la microbiología a la disposición de la producción de … winegar f. barja. 0 2 4 6 8 10 12 ......

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