the effects of chemical preservatives and pasteurization on the microbial spoilage and shelf-life of...

THE EFFECTS OF CHEMICAL PRESERVATIVES AND PASTEURIZATION ON THE MICROBIAL SPOILAGE AND

SHELF-LIFE OF KUNUN-ZAKI

B.J.O. EFIUVWEVWERE' and 0. AKOMA

Department of Microbiology Food & Industrial Division University of Port Harcourt Box 148-Uniport Post Office

Choba, Port Harcourt, Nigeria

Received for Publication April 10, 1997 Accepted for Publication July 6, 1997

ABSTRACT

"Kunun-zaki " was produced using pasteurization alone or in combination with chemical preservatives. Diverse microbial genera (Lactobacillus, Bacillus, Aspergillus and Saccharomyces) dominated the untreated control samples but only two microbial genera (Bacillus and Lactobacillus) were isolatedfiom the samples subjected to preservation treatments with Lactobacillus being the predominant genus. The efects ofpasteurization alone were comparable to the combined efects of pasteurization and chemical preservative treatments in reducing microbial populations. Of all the treatments, sodium benzoate (0.08% w/v) with pasteurization was most efective, reducing the total viable counts by approximately 4.5 log at the end of the storage period. In contrast, the combination of potassium sorbate (O.O6%w/v) with pasteurization was least beneficial, resulting in only 2.6 log reduction. Sheplife (based on sensory overall acceptability and microbial quality) of the samples varied with the treatments, but combination of 0.08% w/v sodium benzoate with pasteurization extended the shey life by approximately 4 d q s whereas samples pasteurized without preservatives showed marginally enhanced shey-life of about 2 dqs . However, untreated control samples exhibited remarkably high microbial loads and were virtually unacceptable 24h after production. Pasteurization alone would therefore be adequate for small scale production and commercialization.

'Corresponding author and current address: Agrotechnological Research Institute (ATO-DLO), Dept.of Food Safety & Applied Microbiology, Bornsesteeg 59, P.O. Box 17, NL-6700 AA Wageningen, The Netherlands, Tel: 3 1-3 17-475048, Fax: 3 1-3 17-475347

Journal of Food Safety 17 (1997) 203-213. AN Rights Reserved, "Copyright I997 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 203

204 B.J.O. EFIUVWEVWERE and 0. AKOMA

INTRODUCTION

Cereal beverages are cherished in many parts of the world particularly in Afiica (Holzapfel 1989). “Kunun-zaki” and other cereal beveragesheers including “Merissa” of Sudan, “Busaa” of Uganda, “Kaffir” of South Africa and “Bouza”of Egypt are popular because of the social, religious and therapeutic values associated with them. However, the short shelf-life of 1 to 3 days is the major problem facing “brewers” and consumers of these products (Odunfa 1985; Efiuvwevwere and Akoma 1995). These deleterious changes are due primarily to the objectionable off-flavor (over-souring) induced by microbial activities.

Use of chemical preservatives and/or mild heat treatment for microbial control in beverages is desirable and continues to generate research interest world-wide (Chichester and Tanner 1972; Okafor 1975; Holzapfel 1989; Shelef and Addala 1994). However, their antimicrobial effectiveness is dependent upon such factors as the type of preservatives, the microbial flora involved and the composition of the food (Banwart 1979).

In spite of the rapid spoilage experienced by the “brewers” and consumers of “kunun-zaki”, virtually no published information is available on the preservation and shelf-life extension of this beverage. Such concern has earlier been expressed by Bolaji and Ja’afari (1988) based on a survey study.

“Kunun-zaki” is a very popular beverage, especially in Northern Nigeria. It is produced by small businesses and is not usually involved in interstate commerce, mainly because of its susceptibility to microbial spoilage and relatively short shelf- life. The objective of this paper is therefore to enhance the shelf stability of the product through pasteurization alone and the incorporation of chemical preservatives, while maintaining the characteristic organoleptic attributes.

MATERIALS AND METHODS

Production and Preservation of “Kunun-zaki”

Kunun-zaki was produced by the traditional fermentation process (Fig. 1) except that chemical preservatives were incorporated and the product was pasteurized.

Stock solutions ( I 0% w/v) of sodium benzoate and potassium sorbate were filter-sterilized (0.2 pm, Gelman Chemical Co., USA) and added alone or in combination to kunun-zaki to obtain the different levels shown in Table 1. The samples (250mL) were then dispensed aseptically (i.e. by flaming the necWopening of the bottles during the process of the dispensation) in 300mL sterile bottles and capped before pasteurization at 70C for 30 min in a water bath (with temperature of 68-70C within the samples; as monitored by a thermometer inserted through

PRESERVATION AND SHELF-LIFE OF KUNUN-ZAKI 205

WHOLE MILLET

CLEANING and WASHING &

& STEEPING (temp/days: 27-33 C/1 and water changed

& at the end of steeping) DECANTING and WASHING

&

& MILLET + DRIED GINGER, WET-MILLING

SIEVING &+Pomace Discarded

SEDIMENTATION &

r 1 COOKED BY ADDITION UNCOOKED OF BOILING WATER

L Mixed 1:l Ratio J

&

&

&

&

&

&

DILUTED 1:3

FERMENTATION 8H

KU"-ZAKI

PRESERVATIVE ADDED

BOTTLED and PASTEURIZED

COOLED and STORED

FIG. 1, FLOW DIAGRAM FOR THE PRODUCTION AND PRESERVATION OF KUNUN-ZAKI

cotton plug). The water bath temperature was obtained before placing the samples and they were occasionally gently agitated manually to achieve uniform heating.

Following pasteurization, the samples were cooled and stored at ambient tropical temperature (27 to 33C) for analysis at regular intervals. Some samples without preservatives were pasteurized while others were unpasteurized (i.e. untreated control samples) and were also analyzed during the storage.

Enumeration and Isolation of the Microorganisms

Samples from the different treatments were prepared serially using 0. I% peptone water as the diluent and pour-plated on plate count agar (PCA) and Rogosa


agar (RA) (Oxoid products, Basingstoke, UK) for total viable and lactobacilli counts respectively. The colonies (30-300) formed after incubation at 30C aerobically for 24-36h on PCA or at 30C anaerobically (Gas Pak, BBL, Cockeysville, USA) for 5 days on RA were enumerated and isolated. Discrete colonies were picked at random, purified and characterized by Gram and spore- staining before being subjected to biochemical and physiological tests (catalase, oxidase, Voges-Proskauer, methyl-red, nitrate reduction, starch hydrolysis, ammonia production, hydrogen sulphide and fermentation of sugars: glucose, arabinose, melibiose, mannitol, sorbitol, glycerol, lactose, maltose and cellobiose). These characterizations and the identification of isolates were carried out according to the methods and criteria of Harrigan and McCance (1976) and Sneath et al. (1 986).

TABLE 1. FORMULATION AND CONCENTRATIONS OF

CI 1EMICAL PRESERVATIVES INCORPORATED INTO KUNUN-ZAKI

Preservative(s) Concentrations incorporated (YO w/v)

KS + NaB 0.03 each

KS + NaB 0.04 each

KS 0.06

NaB 0.06

KS 0.08

NaB 0.08

KS = Potassium sorbate.

NaB = Sodium benzoate.

Measurement of Acidity The pH and titratable acidity (TA) were measured using a referenced pH meter

(Model 291 Mk2, PYE UNICAM, England). To obtain TA values, samples (1OmL) were titrated against 0. IN phenolphathalein to the end-point (pink).

Sensory Evaluation

The sensory attributes (visual apperance, aroma, taste and overall acceptability) of the samples were evaluated by a 10-member panel using a 9-point hedonic scale (Larmond 1977) to assess the shelf-life.


Statistical Analysis

and sensory quality were computed (ANOVA) and the statistical significance determined (Duncan 1955).

Mean differences in microbial population, acidity (pH and titratable acidity)

RESULTS

Microorganisms Isolated from the Different Treatments

The most heterogeneous microflora occurred in the samples subjected to neither preservative treatment nor pasteurization with Bacillus firmus, Bacillus subtilis, Lactobacillus fermentum, L. leichmannii, Aspergillus niger and Saccharomyces spp. being the dominant microorganisms isolated. Conversely, fewer types of microorganisms (Bacillus firmus, Bacillus subtilis, L. fermentum and L. leichmannig were isolated from the samples subjected to both preservative and pasteurization treatments (Data not shown but summarized as a footnote in Table 2). The most diverse flora (bacteria and fungi) were observed in the unpasteurized samples without preservatives (control) while those preserved with 0.08% wiv sodium benzoate and pasteurized showed only lactobacilli and bacterial spore- formers (Bacillus sp.).

Changes in Microbial Populations of the Different Treatments

Of all treatments, the samples neither treated with preservatives nor pasteurized (i.e. control) had the highest microbial populations throughout storage (Table 2). But pasteurization alone resulted in a significant decrease of approximately 6 log (i.e. from log 7.5 to log 1.72) at day 0. Chemical preservative(s) combined with pasteurization reduced the total viable count further but this effect varied with treatments (Table 2). The various magnitudes of microbial reduction in the samples treated with preservatives and pasteurization as compared with the untreatedunpasteurized (control) samples are shown in Table 2. Whereas the efficacy of 0.08% wlv sodium benzoate was most pronounced among the treatments, potassium sorbate (0.06% wlv) was least effective (Table 2).

Chemical Quality

The pH values decreased with storage time in all samples but the lowest pH (2.54) occurred in the unpreserved (control) samples, while the maximum values (pH 3.76) at the end of storage occurred in 0.08% w/v sodium benzoate treated samples (Table 3). Changes in TA were converse of pH values.

TABL

E 2.

EF

FEC

TS O

F PA

STEU

RIZ

ATI

ON

AN

D C

HEM

ICA

L PR

ESER

VA

TIV

ES O

N M

ICR

OBI

AL

(LO

G,,

CFU

hlL

)* Q

UA

LITY

OF

KU

NU

N-Z

AK

I D

UR

ING

TR

OPI

CA

L A

MBI

ENT

STO

RA

GE

N

0

m

~~

~~

~ ~~

Mic

robi

al L

oad

(log,

, CFU

/ml)

from

trea

tmen

t

Qua

lity

Inde

x St

orag

e C

ontr

ol

P-P

P+K

S+N

aB

P+K

S+N

aB

P+K

S P+

NaB

P+

KS

P+N

aB

(day

s)

0.03

%

0.04

%

0.06

%

0.06

%

0.08

%

0.08

%

p

each

ea

ch

Tot

al v

iabl

e cou

nt

0 7.

95a

1.72

b 1.

70b

1.69

b 1.

68b

1.67

b 1.

67b

1.68

b C

4

5.81

a 3.

11b

2.12

c 2.

01c

2.1

3~

2.

1oc

2.06

~

1.9

5~

8 5.

83a

4.01

b 3.

80b

3.65

b 3.

56b

2.54

~

2.50

~

1.9

8~

5 e

12

6.54

a 4.

03b

3.51

b 3.

64b

3.94

b 3.

3 1 b

3.92

b 2.

20c

Lact

obac

illi c

ount

0

7.21

a 1.

60b

1.58

b 1.

59b

1.60

b 1.

58b

1.59

b 1.

58b

I! P 4

5.63

a 2.

86b

1.97

c 1.

98,

2.01

c 1

.89

~

2.00

c 1

.69

~

>

8 5.

61a

3.52

b 2.

4%

1.9

5~

3.

48b

2.2

7~

2.

1%

1.84

~

I >

12

5.65

a 3.

86b

2.7

3~

2.

15d

3.81

b 2.

62~

3.

61b

2.02

d

0”

‘Con

trol s

ampl

es co

nsis

ted

mai

nly

of B

acill

us ,

Lacto

baciZ

Zus, A

sper

gifZ

us an

d Su

ccha

rom

yces

gen

era

but o

nly

Bac

illus

and

La

ctob

acill

us o

ccur

red i

n th

e pr

eser

ved

sam

ples

. V

alue

s (m

eans

of 2

repl

icat

es, n

=4) i

n ro

ws s

harin

g th

e sa

me

lette

r do

not d

iffer

sign

ifica

ntly

at th

e 5%

leve

l. C

ontro

l (ne

ither

past

euriz

ed n

or c

onta

ins p

rese

rvat

ives

). P-

P =

Pas

teur

ized

with

out p

rese

rvat

ives

; P =

Pas

teur

ized

; KS

= P

otas

sium

sorb

ate;

NaE

3 =

Sod

ium

ben

zoat

e.

TAB

LE 3

. EF

FEC

TS O

F PA

STEU

RIZ

ATI

ON

AN

D C

HEM

ICA

L PR

ESER

VA

TIV

ES O

N A

CID

ITY

AN

D S

ENSO

RY

OV

ERA

LL

AC

CEP

TAB

ILIT

Y O

F K

UN

UN

-ZA

KI D

UR

ING

TR

OPI

CA

L A

MB

IEN

T ST

OR

AG

E

Sens

ory

and

Che

mic

al D

ata

Val

ues f

or T

reat

men

ts

71

Qua

lity

Inde

x St

orag

e C

ontro

l P-

P P+

KS+

NaB

P+

KS+

NaB

P+

KS

P+N

aB

P+K

S P+

NaB

3u

(days)

0.03

%

0.04%

0.06

%

0.06

%

0.08

%

0.08

%

z PH

0

4.05

a 3.

93a

4.26

a 4.

28a

4.23

a 4.

15a

4.20

a 4.

27a

5 ea

ch

each

E;

4 3.

1 lb

3.55

b 3.

99a

4.11

b 4.

09a

4.13

a 4.

07a

4.19

a 2: * z U

8 2.

58d

3.21%

3.

65b

3.85

a 3.

49c

3.73

b 3.

91a

3.84

a

12

2.54

d 3.

39c

3.54

c 3.

66b

3.25

~

3.4

1~

3.

85a

3.76

a e x 2 :

TA

0 0.

015a

0.

013a

0.

014a

0.

014a

0.

015a

0.

015a

0.

014a

0.

015a

a

4 0.

045a

0.

031b

0.

018~

0.

018~

0.

018~

0.

019~

0.

018~

0.

016~

n

8 0.

078a

0.

072a

0.

060b

0.

060b

0.

071a

0.

062b

0.

065b

0.

045~

12

0.09

6a

0.09

8a

0.09

1a

0.09

4a

0.09

9a

0.08

1b

0.08

4b

0.07

7b

% 2 0

7.51

a 7.

53a

7.48

a 7.

40a

7.43

a 7.

39a

7.22

a 7.

63a

k SO

A

4 4.

37c

5.46

b 5.

76b

7.13

a 5.

87b

6.04

b 6.

87a

6.84

a ?2

8 3.

64c

5.17

b 4.

19~

6.

24a

4.06

~

4.1

lc

6.72

a 7.

13a

12

ND

2.

74~

N

D

4.72

b N

D

ND

5.

26a

5.74

a

TA =

Titr

atab

le ac

idity

; SO

A =

Sen

sory

ove

rall

acce

ptab

ility

. ND

= N

ot d

eter

min

ed d

ue to

obv

ious

spoi

lage

. Oth

er a

bbre

viat

ions

as

in T

able

2.

N 0

W


Sensory Attributes

The overall acceptability based on sensory attributes (visual appearance, aroma and taste) are presented in Table 3. Unpreserved samples (i.e. neither subjected to preservatives nor pasteurization) were least acceptable within 24 h of storage following production (Table 3). But samples pasteurized without preservatives deteriorated afier about 48 h while those subjected to 0.08% wlv sodium benzoate and pasteurization showed extended shelf-life of about 4 days (Table 2).

DISCUSSION

The rapid deterioration in shelf-life of traditionally produced ‘kunun-zaki’ and other African beverages is widely acknowledged and is of great concern (Odunfa 1985; Dirar 1993; Efiuvwevwere and Akoma 1995). The occurrence of diverse microbial genera and the remarkably high microbial load in the preservative-free and unpasteurized control samples (Table 2) is a major cause of the accelerated spoilage commonly experienced by the brewers and consumers of these products.

Whereas pasteurization destroys pathogenic and spoilage vegetative microorganisms in foods (Anon 1980)’ its application alone in the present study showed comparable efficacy with the combination of pasteurization and chemical preservative treatments. But invariably, the magnitude of effectiveness differed (Table 2). This indicates that the pasteurization process alone was adequate in destroying most of the microorganisms in kunun-zaki except the Bacillus species and the lactic acid thermo-resistant organisms. The latter have been reported to be among the nonsporulating species that survive pasteurization (Jay 1986). Similar findings had been reported by Okafor ( 1 975) during palm wine preservation. However, the combination of pasteurization with sorbic acid (sodium benzoate was not evaluated) was found most suitable for the preservation of palm wine.

In general, potassium sorbate and sodium benzoate have comparable antimicrobial activities (Jay 1986). But the more appreciable inhibitory effect exhibited by sodium benzoate (Tables 2 and 3) may be attributed to the differences in their chemical structures and the enhanced potency of sodium benzoate at the lower pH range of 2.05- 4.20 (Eklund 1989) which is similar to that of kunun-zaki. In addition, sodium benzoate tends to exert a more pronounced cell membrane interference and disruption than potassium sorbate (Eklund 1989). But the effects tended to wane with storage time and this could be due to microbial degradation particularly in the presence of high lactobacilli load (De Boer 1988; Efiuvwevwere and Oyelade 1991).

l h e sharp decrease in pH within 8 days of storage of the untreated control samples ( i.e. neither treated with preservatives nor pasteurized) (Table 3) suggests greater microbial activities since this decrease was not observed in the preserved samples. Thus, the accentuated microbial activities in the control samples must

PRESERVATION AND SHELF-LIFE OF KUNUN-ZAKI 21 1

TABLE 4. EFFECTS OF PASTEURIZATION AND CHEMICAL PRESERVATIVE TREATMENTS ON

KUNUN-ZAKI DURING TROPICAL AMBIENT STORAGE

Reduction of microbial populations (log,,) at storage time (days)

0 4 8 12

TVC LAC TVC LAC TVC LAC TVC LAC Treatments

P-P _ _ - -- -- -- 6.23 5.61 2.70 2.77 1.82 2.09 2.51 1.79

P+KS+NaB (0.03% each) 6.25 5.63 3.69 3.66 2.03 3.16 3.03 2.92

P+KS+NaB (0.04% each) 6.26 5.62 3.80 3.65 2.18 3.66 2.90 3.46

P+KS (0.06%) 6.27 5.61 3.68 3.62 2.27 2.13 2.60 1.84

P+ NaB (0.06%) 6.28 5.63 3.71 3.74 3.29 3.34 3.23 3.03

P+KS (O.O8YO) 6.28 5.62 3.75 3.63 3.33 3.46 2.62 2.04

P +NaB (0.08%) 6.27 5.63 3.86 3.94 3.85 3.77 4.34 3.63

*Reduction was computed based on comparison with the data obtained from untreated control samples (i.e. unpasteurized, not containing preservatives; see Table 2). Abbreviations oftreatments are the same as in Table 2. TVC= Total viable counts; LAC= Lactobacilli counts.

have resulted in the early manifestation of unacceptable sour flavor often associated with the spoilage of traditional kunun-zaki (Abasiekong et al. 1988) within 24-48 h of production.

The apparent beneficial antimicrobial effects observed using 0.08% (w/v) sodium benzoate without inducing adverse sensory qualities is highly desirable since 0.1% is the common maximum limit for beverages (Speck 1984). Nevertheless, in Nigeria, the maximum limit in beverages is 0.3% (w/v) (SON 1985). Whereas this level was more beneficial from the microbiological stand point, consumer acceptability was detrimental, confirming the off-flavor that occurs when used at greater than 0.1% (w/v) concentration (Speck 1984; Jay 1986).

The microbial population of beverages and foods help predict the shelf-life and potential safety (Banwart 1979; Walker and Jones 1994). The commonly recommended microbial load for beverages and liquid foods is between log,, 4 and 5 (Speck 1984 ). It is therefore evident that the dramatic microbial reduction by approximately 4.5 log (i.e. from log 6.54 to log 2.20) on day 12 in untreated control kunun-zaki as compared with combination treatment of 0.08% sodium benzoate with pasteurization (Table 2) is highly significant and provides a wide margin of safety especially in the presence of high acidity. But the probability of germination and outgrowth of the heat resistant spores under the temperature abuse conditions must be given serious consideration. Consequently, combination treatment of 0.08% (wh) sodium benzoate with pasteurization extended the shelf-


life by about 4 days. This combination is therefore recommended if a more extended shelf-life of kunun-zaki is desired in order to enhance its commercial potential; by allowing its sale beyond the locality of its production. Nevertheless, judging from the results (Tables 2 and 3) and the fact that most of the brewers do not have modem facilities to preserve the product, pasteurization alone (if adequately carried out) is all that is needed to extend the shelf-life by about two days.

REFERENCES

ABASIEKONG, S.F., EDWARD-INATIMI, E.B. and WAKDOK, J. 1988. Improving some inorganic nutrients of kunun by fermentation. Nigerian J. Biotechnol. 6, 80-82.

ANON. 1980. Microbial Ecology of Foods, Vol. 11, Food Commodities. Academic Press, New York.

BANWART, G.J. 1979. Basic Food Microbiology. Chapman & Hall, New York. BOLAJI, P.T. and JA ‘AFARI, M. 1988. Up-grading the traditional technology of

‘kunun-zaki’ production. pp. 43-44, 12th Annual Conference of the Nigerian Inst. Food Sci. Technol. Abst.

CHICHESTER, D.F. and TANNER, F.W. 1972. Anti-microbial food additives. In Handbook ofFoodAdditives, Vol. 1 2nd Ed.(T.E. Furia, ed.) pp. 268-273, CRC Press, Cleveland, OH.

De BOER, E. 1988. Food Preservatives. In Introduction to Food-borne Fungi, 3rd Ed. (R.A. Samson and E.S. van Reenen-Hoekstra, eds.) pp. 268-273, Centraalbureau Schimmelcultures, Baam.

DUNCAN, D.B. 1955. Multiple range and multiple F-tests. Biometrics I ! , 1-42. EFIUVWEVWERE, B.J.O. and AKOMA, 0. 1995. The microbiology of ‘kunun-

zaki’ ; a cereal beverage from northern Nigeria, during fermentation (production) process. World J. Microbiol. Biotechnol. 11,49 1-493.

EFIUVWEVWERE, B.J.O. and OYELADE, J.A. 1991. Biodeteriorative and physico-chemical changes in modified atmosphere packaged oranges and the microbial quality of the preserved and unpreserved juice. Trop. Sci. 31,325- 333.

EKLUND, T. 1989. Organic acids and esters. In Mechanisms o j Action of Preservation Procedures (G. W. Gould, ed.) pp. 161-200, Elsevier Applied Science, London.

HARRIGAN, W.F. and McCANCE, M.F. 1976. Laboratory Methods in Food and Dairy Microbiology. 2nd Ed. Academic Press, London.

HOLZAPFEL, W.H. 1989. Industrialization of Africa’s indigenous beer brewing. In Industrialization of Indigenous Fermented Foodrs. ( K.H. Steinkraus, ed.) pp. 191-283, Marcel Dekker, New York.

JAY, J.M. 1986. Modern Food Microbiology. 3rd Ed. Chapman & Hall, New York.


LARMOND, E. 1977. Laboratory Methods for Sensory Evaluation of Food. Canada Department of Agriculture, Ottawa.

ODUNFA, S.A. 1985. African fermented foods. In Microbiology of Fermented Foods. Vol. 2 (B.J. B. Wood, ed.) pp. 155-191, Elsevier Applied Science, London.

OKAFOR, N. 1975. Preliminary microbiological studies on the preservation of palm wine. J. Applied Bacteriol. 38, 1-7.

SHELEF, L.A. and ADDALA, L. 1994. Inhibition of Listeria monocytogenes and other bacteria by sodium diacetate. J. Food Safety 14, 103- 1 15.

SNEATH, P.H.A., NAIR, N.S., SHARPE, N.E. and HOLT, J.G. 1986. Bergey’s Manual of Systemic Bacteriology. Vol. 2, Williams and Wilkins, Baltimore, MD.

SON (Standard Organisation of Nigeria) 1985. Revised Standard for Soft Drink. Ministry of Industries, Lagos.

SPECK, M.L. 1984. Compendium of Methods for the Microbiological Examination of Foods, 2nd Ed. American Public Health Association, Washington, DC.

WALKER, S. and JONES, J. 1994. Microbiology modelling and safety assessment. pp. 25-29, Food Technol. Int. Europe 1994.

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