rays in charging pipe,separation of vinegar produced on Acetator and generator
Sterile filtrationStrict hygiene
Laboratory of Bioenergetics and Microbiology
Marie-Louise ChappuisSybille Schilleman
Aurelia weber
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