Microbiological Research on Soft Drinks.discolouring of NaturalFlavoured

8/11/2019 Microbiological Research on Soft Drinks.discolouring of NaturalFlavoured

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ZentralbI. MikrobioI.

7

1992),

51 6

Gustav Fischer Verlag lena

[Istituto di Microbiologia Agraria e Tecnica, Universita di Bologna, Italia, Istituto di Industrie Agrarie,

Universita di Bologna]

Microbiological Research on Soft Drinks: Discolouring of Natural-

Flavoured Products

Grur ro CES RE

TURTUR

and A

TTILE

MINGUZZI

With 2 Figures

ey words discolouring of soft drinks, effects of sunlight, heat and yeasts

ummary

The results of experiments testing the effects of yeasts, sunlight, and temperature on the food dyes

tartrazinc, ponceau 4R, indigotin and azorubine used for colouring ginger soft drink) are reported. Light was

found to exert a greater influence than heat, and yeasts growth hastened colour degradation. Yeasts assimila

ted to varying extent the colouring compounds and, when failing to do it, showed a certain power of

adsorption by the no longer viable cells.

usammenfassung

Experirnentell wurde der EinfluB von Hefen, Sonnenlicht und Temperatur auf die Lebensmittelfarbstoffe

Tartrazin, Ponceau 4R, Indigotin und Azorubin gepriift, die Erfrischungsgetranken zugesetzt werden. Licht

zeigte einen starkeren EinfluB als Warme. Hefewachstum beschleunigte den Farbstoffabbau. Hefen assimilier

ten die getesteten Farbstoffe in unterschiedlichem AusmaB. Waren sie dazu unfahig, so konnten sie nicht mehr

lebcnsfahigen Zellen den Farbstoff absorbieren.

The legislation governing the manufacture and sale of non-alcoholic beverages in Italy

D.P.R. 19 maggio 1958 n 719) includes an extensive array of products whose composition

ranges from the richest in fruit juices to the richest in fruit juices to the poorest ones,

containing sugar, citric or tartaric acid and sundry flavourings

TURTUR

and S M J

19 75a ). Practically c ol ourne ss, soft drinks are usually enric hed with c ol ouri ng agents to

enhance their appeal. The food colouring substances permitted by the country s legislation

D .M. 22 di cem bre 1967) must re spond to given purity requirements that, effectively, ban

mineral and organic c ompounds d ee me d harmful to huma n healt h

ZONT

A and

ST NCHER

1989).

Non-alcohol ic beverages can be altered by acidophilic microorganisms - yeast espe

cially

TURTUR

and S M J 975b when their generally is higher than 2.5

TURTUR and S M J 1978a). Obviously the intensity of microbial growth is related to the

richness of t hei r nutrient content TURTUR and

S M J 978b; TURTUR

and

S M J

1978c;

TURTUR

and

S M J

1978d;

TURTUR

and

S M J

1979;

TURTUR

and

S M J

1984; TURTUR and M SS 1987; TURTUR et al. 1988).

A

discolouring of non-alcoholic beverages is a common occurrence, even when colou

ring agents other than those permitted today which must be listed on labels) are used.

Physico-chemical and microbiological factors are involved: among the former are molecular

instability of colouring agents to light, heat and oxygen and their insolubilization when metal


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52 G

C. TURTURA und A. MINGUZZI

ions (iron in particular) are present in water; among the latter are degradation and adsorption

by contaminating microorganisms.

The present study aimed to investigate the stability of the four most common colouring

agents used by the italian industry, i.e., tartrazine, azorubine, ponceau

and indigotin, for

the production of naturally flavoured, artificially coloured soft drinks. Exposure to sunlight,

temperature and contaminating yeasts were considered.

teri ls nd ethods

Sample testing

a) A first set of twelve samples AI -Ad from a lot of a carbonated (pH 3.5) ginger soft drink, in plain

glass, 120-ml commercial bottles, sealed with crown caps and kept in light at room temperature, were assayed

six months after bottling, i.e. immediately before shelf-life deadline date. Table I shows the label-listed

colouring agents and their concentrations, employed to give the beverage its original dark red hue.

All the samples showed evident signs of decolouration, ranging from a slight fading of the original hue to a

greenish-yellow of varying shade and intensity. Samples A

A

10

showed a sediment formed by dark red lumps

of variable number and size; there was no sediment in samples All and A

lz.

Because of blastomycetic cells were seen in the deposited material, microbiological analysis of the twelve

samples was made using yeast extract - malt extract agar WICKERHAM 1951) adjusted to pH 5. Isolated strains

were cultivated in malt extract LODDER and KREGER-VAN

Rtr

1952) at pH 5 and identified according to WALT

and YARROW (1984). The liquid of these samples was subjected to spectrophotometry analysis.

b) A second set of twelve samples (BI- B12) was taken from a lot of freshly bottled ginger drink showing no

visible signs of alteration. The samples BI- B

6

were exposed to sunlight, while samples B

7

-

B

12

were similarly

exposed, but covered with a thin sheet of aluminium. This exposition lasted two months.

Preparation of colouring-agent solutions

The individual colouring agents were prepared separately in aqueous solutions, adjusted with citric acid to

pH 3.5, and blended in the proportions most commonly employed in the soft-drink industry to colour ginger

beverage. These solutions were used for maximal adsobancy determination and for studying the effects of light

and temperature on their degradation. The solutions were therefore divided into two batches, which were

exposed as indicated before.

The tests were carried out in June, July and August - a period with a maximum temperature difference of

nearly 20 QC Through the test period all samples were measured for colouring agent intensity, the readings

being taken at peak-absorption wavelenght at four day intervals. The pH and the amounts of the dissolved

oxygen were measured too, but their levels remained unchanged.

Microbiological assays

The effects of the yeasts on the colouring agents were studied using the synthetic medium of

WILLIAMS

et

a . (1941) modified as follows: sucrose 100g, monopotassium phosphate (KH

2P04

)

I g, magnesium chloride

(MgCl

z

6H

20

0.25 g, calcium chloride (CaCl

z)

0.25 g, boric acid (H

3B 03

) 0.001 g, manganese chloride

(MnCh 4H

z

O 0.001 g, iron chloride (FeCI

3

6H

z

O 0.0005 g, potassium iodide (KJ) 0.0001 g, zinc chloride

(ZnCh) 0.0001 g, traces of copper sulphate (CUS04 5H

z

O), traces of thallium chloride (TaCl

s),

biotin

0,025 mg, distilled water 1000 m .

Adjusted with citric acid to pH 3.5 , this NF (nitrogen-free) medium was enriched either with

2 4g

ammonium nitrate per liter (AN medium) or with

6g

yeast extract (Difco) per liter (YE medium). The four

colouring agents under study were added to the three nutrient solutions at the concentrations used in the industry

and in all possible combinations: 15 solutions were thus obtained and studied.

The nutrient solutions were inoculated with a strain of

cch romyces cerevisi e

(10

4

cells/ml) which had

been isolated from the flocculent sediment of a contamined sample. Cultured repeatedly in liquid malt extract,

this strain showed the formation of a pulverulent sediment typical of

cch romyces cerevisi e

ZAMBONELLI

1988). The nutrient solutions were examined after two months incubation in the dark at

25C

for sediment and

discolouring, and then measured at the peak absorption wavelength of each dye.


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54 G. C. TURTUR und A. MINGUZZI

The differences in discolouration of the three nutrient solutions were compared in a series of Wilcoxon s

sample-dependent parameter tests SIEGEL1980). The data from the spectrophotometry and appearance analyses

were processed separately. For the latter the quality rating were translated into values on the following scale: 0

uncertain discolouring; I-very slight discolouring; 2-slight discolouring; 3-discolouring; 4-marked discolou

ring; 5-total discolouring.

esults and iscussion

Identification of microorganisms

The sediments of the altered samples Aj-A

lO

showed yeast cells with a degraded structure;

nonetheless numerous strains were isolated and ascribed to Saccharomyces cerevisiae.

Sucrose was one of the sugars fermented. Given its geographic distribution in Italy C STELLI

1954 Saccharomyces cerevisiae is one of the yeasts most often responsible for the

contamination of both fruit juices and aroma-based soft drinks LLOYD 1975; TURTUR and

S M J 1978a; COMI et al. 1981). The isolation of yeasts from the sediment-free samples

ll

and

A

l

was negative. These samples were commercially sterile and free even of previous,

aborted microbe growth.

Dyes solutions: effects of light and temperature

Light and heat markedly influenced discolouration, the extent of their effects obviously

depending on dye Tab. 2 and 3). Table 2 shows that their influence is fairly strong on

tartrazine and azorubine and devastating on indigotin and ponceau R Comparing tables 2 and

Table 2. Influence of light and temperature on colouring agent degradation.

Colouring agent

max

Absorbance

initial final

Tartrazine

425 2.000 1.852

7.4

Ponceau 4R

505 1.300 0.000 100.0

Indigotin

610 2.150 0.000 100.0

Azorubine

515 1.480 1.288

13.0

Table 3. Influence of temperature on colouring agent degradation.

Colouring agent

max

Absorbance

initial

final

Tartrazine

425

2.000 1.980

1.0

Ponceau 4R

505

1.300 1.270

2.3

Indigotin

610

2.150 0.000

100.0

Azorubine

515 1.480

1.435

3.0

3 one sees that colour degradation is almost exclusively to be attributed to light exposure,

being very weak the effect of heat. Indigotin is the only exception, the effects of light and heat

matching only apparently see faster degradation patterns in Figs. 1 and 2 when both are


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Microbiological Research on Soft Drinks 55

c 0 0 I

2.0

1.5

W

0

Z


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56

G. C.

TURTURA

und A. MINGUZZI

Table 4.

Colouring agents compared a different stage of alteration.

Groups

Colour

Tartrazine Ponceau

4R Indigotin

Azorubine

of samples

Liquid

Sediment

AI)

AI)

AI)

AI)

AI, A

6

Yellow-orange Dark red

0.569

25 0.081

77

0.000

0.182

50

A

z.

A

7

, A

9

Yellow Dark red 0.523

31

0.046

87 0.000 100 0.189 48

A

3

, As As

Dark yellow Dark red 0.553

27

0.041

88

0.000

0.175 52

A

4

, A

IO

Greenish-yellow Brownish

0.553

27 0.007

98

0.000

100

0.153

58

All

Pale pink Absent

0.605

20

0.138 60 0.000 100 0.218 40

A

l2

Orange Absent 0.582 23 0.128

63

0.000

100

0.226

38

B -B

6

)2)

Unchanged

Absent

0.658 13 0.239 31 0.009

93

0.346

5

B

7 B 2 3

Unchanged Absent

0.756 a

0.346

0

0.132

a 0.364 a

I) Absorbance: the values are the ari thmetical mean

of

the groups

2) Samples exposed to light for two months

3) Samples kept in dark for two months

concomitant). Whereas colour vanishes under the combined influence of light and heat in about

15 days, it diminished by only about 2.5 in 10 days and did not vanish until about 70 days

under heat alone; ponceau 4R, on the other hand, remained unchanged over the first 10 days,

vanished after about 67 days under heat and light Fig. I), and remained unaltered throughout

the test period under heat alone Fig. 2). Tartrazine and azorubine, by contrast, showed a

remarkable stability under all test conditions.

Marketed samples: effects

of

light

Table 4 lists the results of the spectrophotometry analyses. The samples in the first set

showed amore or lessmarkeddiscolouration with

A i A

w

and without AI i and A 12 sediment;

those in the second set which were exposed to light B

j B

6

and dark

r

1z

for two months

showed no visible alterations

These findings evidence that: i) the decolouration pattern here closely coincides with that for

the pure colouring agents, i.e. degradation is limited for tartrazine and azorubine, more marked

for ponceau 4R and almost complete for indigotin; ii) tartrazine shows the greatest stability in

light sediment-free samples All and A d and to microorganisms AI-A

w

).

That the B

j B

6

samples showed no evident colour alterations although the extent of

tartrazine, azorubine and ponceau 4R degradation is considerable; evidently the complete

destruction of indigotin is without effect so as to raise the question of its necessity as a colouring

additive.

Yeast behaviour

Table 5 and 6 show the results of spectrophotometry and visual rating colour and sediment)

of the yeast-inoculated nutrient solutions. In spite od certain discrepancis, the two sets of values

can be easily integrated and their significance evaluated.

The Saccharomyces cerevisiae strain showed a flocculent growth: in the inoculated nutrient

solutions the coloured aggregates cannot be easily dispersed on stirring. The state of cell

aggregation was similar in appearance and density to that found in samples

A

j

lO

;

this is a

character difficult to quantify and for which specific research is lacking: deposits of varying

consistency are formed in relation to nutrient medium condition, i.e. the lack of nutrients and the

lowering of the

values to the limits of yeast development

TuRTuRA

and

SAMAJA

1975a).


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Microbiological Research on Soft Drinks

57

Table 5. Spectrophotometric analysis of media NF nitrogen-free), AN with ammonium nitrate), YE with

yeast extract) enriched with tested gyes and inoculated with a strain of cch romyces cerevisi e after two

months incubation at 25T

Colouring

Nutrient solution NF Nutrient solution AN

Nutrient solution YE

agents )

Absorbance

Absorbance Absorbance

Control Inoculated

Control Inoculated Control Inoculated

T

0.959 0.959 0

0.959 0.959 0 0.959 0.237 75

P

0.482 0.444 8

0.482 0.208 57 0.456 0.222 51

I

0.061 0.000 100

0.061 0.000 100

0.061 0.000

100

A 0.036 0.027

26 0.036 0.009 76

0.066 0.009

87

T

0.886 0.886 0 0.921

0.854 7 0.921 0.638 31

A

0.041 0.036 12 0.041

0.018 57 0.046 0.009 81

T

1.000 1.000 0

0.959 0.721 25

0.921

0.553 10

P 0.495 0.495 0

0.495

0.260 48 0.495

0.125 75

T 0.921

0.854

7

0.921

0.745 19 0.921 0.553 40

I

0.071 0.004 94 0.066

0.004

93

0.066 0.004

93

A

0.044 0.044 0

0.044 0.035 20

0.044

0.035

20

P 0.509 0.509 0

0.509 0.310 39

0.538 0.347

36

A

0.046

0.046

0

0.046 0.027

41 0.051

0.022 56

I

0.076

0.009

88 0.071

0.004 94 0.071

0.004

94

P

0.495 0.456 8

0.495 0.208 58 0.482 0.208 57

I

0.056 0.004

92

0.051

0.004 91 0.051 0.000 100

T 1.000 1.000 0 0.959 0.678 29 1.000 0.678 32

A

0.044

0.044

0

0.044 0.038 14 0.044 0.031 30

P

0.523 0.523 0 0.538

0.301 44 0.523 0.301 43

T

0.921 0.921

0

0.886

0.886

0

0.921

0.509

45

A

0.046 0.004

90

0.046 0.004 90

0.046 0.004

90

0.066 0.004 93 0.071

0.009

88

0.071

0.001 94

T

0.92[

0.921 0 0.886

0.886 0 0.921

0.509 45

P 0.496 0.099 80

0.496 0.064 87 0.496 0.064 87

I 0.066

0.004

93 0.071

0.009 88

0.07[

0.004 94

P

0.456 0.456 0 0.456 0.168 63 0.456

[49

67

A

0.046 0.046

0

0.046 0.021 54 0.046

0.024

48

I 0.076

[3

83 0.076 0.009 88 0.076 0.009 88

T

1.000 1.000

0

0.921 0.745 19 1.000 0.638 36

A 0.048 0.048 0 0.048 0.037 28

0.050

0.030 40

P 0.509 0.509 0 0.523 0.292 44 0.553 0.319 12

I

0.066

0.009 87

0.066 0.009

87

0.076

0.009 88

Letter indicates colouring agent: T

=

tartrazine, P

=

ponceau 4R, ]

=

indigotin, A

=

azorubine

Type and amount of the nitrogen source in the nutrient solutions affected yeast growth, and

assimilation or destruction of the dyes.

The individual colouring agents were assimilated differently by the yeast strain tested:

indigotin completely, azorubine and ponceau 4R partially and tartrazine slightly or not at all;

thus, in a nitrogen-poor soft drink like ginger, in which the yeast behave as oligonitrophile

SACCHETTI and SAMAJA 1972), certain dyes enhance their growth, a fact which is apparently

linked to the nitrogen availability in the colouring agent s chemical structure.


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58 G. C.

TURTURA

und A.

MINGUZZI

Table 6. Visual rating of media NF (nitrogen-free), AN (with ammonium nitrate), YE (with yeast extract)

enriched with testeddyes and inoculatedwith a strain of

cch romyces erevisi e

after two months incubation

at 25C

Colouring

Nutrient solution NF

Nutrient solution AN Nutrient solution YE

agents )

Liquid and sediment Liquid and sediment Liquid and sediment

T

Uncertaind iscolouring Uncertain discolouring

Uncertain discolouring

No sediment

No sediment Yellowish sediment

P

Uncertain discolouring Discolouring Marked discolouring

Very slight red sediment Red sediment

Red sediment

Total discolouring

Total discolouring Total discolouring

Greyish sediment Greyish sediment

Greyish sediment

A

Slight discolouring Discolouring Marked discolouring

Very slight pink sediment

Pink sediment Pink sediment

TA Uncertain discolouring

Very slight discolouring Discolouring-)

No sediment Pink sediment Pink sediment

TP


Discolouring) Discolouring )

No sediment Red sediment Red sediment

TI

Uncertain discolouring Very slight discolouring Slight discolouring

No sediment Greyish sediment

Greyish sediment

AP Slight discolouring Discolouring Marked discolouring

No sediment

Red sediment Red sediment

AI

Very slight discolouring Marked discolouring Total discolouring

No sediment Reddish sediment Reddish sediment

PI Uncertain discolouring Slight discolouring Marked discolouring

No sediment Reddish sediment Red sediment

TAP

Slight discolouring

Discolouring-)

Discolouring-)

Slight red sediment Red sediment Red sediment

TAl Slight discolouring

Discolouring-)

Discolouring-)

No sediment Red sediment

Red sediment

TPI Slight discolouring Discolouring ) Discolouring-)

Slight red sediment Red sediment Red sediment

PAl Uncertain discolouring Marked discolouring Marked discolouring

No sediment Red sediment Red sediment

TAPI


Discolouring-) Discolouring-)

No sediment

Red sediment

Red sediment

Letter indicates colouring agent: T = tartrazine, P = ponceau 4R,

indigotin, A= azorubin

2)

Green liquid,

3)

Yellowish liquid,

4)

Greenish-yellow liquid,

5)

Greenish-orange liquid

A cause of decolouration is linked to the adsorption of the dye by the dead yeast cells: this

is true expecially for ponceau 4R. It cannot be ruled out however that some of the dye

disappears, because of the formation of the Ieuco structure as effect of a long stay in a

reducing environment, like the one which is formed in the soft drink by the strongly

deoxygenating saccharomycetic growth.

The statistical data of both spectrophotometry and appearance values show significant

differences in the extent of the discolouring found with the dyes in the three nutrient solutions.

The comparison NF AN NF YE and ANIYE were highly significant for the spectrophotomet

ric data at P=O.00020,

P=O OOOOl

P=O.00070 respectively. The appearance ratings showed

that the statistical significance of the discolouration differences between solutions was less

than for the spectrophotometry: P=O.015 for

NF AN

P=OOlOfor

NF YE

P=O.0209 for N

YEo


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Microbiological Research on Soft Drinks 59

Conclusions

Light, heat and yeasts are the most frequent causes of discolouration in aroma-based soft

drinks. The combined effects of light and heat (the latter acting less actively) induced a

complete decolouration of indigotin and ponceau 4R and a partial one of tartrazine and

azorubine, the four colouring agents studied.

Yeast development was affected by the dyes, which were assimilated to varying extent:

indigotin completely, azorubine and ponceau 4R partially and tartrazine not at all. The failure

of the yeasts to utilize the dyes does not preclude their adsorption by dead cells to an extent

depending on colouring agent.

It must be noted the synergism of light and yeast metabolic

activity in the colour degradation processes.

Protecting soft drinks from microbial vegetation - yeasts especially - and from direct

exposure to sunlight in the best way of preventing discolouration.

References

CASTELLI T.: Les agents de la fermentation vinaire. Arch. Mikrobiol. (1954)

323 342

COMI G., DENOZZA D., CANTONI C SuI deposito di lieviti in bibite analcooliche. Ind. Bev. 11, (1981)

346 348

Decreto Ministeriale 22 dicembre 1967. Disciplina delI impiego e approvazione dell elenco deJIe materie

coloranti autorizzate nella lavorazione delle sostanze alimentari, delle carte e degli imballaggi di sostanze

alimentari, degli oggetti di usa personale e domestico. Gazzetta Ufficiale Repubblica Italiana n 28 deII I

febbraio 1968.

Decreto del Presidente della Repubblica 19 maggio 1958 n 719. Regolamento per la disciplina igienica della

produzione e del commercia delle acque gassate e delle bibite analcooliche gassate e non gassate

confezionate in recipienti chiusi. Gazzetta Ufficiale Repubblica Italiana n 178 del 24 luglio 1958.

LLOYD

A.

c.:

Osmophilic yeasts in preserved ginger products. J. Food Techn.

1

(1975)

575 581

LODDER

J.,

KREGER VAN

Rn, N. T. W.: The yeasts, a taxonomy study. North-Holland Publ. Co., Amsterdam.

1952.

SACCHETTI M.,

SAMAJA

T.: Aspetti microbiologici delle bevande analcooliche. L imbottigliamento n 3

(1972)

5 12

SIEGEL

S.: Statistica non parametrica. OS, Firenze. 1980.

TURTURA G. c

MASSA

S.: Microbiological research on soft drinks. VIII. A case of alteration of fruit juices

packaged in flexible multilayer containers. Microbiol. Alim. Nutr. 5, (1987)

17 20

-

CICCARONE

c.: Microbiological research on soft drinks. IX. A study of fruit juices in soft-pack,

multilayer containers. Microbiol. Alim. Nutr. 6 (1988) 201208

SAMAJA

T.: Alterazione da blastomiceti delle bevande anaIcooJiche a base di aromatizzanti vegetali:

importanza dell acqua impiegata. Riv. Sci. Teen. Alim. Urn.

5

(1975a) 183 187

- Alterazioni blastomicetiche delle bevande analcooliche a base di aromatizzanti naturali. Considerazioni

sulle possibilita di prevenzione. Atti XVIII Congresso Nazionale Soc. It. Microbiol., Padova, (1975b)

881 889

- Ricerche microbiologiche suIle bevande analcooliche. I Identificatione di blastomiceti isolati da bibite

preparate con aromatizzanti naturali. Ann. Microbiol.

28

(1978a)

1 9

- Ricerche microbiologiche sulle bevande analcooliche. II. Influenza degli aromatizzanti naturali sullo

sviluppo dei blastomiceti. Ann. Microbiol.

28

(1978b) 15 23

- Ricerche microbiologiche sulle bevande anaIcooIiche. III. L acqua oligominerale nella preparazione delle

bibite a base di aromatizzanti naturali. Ann. Microbiol. 28 (1978c) 85 90

- Ricerche microbiologiche sulle bevande analcooliche. IV. Azione antifermentativa dell acido benzoico.

Ann. Microbiol.

28

(1978d) 135 142.

- La conservazione delle bevande analcooliche. Ind. Bev.

9

(1979) 337 338

- Ricerche microbiologiche sulle bevande anaIcooJiche. VII. Preparazione industriale dei succhi di mela.

Ann. Microbiol. 34 (1984) 127 136

WALT J. P. van der,

YARROW

D.: Methods for the isolation, maintenance, classification and identification of

yeasts. In The Yeasts, a taxonomic study ,

KREGER VAN Rrr

N. J.W. (Ed.), p. 45. Elsevier Sci. Publ. B.

V., Amsterdam. 1984.


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60 G. C. TURTURA und A.

MINGUZZI

WICKERHAM

L. J.:

Taxonomy of yeasts. Techn. Bull. 1029, U.S. Dept. Agr., Washington, D.C. 1951.

WILLIAMS

R. 1.,

ATKIN L.,

FREY C.

N SCHULTZ

A. A.: Assay method for inositol. Univ. Texas Publ. n

4137.1941.

ZAMBONELLI c : Microbiologia e biotecnologia dei vini, p. 148. Edagricole, Bologna. 1988.

ZONTA F.,

STANCHER

B.: Aggiornamento merceologico sui coloranti alirnentari: caratteristiche chimiche,

impieghi, normativa comparata CEE-USA. Ind. Alim.

8

1989

913 920

Authors addresses: Prof Dr

G. C.

TURTURA

Istituto di Microbiologia Agraria e Tecnica, Universita di

Bologna, Via Filippo Re 6, Bologna, Italia. Prof. Dr. A.

MINGUZZI

Istituto di IndustrieAgrarie, Universita di

Bologna, Via S. Giacomo 7, Bologna, Italia.

Documents

Microbiological Research on Soft Drinks.discolouring of NaturalFlavoured