Effect of ionizing radiation on the hygienic quality and shelf - life of Turkish fermented sausage : FAO/IAEA research Co-ordination Meeting on Food Irradiation for the Middle East and European Countries Lalahan-Ankara, Turkey, 6-10 May 1991

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F A O /IA E A Second Research Co- ordination Meeting on Food Irradiation for the M iddle East and European Countries

L a la h a n -A N K A R A , T U R K E Y , 6 -1 0 May 1991

R E P O R T

(Research Contract N o: 5552/CF)

EFFECT OF IONIZING RADIATION ON THE

HYGIENIC QUALITY AND SHELF - LIFE OF

TURKISH FERMENTED SAUSAGE

izzet Ö Z T A Ş IR A N Seniha A K IN

Sıtkı ERSEN Harun Ç E R Ç İ

T U R K IS H A T O M IC E N E R G Y A U T H O R IT Y Lalahan Nuclear Research Institute in Animal Health

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F A O /IA E A Second Research Co- ordination Meeting on F ood Irradiation for the M iddle East and European Countries

L a la h a n -A N K A R A , T U R K E Y , 6 -1 0 May 1991

R E PO R T

(Research Contract No: 5552/CF)

EFFECT OF IONIZING RADIATION ON THE

HYGIENIC QUALITY AND SHELF - LIFE OF

TURKISH FERMENTED SAUSAGE

İzzet Ö Z T A Ş IR A N Seniha A K IN

Sıtkı ERSEN Harun Ç E R Ç İ

T U R K IS H ATO M IC E N E R G Y A U T H O R IT Y Lalahan Nuclear Research Institute in Animal Health

Lalahan - A N K A R A

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INTRODUCTION

Turkish fermented sausage is one of the most popular meat products in Turkey. The production and storage of this product involves considerable problems both for manufactures and for consumers. The quality of the raw mate rial, itTs production technological and production hygienic conditions greatly influences the microbiological quality of the product. Unsatisfactory micro biological quality leads to a short shelf-life and presents hygienic risks for the consumer. Irradiation has a potential use in the production of fermen ted sausage, which relies on the dominance of lactic acid bacteria in the microflora to produce the desired flavor and inhibit the growth of pathogenes. Furthermore, irradiation would ensure the safety of the product by eliminating pathogenic bacteria in the vegetative state.

Irradiation as a food preservation method has already been accepted in many countries. The wholesomeness tests with irradiated food have brought favourable results.

The purpose of this investigation was to determine the effect of ionizing radiation on the hygienic quality and shelf-life extension of the Turkish Fermented Sausage.

MATERIALS AND METHODS:

All the materials expect starter culture used in production of the Turkish fermented sausage were provided from the local market in Ankara. After being kept at -18 °C for 12 hours the frozen mixture of minced beef

(90 %) and tail fat (10 %) were irradiated in the Cs-137 source (Mark 1-22 Irradiator, 10.000 Ci, JL Sheppard and Associates) at 1 kGy, 2 kGy and 4 kGy doses. Additives for sausage that are blackpapper redpepper and cumin were sterilized in the Cs-137 source with 10 kGy dose. The frozen mixtures of minced meat and fat were melted at refrigerator temperature and homogenized with cutter by adding of additives and starter culture according to the requirements in Table 1.

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Table 1. Composition and separation of sausage samples.

Sausage samples Ingredient Quantity (kg)

A (ControO : Beef 9,0 Tail fat 1,0 Sodium chloride 0,2 Sodium nitrate 0,005 Garlic 0,1 Blackpepper 0,05 Redpepper 0,05 Cumin 0,05 Sucrose 0,05

*B (0 kGy) : A-f Starter culture

C (1 kGy) : A+ 1 kGy + Starter culture D (2 kGy) : A-f 2 kGy + Starter culture E (4 kGy) : A-f 4 kGy + Starter culture

Meat batter of both control and test groups were stored in special vessels at 4- 2 °C for one night and then were filled in artificial cases with fill ing machine. Sausages of both control and test groups were left for fermenta tion and draying in the environmental chamber in accordance with the bases in

Table 2.

Table 2. Fermentation and drying conditions of Turkish fermented sausage.

Days Temperature (° C) Relative humidity (%)

0-3 24 + 2 95 + 3

3-7 22 + 2 85 + 3

7-14 18 + 2 75 + 3

14-28 15 + 2 65 + 3

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2-After completion of fermentation and draying, sausage samples were stored at 10 °C in the refrigerator as being vacuum packed. The changes of microbiolo gical, chemical and sensory quality of samples both during and after fermen tation were determined by means of following tests:

Microbiological evaluation :

The microbial analyses were done in parallel to chemical and sensory testing. From each samples, 10 g. was weighed under sterile conditions and homogenized after addition of tryptone solution (0,1 % pepton of the casein and 0,04 % Agar No 1) so as to form a 10 % dilution in a Stomacher 400 for

—2 —8

2 minutes. From this homogenate a series of dilutions (10 to 10

were prepared. The number of colony forming units (CFU/g) was determined for microflara as follows (Table -3):

Total aerobic count was obtained by using Plate Count Agar (Oxoid CM-325)

Incubation lasted 48 hours at 37 °C (9). For total coliforms, were detected

by the 3- tube MPN method using Lauryl Tryptose Broth (Oxoid CM-451) fol

lowed by incubation 48 hours at 37 °C). Aliquats from each positive tube were transferred to Brillant Green Bile Broth 2 % (BBL-11080) for coliform confir mation and to EC Buyyon (Difco -0314) for detection of Escherichia coli. Tubes were incubated for 48 hours at 37 °C and 45,5 °C, respectively. Tubes of EC Buyyon showing growth were streaked on Eosin Methylene Blue Agar (Difco- 0076) plates and incubated at 37 °C for 24 hours. Each presumptive colony was confirmed as Escherichia coli if it exhibited a positive indole and nega tive citrate reaction. Fecal streptococci were enumerated using KF Streptococ

cus Agar (Oxoid -CM-701) to which 10 ml/L 1% (W/V) aqueous triphnyltetrazolium chloric (TTC) was added. Plates were incubated at 37 °C for 48 hours and all red or pink

colonies were counted (5) . Salmonella : Each samples were homogenized in 90 ml PBS. Homogenates were inoculated at 37 °C for 18-24 hours. This homogenates were inoculated into Tetrathionate Broth Base (Oxoid-CM-29) and incubated at 43 °C for 24 hours in to water both. After incubation this culture were passed on to Brillant Green Agar (Oxoid CM-263).

Staphylococcus aureus : Homogenate were inoculated on Baird Parker Medium (Oxoid CM-275) with Egg Yolk Tellurite (Oxoid SR -54). Black, convex, narrow white entire margin and surrounded by a clear zone’s colonies were investigated. Gram strain was prepared and observed gram positive staphylococci. Then coagulase

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test was performed (3).

Mold and Yeast : All samples were inoculated on to Oxytetrocycline Gentamisin Yeast Extract Glucose Agar. Plates were incubated at 20-23 °C for 3-5 days. After this periods colonies were counted.

Microccoccus/Staphylococcus were obtained by using Baird Parker Medium and incubated at 37 °C for 48 hours (4). Lactobacilli were determined on MRS Agar

(Oxoid CM-361) and incubation at 30 °C in a anaerobic condidion (Oxoid Gas generating kit No BR-38) for five days (6). Estimation of sulfit reducing colostridia were studied using SPS Agar (Difco-0845) as culture media and incubated at 37 °C for 72 hours (4).

Biochemical properties :

Thiobarbituric acid (TBA as mg malonaldehyde/1000 g. samples) was determined as spectrophotometrically using the extraction and filitration procedure with trichloracetic acid (7). pH values of samples were determined with the EMAF pH Meter (Model Em 78 x) (1).

Sensory evaluation :

The sensory evaluation tests were carried out with the method of American Society for Testing Materials Committee (2) and flavaur, texture, appearance and general acceptance of the sausage samples were judged by a trained panel of 5-6 and scored an a five poind scale (Table 4).

Table 4. The sensory evaluation test card

Name Date Samples No:

Criteria Poor

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Below medium Above poor (2) Medium (3) Good (4) Very Good (5) 1. Flavour 2. Texture a . Chew b. Hardness 3. Appearance a. Inside b. Outside 4.Preference sore

Remarks an suggestions :

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Statistical analysis:

Two-way analysis of variance was carried out to study the differences among treatment groups (8).

RESULTS AND DISCUSSION

1. The Changes in Microbiological, Chemical and Organoleptical Quality of Samples Before Fermentation and During Ripening:

As it is seen Table 5, at the microbiological analyses of the control and treated samples just before fermentation, total aerobic counts were found as 6,2.105/g., l,6.106/g., 5,8.105/g., l,8.105 /g. and 4,7.104/g for the A, B, C, D and E samples, respectively. The Micrococcus/Staphyiococcus and Lactobacillus counts in A samples were found lower than the B,C,D andE samples in which star ter culture was used. While the coliform counts in A and B samples were found

with the order o f 93/g. and 240/g., 9/g. in the C samples, in the D and E samples

< 3 / g were detected. In the C and D samples of Fecal streptococci lower counts were detected than A and B control samples due to the effect of radiation at 4 kGy doses. As it is seen in Table 5 and Figure 1 total aerobic counts in all samples increased by the 7 1 th day of ripening, then irregular decreases were observed except B samples. In addition, Lactobacilli counts in control and tre ated samples increased by the 7 ! th day of ripening process and later a regular and slow decrease was observed (Table 5, Figure 2). Again increase was seen in the Micrococcus/Staphyiococcus counts in all samples by the 3'rd day of the ripening

as seen in Figure 3. However irregular decreases were seen later. Fecal strepto cocci counts increased in all samples except E samples during the first three days of ripening. Coliforms and E.coli were not present in D and E samples before and during the process of ripening. Besides, these microorganisms were not detected in the C,B and A samples after the 2 1 Tst day of ripening (Table 5). Although no marked differences were seen between the pH values of the control and treated samples before the ripening process, marked decreases were observed in the pH values of the B,C,D and E samples in the first three days of the

ripening, pH values of A control samples have been found in higher level compared to the B,C,D and E samples during the ripening (Table 9, Figure 8).

The results of sensory assessments and quality indices of treated and control samples have been presented in Table 7. No marked difference was found between the sensory assessment of treated and control samples during the ripening.

At the sensory tests by the panelists which samples were prepared from the

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-6-radiated minced beef couldn’t be determined. Panelists were able to identify the samples in which the starter culture added because of their tastes specific to themselves.

The pH and TBA values of C,D.E and B samples were shown to be significantly different (p<C0,05) before the fermentation. It was found that the differences

between the TBA values of the C,D,E and B samples were significantly (p 0,05)

during the first three days of ripening thereafter which were found not to be significantly different (p^> 0,05). The pH values of treated and B control samples were found significantly different (p<^ 0,05) by the 14’th day of ripening and

later which were found to be not significantly different (p^OjOS).

2. The Changes in Microbiological, Chemical and Organoleptical Quality of Samples During the Storage at 10 °C:

The microbiological changes in treated and control samples as being vacuum packed during storage at 10 °C are shown in Table 6. Begining from the first day of the storing which is the 28th day of ripening, irregular decreases were seen till the 120 th day on which organoleptic spoil was determined with

sensory tests in the total aerobic and lactobacilli counts of A and B samples. It was detected that the counts of the same bacteria species were less in the C,D and E samples when compared to B samples during storage. While the total aerobic counts of A and B samples on the 120 th day of storing meeting with the spoiling of A and B samples were 4.0.10^/g and 7,2.10^/g respectively, on the 150 th day, when C,D and E samples were spoiled the counts of same bacteria

c c c

were 2,5.10 /g., 8,3.10 /g. and 2,8.10 /g. And it was observed that the total aerobic count may not be used to determine the loss of acceptability or the end of shelf-life of Turkish fermented sausage. Micrococcus/Staphylococcus

counts in all samples were showed irregular decreases during storage (Figure 6). The counts of these microorganisms of B control samples were obtained markedly high than the other samples (Table 6, Figure 6). On the other hand no connec tions have been found concerning the dimensions of the applied doses between the counts of Micrococcus/Staphylacoccus of the C,D and E samples. It was determined that Fecal streptococci counts of A samples were higher than the B samples during storage (Table 6). In addition when Table 6 was examined, it was obtained that in C and D samples, increase rate of this group microorganisms were slower than that in B samples. Furthermore Fecal streptococci were not present in the E samples to which 4 kGy dose was applied during storage. Coli- forms and E.coli were found under the limits of detection (<^0,3/g) in the

treated and control samples during storage. Sulfit reducing Clostridia were

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detected from time to time even though in small numbers. According to the results taken, it was seen that due to using radiation and starter culture, these microorganisms couldnft be eliminated completely. No markedly differences have been found between the increase of the TBA values of B samples and A samples as it can be seen in Table 10, Figure 9.

It was seen that the TBA values of treated and control samples haven’t reached to the threshold value for the human concumption which is 1 mg/kg. The changes in the pH values of the treated and control samples are shown in Table 10 and Figure 10. When Table 10 and Figure 10 are examined, the pH values of A samples were found higher than the B,C,D and E samples. However it was seen that the pH values of C,D and E samples were lower than the B samples. No relation was detected between the increase of the doses and the decrease of pH values. In addition a direct relation between the changes of pH values and total aerobic and lactabacilli counts of treated and control samples couldn’t be found. As it is seen in Table 8, no markedly differences were found related with the flavour, texture, appearance and general preference between the B and A samples during 120 days of storage period. It was seen that highest score was given to the D samples in relation with the general preference among the C,D and E samples. No markedly differences were found between flavour, texture, appearance and

general preferance of B control and C,D and E treated samples during 90 days of storage.

The both of A and B samples spoiled after 120 days of storage (Table 8, Figure 12). In the 150 th day of storage the spoiling in the C, D and E samples was detected by the panel.

As a result, it was determined that the shelf-life of Turkish fermented sausage prepared by using starter culture after irradiation with 1 kGy dose, can be extended at least for one month more over the untreated one at 10 °C.

CONCLUSION

It is apparantly seen that the shelf-life of the vacuum packed Turkish fermented sausage which is prepared by using irradiation and starter culture can be preserved at least 4 months at 10 °C. In addition, with this process it is possible to produce products of high hygenic quality.

This study, on the other hand, showed that total aerobic count, pH and TBA values are not sufficient indices in determination of shelf-life and renewable of product while organoleptic tests are trustworthy indices.

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-8-Table 5. Resulls of microbiological studies of control and treated sausage samples during ripening

D A Y S cn H W GROWTH MEDIUM < w Eh p c a BP MRS LST EC KF SPS (Mold) YEGA (Yeast) YEGA pH A 6.2.105 1.5.105 5.1.104 93 4 2,2.104 9,5.1ü1 > 3 0 0 ₁₅ 5,88 B 1.6.106 9.6.105 6.3.105 240 4 2.0.104 6.0.IO1 > 3 0 0 15 5,88 0 c _5.8.105 _6.7.1QS _5.7.105 ₉ ₄ _3,9.103 _- _{> 3 0 0} ₃₀ _5,91 D 1.8.105 3.7.105 3.8.105 _<3 _<3 1,5.103 - 2190 25 5,89 E 4,7.104 8.0.105 6,3.105 <3 <3 - - ₁₉₀ ₂₅ _5,88 A 1,6.108 4,1.104 5,4.107 1,1.10 2 15 6,9.104 7,0.IO1 70 60 4,80 B 8,9.107 6.5.106 1,8.108 460 9 2,5.IO4 - ₇₀ ₁₀ _4,60 3

c

6,7.107 1,9.106 4,1.107 240 4 1,8.104 2,0.IO1 200 15 4,54 D 1,6.108 8,3.105 1,4.108 _<3 _<3 3,7.IO3 - ₁₅₀ _- _4,20 E 3,9.107 8,6.105 1.3.108 _<3 _<3 - - - _4,20 A 7,1.108 6,1.105 2,8.108 1,1.10 2 4 3,1.104 1,5.1ü1 - ₃₀ _4,67 B 2,1.108 9,6.105 7,1.108 1,1.10 2 4 6,1.104 2,5.104 - - 4,62 7 C 3,8.108 6,1.105 1,9.108 4 _<3 2,3.IO4 <10 - - _4,54 D 1,9.108 7,9.105 1,7.108 _<3 _<3 3,6.103 2,5.IO1 - - _4,39 E 3,7.108 4,3.105 1,5.108 _<3 _<3 - - _4,36 A 1,0.108 2,7.105 1,1.108 43 4 6,1.104 <10 - ₁₀ _4,65 B 1,4.108 5,3.105 1,1.108 23 9 4,5.104 - - ₃₀ _4,37 14

c

2,2.108 1,8.105 1,8.108 4 4 2,4.104 <10 - - _4,18 D 9,0.107 8,1.104 1,1.io8 <3 <3 3,8.103 - - - _3,99 E 8,7.IO7 1,1.105 7,8.108 _<3 <3 - - - - _4,11 A 1,3.108 1,2.104 1,9.108 <3 <3 4.1.104 - - ₁₅ _4,53 B 3,6.108 9,6.104 3,7.108 4 _<3 3.5.104 <10 - ₁₅ _4,44 21

c

3,3.107 1,8.104 4,1.107 <3 <3 3,2.103 1 ,0.io1 - ₁₀ _4,24 D 2.2.107 9,0.103 2,5.107 < 3 <3 1,4.IO3 - - 4,17 E 4,0.107 9,0.103 3,3.107 <3 <3 - - - - 4,22 A 5,4.108 6,6.103 5.0.108 < 3 _<3 5,3.104 1,5.1ü1 - - _4,63 B 3,8.108 1,2.105 2,2.108 < 3 <3 3,0.104 - - - _4,47 28

c

5.2.107 rH O 4,5.107 <0,3 <0,3 2,6.IO3 <10 - - 4,50

D

_{2,0.107 2,6.104 4,2.10? <0,3 <0,3}

2,1.103

- - - _4,18

E

1,4.107

9,1.103 1,1.107 <0,3 <0,3 - - - - _4,06

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Table 6 : Results of microbiological studies of control and treated sausage samples during storage at 10 °C.

DA

YS

CA

H

W

_{g -}

GROWTH MEDIUM

<

H

PGA

BP

MRS

LST

EC

KF

SPS

(Mold)

_YEGA

(Yeast)

_YEGA

PH

A

5,4.108 6,6.103 5,0.108

<3

_<3

5,3.104 1,5.1ü1

— _

4,63

B

3,8.108 1,2.105 2,2.108

<3

3.0.104 -

-

4,47

0 c

5,2.107 4,9.104 4,5.107

<0,3 <0,3

2,6.103 <10

-

4,50

D

2,0.107 2,6.104 4.2.107

<0,3 <0,3

2,1.103

-

- -

4,18

E

1,4.107 9,1.103 1,1.107

<0,3 <0,3

-

4,06

A

_{3,5.108 4,2.103 4,8.108}

_{<0,3 <0,3}

_4,2.104

₁

_,

₀

_.io1

_-

₅

_4,69

B

3,1.108 8,2.104 2,3.108

<0,3 <0,3

2,7.104 s.s.io1

-

5 4,54

30 c

3,6.107 1,4.104 2,5.107

<0,3 <0,3

3,3.103

1

,

0

.io1

-

4,39

D

1,3.107 1,8.104 1,6.107

<0,3 <0,3

4,0.102

-

4,17

E

8,6.106 7,3.104 1,1.107

<0,3 <0,3

-

2,0.1ü1

-

4,16

A

2,3.108 5,3.103 1,8.108

<0,3 <0,3

7,3.104

- -

4,64

B

7,4.107 6,8.104 5,8.107

<0,3 <0,3

2,3.104

- - -

4,46

60 C

9,9.106 1,6.104 4,7.106

<0,3 <0,3

9,9.103

- - -

4,36

D

5,8.106 9,1.103 4,0.106

<0,3 <0,3

1,2.103

- - -

4,10

E

5.1.106 7,8.103 2,4.106

<0,3 <0,3

-

<10

-

4,10

A

1,4.108 1,5.103 1,2.108

<0,3 <0,3

6,2.104 ı.s.ıo1

- -

4,72

B

9,8.107 3,7.104 8,9.107

<0,3 <0,3

1,1.io4 1,5.1ü1

-

4,54

90 C

1,1.107 1,3.104 1,1.io7

<0,3 <0,3

1,2.103

- - -

4,30

D

1,3.106 5,1.103 1,2.106

<0,3 <0,3

7,0.102 <10

- -

4,19

E

4,5.105 6,4.103 1,1.io6

<0,3 <0,3

- - - -

4,10

A

4,0.107 1,1.103 6,3.107

<0,3 <0,3

1,2.104

- -

4,65

B

7,2.107 3,4.104 7,1.107

<0,3 <0,3

9,3.103

- - ~

4,57

120 C

4,1.106 7.9.103 6.5.106

<0,3 <0,3

5,9.103

- - -

4,33

D

2,0.106 3,7.103 4,7.106

<0,3 <0,3

9,0.102 <10

- -

4,20

E

5,9.105 3,9.103 8,0.10S

<0,3 <0,3

-

“

4,08

A

B

150 c

2,5.106 2,5.103 5,2.106

<0,3 <0,3

-

<10

- -

4,35

D

_{8,3.10s 8,0.102 3,2.106}

_<0,3

- M - -

4,33

T7 O Q 1 A ^ i o n a

3

O O 1 A “* / a o /. 1 A

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-10-Table 7 : Results of sensory and biochemical indices of treated and untreated samples during ripening.

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Table 8: Results of sensory and biochemical indices of treated and untreated samples during storage at 10 °C.

Mean Sensory Scores (5-point hedonic scale)

C/3 H 5 3 W g Texture Appearance DAYS 1 1 C W c*5 H

Flavour Chew hardness Inside 0utside Preferenei p H

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Figure 1: Changes in total aerobic count of control and treated samples during ripening Colony Count/g.(Log.)

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Figure 5: Changes in lactobacillus count of control and treated samples during storage at 10 C.

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Figure 6: Changes in micrococcus/staphylococcus count of control and treated samples during storage at 10 C.

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Figure 9: Changes in TBA values of control and treated samples during storage at 10 °C.

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-20-REFERENCES :

1. Acton,J.C., Williams,J.G. and Jonson,M.G.(1972): Effect of fermentation

temperature on changes in meat properties and flavor of summer sausage,J.Milk Food Technol., 35(5): 264-268.

2. American Society For Testing Materials Committee (1976): "Manual On Sensory Testing Methods" Technical Puplication 434, American Society for Testing Materials. Philadelphia.

3. Food And Drug Administration (1978): Bacteriological Analytical Manual For Foods, Association of Official Analytical Chemists, Washington.

4. Henner,S., Hartgen,H., Kleih,W. and Schneiderhan,M. (1983): Microbiologischer Status von Gewlirzen für Fleischerzeugnisse, Fleischwirtsch. 63(6): 1051-1052. 5. Holley,R.A., Lammerding,Anna M. and Trittiger,F. (1988): Microbiological

safety of traditional and starter mediated processes for the manufacture of Italian dry sausage, Int.J. Food Mirobiol. (7):49-62.

6. Holley,R.A., Lammerding, AnnaM. and Trittiger,F. (1988): Occurence and signi ficance of streptococci in fermented Italian type dry sausage, Int.J. Food Microbiol. (7):63-72.

7. Mwansyemala,N.A.K.G. (1973): "Report Studies of Routine Analysis for Food Chemistry", Institute For Fishery Products, TNO. Rep.No: 1-145. Ijmuiden, Holland.

8. Remington,D.D. and Schork,M.A.(1970) : "Statistics with Application to Biological and Health Science" Prentice Hall, Inc.New Jersey.

9. Silliker,J.H. (1980): Microbial Ecology of Foods I Academic Press, New York. 10. Speck,M.L. (Ed.)(1976): "Compendium of Medhods for the Microbiological

Examination of Foods" American Puplic Health Association, Washington.