EJPAU 2005. Zaborowska Z. , Uchman W. , Bilska A. , Jeleń H. , Rudzińska M. , Wąsowicz E. EFFECT OF STORAGE ON CHOLESTEROL OXIDATION PRODUCTS IN THE "PIWNA" AND "MORTADELA" TYPES OF SAUSAGE

Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.

2005
Volume 8
Issue 2

Topic:

Food Science and Technology

ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES

Zaborowska Z. , Uchman W. , Bilska A. , Jeleń H. , Rudzińska M. , Wąsowicz E. 2005. EFFECT OF STORAGE ON CHOLESTEROL OXIDATION PRODUCTS IN THE "PIWNA" AND "MORTADELA" TYPES OF SAUSAGE, EJPAU 8(2), #14.
Available Online: http://www.ejpau.media.pl/volume8/issue2/art-14.html

EFFECT OF STORAGE ON CHOLESTEROL OXIDATION PRODUCTS IN THE "PIWNA" AND "MORTADELA" TYPES OF SAUSAGE

Zofia Zaborowska¹, Waldemar Uchman², Agnieszka Bilska², Henryk Jeleń³, Magdalena Rudzińska³, Erwin Wąsowicz³
¹ Institute of Agricultural and Food Biotechnology, Departament of Food Concentrates, Poznan, Poland
² Institute of Meat Technology, University of Life Sciences in Poznań, Poland
³ Institute of Plant Products Technology, August Cieszkowski Agricultural University of Poznan, Poland

ABSTRACT

The effect of storage on the cholesterol oxidation products (COPs) content, lipid oxidation, hydrolytic changes, pH, ascorbic acid and nitrite contents in "piwna" and "mortadela" type sausages was studied. All the parameters were examined after 1, 3, 6, 8, 10 days of storage at 4°C in the case of the "mortadela" type sausage, and after 1, 3, 8, 10, 13, 15, 17 days of storage at 4°C in the case of "piwna" sausage. A statistical analysis showed an influence of storage time on the peroxide value, pH, ascorbic acid level, 25-OHC and sum of oxysterols in "mortadela" type sausage. In the "piwna" type sausage the storage time has a statistical influence on all the parameters except for its pH value. A significant increase (p = 0.05) oin the peroxide value and the pH value in the "mortadela" type sausage was noted. In the case of the "piwna" type sausage a significant increase of the peroxide value, acid number was noted. Storage time had a significant effect (p = 0.05) on the decrease in ascorbic acid in both types of sausages and nitrite level in the "piwna" type sausages. The content of particular oxysterols statistically increased (p = 0.05) in the "piwna" and the "mortadela" type sausages. The total sum of oxysterols increased (p = 0.05) in both types of sausages.

Key words: cholesterol oxidation products; peroxide value; acid number, cooked sausages; time storage.

INTRODUCTION

Cholesterol oxidation products (oxysterols; COPs) have received considerable attention in recent years because of their biological activities associated with human diseases. The implications of adverse biological effects such as atherogenesis, cytotoxicity, mutagenesis, and carcinogenesis from oxysterols have been reviewed [6, 8]. Animal studies suggested that COPs in the diet could be associated with heart and vascular diseases. Human studies also showed that the quantity of oxidized lipids in the diet was directly related to the level of oxidized lipids in serum postprandial chylomicrons [22], which provides a mechanism by which dietary oxidized lipids can affect the oxidative states of endogenous lipoproteins. Staprans et al. [22] showed that oxidized cholesterol in the diet could be directly absorbed into the circulation, and that COPs accelerated the development of atherosclerosis in rabbits. It is also convincing that oxysterols associated with lipid oxidation in meat arise from heating [7, 11], during storage [7], at various stages of processing, and type of meat product [5]. Moreover, cholesterol readily undergoes oxidation in the presence of oxygen, light, metal ions, radiation and other compounds which could generate reactive components [10, 12, 14, 23]. During food processing and storage, polyunsaturated fatty acids tend to be oxidized. Cholesterol can be oxidized by the same mechanism as fatty acids. Therefore, as Smith [21] suggested, hydroperoxides of polyunsaturated fatty acids formed during lipid oxidation can accelerate the formation of oxysterols from cholesterol.

Oxidation is a major cause of deterioration of food because of its negative effect on organoleptic qualities (flavor, color, etc.). Oxidation of lipids can also have a marked negative effect on nutritional value, and may be responsible for the production of toxic compounds [4].

Despite the widespread existence of oxysterols in foods and their adverse effect on health, little work has been done on meat products. Particularly, in the "piwna" and the "mortadela" types of sausage the presence of oxysterols has not been investigated.

The objective of this study was to determine the effect of storage time on lipid fraction (peroxide value, acid number), pH, ascorbic acid, nitrite and cholesterol oxidation products (COPs, oxysterols) content in "piwna" and "mortadela" types of sausages.

MATERIALS AND METHODS

Both of the "piwna" and "mortadela" sausages were produced according to Good Manufacturing Practice on a commercial scale in a large plant located in Poznań. Freshly prepared, processed sausages were purchased in the local supermarkets. The analysis of the sausages took place two days after their production date and immediately upon their acquisition. All parameters were examined after 1, 3, 6, 8, 10 days of storage in the case of the "mortadela" sausage type, and after 1, 3, 8, 10, 13, 15, 17 days of storage of the "piwna" sausage type. Both of the sausages were stored at 4°C in a dark place.

Reagents
Hexane, acetonitrile, isopropanol, methyl tert-butyl ether (MTBE), dichlorohydroxy-N-(1-naftylo)-etyldiamine and a 30% methanolic solution of sodium methylate and pyridine, derivatization reagent, BSTFA (Bis(trimethylsilyl)trifluoroacetamide), were purchased from Sigma - Aldrich Inc.. 2,6-Dihydrofenoloindofenol was purchased from Loba (Germany). All the rest of chemicals used were obtained from POCH (Gliwice, Poland) and PPH (Lublin, Poland).

Standards
7b-hydroksycholesterol (7b-OHC), 20a-hydroksycholesterol (20a-OHC), 25-hydroksycholesterol (25-OHC), cholesterol a-epoksy (a-epoksy-C), cholesterol b-epoksy (b-epoksy-C) 7-ketocholesterol (7-keto-C), cholestane-3b-5a,6b -triol (triol-C), 27-hydroksycholesterol (27-OHC) and the internal standard 19-hydroksycholesterol (19-OHC) were obtained from Sigma. 7a-hydroksycholesterol (7a -OHC) was obtained from Steraloids Inc.

Equipment
A Hewlett-Packard 6890 gas chromatograph with split/splitless injector and a FID detector was used for the analyses. Compounds were separated using DB-5 column (J&W, 30 m x 0.25 mm x 0.25 µm). The identity of oxysterols was confirmed on a Hewlett-Packard HP 5890 II gas chromatograph coupled to an quadrupole mass spectrometer.

Determination of cholesterol oxidation products
The determination of cholesterol oxidation products was made according to Przygoński et al. [18] with the following modification:

Lipid was extracted using the Folch (1957) method [2]: a homogenized sample (1.000 g) was placed in an Erlenmeyer flask and an internal standard (250 µg 19-OHC) was added. Subsequently, 50 ml of chloroform/ methanol mixture (2:1) containing 0.006% BHT was added and the sample was homogenized for 3 min (8.000 rot/min.) using Ultra - Turrax T 25. The sample was then shaken in a separatory funnel for 6 min. Afterwards the sample was filtered and transferred into a separatory funnel into which 15 ml of water was added. The lower, chloroform layer was filtered over anhydrous sodium sulfate (5 g) and the funnel was washed with chloroform (5 ml). The chloroform fraction was collected in a 100 ml flask, 1 ml of anhydrous ethanol was added and the sample was evaporated to dryness at 30°C under nitrogen.

The following main products of cholesterol oxidation were determined: 7b-hydroksycholesterol (7b-OHC), 25-hydroksycholesterol (25-OHC) and b-epoxycholesterol (b-epoxy-C). Additionally, the sum of oxysterols was measured as the total amount of following compounds: 7b -hydroksycholesterol (7b-OHC), 20a-hydroksycholesterol (20a-OHC), 25-hydroksycholesterol (25-OHC), cholesterol a -epoksy (a-epoksy-C), cholesterol b-epoksy (b-epoksy-C) 7-ketocholesterol (7-keto-C), cholestane-3b -5a,6b-triol (triol-C), 27-hydroksycholesterol (27-OHC) and 7a-hydroksycholesterol (7a-OHC).

Measurment of peroxide value and acid number
The lipid peroxidation was measured in accordance with PN-ISO 3960 [15] and expressed in terms of miliomols of O₂ /kg of sample. The acid number was measured in accordance with PN- 84/A - 85803 and expressed in milligrams of KOH/ 1 g of fat.

Nitrite determination
Nitrite levels were determined in triplicate in accordance with PN-74/A-82114 [17] and expressed in mg/ kg of sample.

Ascorbic acid determination
Ascorbic acid levels were determined using the method of Tillman's for colourless products [9].

Measurment of pH
PH levels were determined using pH - meter N-512 connected with the ERH 111 electrode [9].

Statistical analysis
The experimental design was intended to determine the influence of storage time on the lipid fraction, selected chemical factors and cholesterol oxidation products contents in Polish cooked sausages of the "mortadela" and "piwna" type. The correlations between storage time and all the parameters were calculated. Data was analyzed using a simple regression analysis. The significance was defined at p = 0.05. The experiment was carried out in three replications.

RESULTS AND DISCUSSION

Mean values of the experimental peroxide value, acid number, pH, ascorbic acid and nitrite dates are presented in Table 1 for the "mortadela" and "piwna" type of sausages respectively.

Table 1. The effect of storage (at 4°C) on peroxide value (PV), acid number (AN), pH, ascorbic acid and nitrite value in the "mortadela" and the "piwna" type sausages

	Storage time [days] Type of sausage	1	3	6	8	10	13	15	17
PV^*	mortadela	2.25^a	2.59^b	3.33^c	4.16^d	4.59^e	x	x	x
PV^*	piwna	3.16^a	3.66^b	x	3.90^b	4.51^c	6.05^d	7.00^e	4.71^fc
AN^*	mortadela	2.42^ab	2.30^a	3.03^b	7.01^c	14.18^d	x	x	x
AN^*	piwna	2.20^a	3.25^b	x	3.74^c	4.15^c	5.42^de	5.76^d	6.11^de
PH	mortadela	6.10^a	6.39^bc	6.40^be	6.55^b	6.70^bdf	x	x	x
PH	piwna	6.35^a	6.40^b	x	6.46^ca	6.48^ca	6.52^d	6.45^ec	6.45^ec
ascorbic acid^*	mortadela	20.40^a	17.00^b	15.87^b	11.33^c	9.53^c	x	x	x
ascorbic acid^*	piwna	21.12^a	18.47^b	x	18.47^b	18.47^b	18.47^b	18.47^b	15.80^c
Nitrite^*	mortadela	11.68^a	5.89^b	3.86^c	3.68^c	3.22^c	x	x	x
Nitrite^*	piwna	6.12^a	5.26^b	x	4.91^c	4.89^c	2.78^d	2.85^d	2.25^e

^* Peroxide values were expressed in mmol O₂/kg of sample, acid numbers in mg KOH/ 1 g of fat , nitrite content in mg/ kg of sample, ascorbic acid content in mg/ 100 g of sample
^a,b,c - the mean values in the same line are statistically different

The most serious change that takes place in stored meat products is the auto-oxidation of lipids. However, a number of products may be formed during the oxidation process.

Peroxide value is the most common method for measuring hydroperoxides, which are the primary products of lipid oxidation. The hydrolysis changes of samples were measured, namely by determination of acid number.

Statistical treatment of analytical data showed a significant influence storage of time on peroxide value in both sausages and acid number in "piwna" type sausage.

The peroxide value and the acid number increased markedly during its storage at 4°C for the whole time. Only in the "piwna" type of sausage the peroxide value increased for 15 days and next, decreased statistically. In both sausages the peroxide value doubled during storage time.

The initial value of the acid number (mg KOH/ 1 g of fat) in the "piwna" type sausage was 2.20 and it increased nearly 3 times.

The pH analysis is the most commonly method used for determine the quality of meat and meat products.

Storage time influenced on increase of pH values in the "mortadela" sausage.

Ascorbic acid level decreased during the whole storage time in both sausages, but in the "mortadela" type of sausage this process undergoes faster. In "mortadela" as well as in "piwna" this decrease was statistically significant. The reason for the decrease in ascorbic acid level is its oxidation to dehydroascorbic acid and other intermediate products.

Both nitrite level and ascorbic acid level decreased all the time in the "mortadela" sausage.

As previously shown, different researches of oxidative changes of lipids are closely associated with cholesterol oxidation [3] and therefore in the experiment we determined the degree of cholesterol oxidation products formation.

In the "piwna" and "mortadela" types of sausages we determined the content of 25- hydroxycholesterol (25-OHC), 7b- hydroxycholesterol (7b-OHC), b-epoxycholesterol (b epoxy-C) and the sum of oxysterols (Table 2). The sum of oxysterols was the sum of 7b-hydroxycholesterol, 7a-hydroxycholesterol, b-epoxycholesterol, a-epoksycholesterol, 25- hydroxycholesterol (25-OHC), 20 a -hydroxycholesterol, 7 ketocholesterol, 27-hydroxycholesterol, cholestanetriol.

Table 2. The content of the selected COPs [µg/ 1 g of sample] in the "mortadela" and the "piwna" type of sausages

	Storage time [days] Type of sausage	1	3	6	8	10	13	15	17
7b -OHC	mortadela	1.10^a	0.82^a	2.21^b	1.30^ca	3.83^d	x	x	x
7b -OHC	piwna	0.08^a	0.07^a	x	0.33^b	2.09^c	2.04^d	2.33^e	1.82^f
b -epoxy-C	mortadela	1.70^a	2.17^b	2.62^c	2.10^dab	1.90^eab	x	x	x
b -epoxy-C	piwna	0.20^a	0.14^a	x	0.16^a	0.17^a	0.55^b	0.36^c	0.81^d
25-OHC	mortadela	0.11^a	0.02^a	1.41^b	1.10^b	3.10^c	x	x	x
25-OHC	piwna	0.28^a	0.60^b	x	0.45^c	0.53^cb	0.58^cb	1.61^c	1.85^d
Sum of oxysterols	mortadela	8.04^a	8.74^a	12.41^b	13.73^c	26.45^d	x	x	x
Sum of oxysterols	piwna	11.89^a	11.02^b	x	12.50^c	14.54^d	13.94^e	17.43^f	16.61^g

^a,b,c - the mean values in the same lines are statistically different

A statistical analysis showed significant increase in all oxysterols (Fig. 1, 2, 3, 4) in the "piwna" type of sausage. In the "mortadela" type of sausage storage time had influence on 25-OHC (Fig. 3) and on the sum of oxysterols (Fig. 4). All the time the level of oxysterols in this sausage was higher, and cholesterol oxidation underwent more rapidly than in the "piwna" type of sausage. It may be connected with the more advanced technological processing of the "mortadela" type of sausage. The sum of oxysterols showed the same behavior as in the individual ones.

Fig. 1. The correlation of storage time with 7bb-OHC for the "piwna" type of sausage

Fig. 2. The correlation of storage time with b epoksy-C for the "piwna" type of sausage

Fig. 3. The correlation of storage time with 25-OHC for the "mortadela" and the "piwna" types of sausages

Fig. 4. The correlation of storage time with sum of oxysterols for the "mortadela" and the "piwna" types of sausages

Cholesterol has one double bond between 5^th and 6^th carbon atom and undergoes oxidation via a free radical mechanism [20]. It was also observed that increased during long - term storage of foods [13]. The exposure of cholesterol for a long period of time to the presence of oxygen may initiate the allylic free - radical reaction at the 7^th carbon atom [20]. In addition, the formation of b epoxycholesterol in sausages might mainly come from the secondary oxidation of cholesterol.

The concentration of oxysterols increased dynamicall, in cooked sausages ("mortadela" and "piwna"). The concentration is the same as in Pie et al. (1990) [14]. Engeseth and Gray (1994) [1] showed that cooked meat samples oxidized more rapidly than the raw ones. Thisobservation is well documented.

Decrease in COPs could have been caused by their destruction or by their reaction with other molecules [19].

The data collected in Table 1 and Table 2 were used to calculate the correlation coefficient between all the mentioned parameters. The obtained results have been presented in Table 3 and Table 4, for the "mortadela" and "piwna" sausages respectively.

Table 3. The correlation coefficients for the analyzed parameters in the "mortadela" type of sausage

	Storage time	PV	AN	7b -OHC	b epoxy-C	25-OHC	Sum of oxysterols	pH	nitrite	Ascorbic acid
Storage time	x	0.984	0.731	0.587	0.038	0.800	0.762	0.901	0.726	0.950
PV		x	0.774	0.549	0.009	0.773	0.760	0.864	0.625	0.965
AN			x	0.721	0.083	0.826	0.950	0.685	0.288	0.764
7b-OHC				x	0.0001	0.943	0.882	0.469	0.444	0.463
B epoxy-C					x	0.002	0.016	0.038	0.380	0.006
25-OHC						x	0.943	0.656	0.500	0.684
Sum of oxysterols							x	0.703	0.423	0.727
pH								x	0.802	0.945
nitrite									x	0.683
Ascorbic acid										x

Table 4. The correlation coefficients for the analyzed parameters in the "piwna" type of sausage.

	Storage time	PV	AN	7b -OHC	b epoxy-C	25-OHC	Sum of oxysterols	pH	nitrite	Ascorbic acid
Storage time	x	0.608	0.964	0.757	0.596	0.628	0.830	0.516	0.896	0.593
PV		x	0.669	0.710	0.201	0.328	0.603	0.404	0.636	0.106
AN			x	0.716	0.632	0.661	0.753	0.492	0.966	0.612
7b -OHC				x	0.310	0.339	0.759	0.524	0.611	0.235
B epoxy-C					x	0.528	0.416	0.145	0.747	0.484
25-OHC						x	0.730	0.032	0.644	0.537
Sum of oxysterols							x	0.203	0.666	0.324
pH								x	0.416	0.273
Nitrite									x	0.558
Ascorbic acid										x

The presented results indicate a high correlation between the peroxide value and the level of acid number and the sum of oxysterols in both types of sausages (Fig. 5, 6). In the "mortadela" type of sausage additionally a correlation between the peroxide value and 25 - OHC, pH and ascorbic acid was found. In the "piwna" type of sausage, namely, the correlation peroxide value with 7 b - OHC and nitrite was found. The increase in the peroxide value was parallele with the increase in acid number, the sum of oxysterols, 25 - OHC, pH and 7 b - OHC. At the same time ascorbic acid and nitrite level decreased. Polyunsaturated fatty acids and cholesterol are integral components of membrane structure and are susceptible to autooxidation by free radicals into phospholipids. Oxidation may initiate cholesterol oxidation in the tissue membranes of processed meat. The formation of fatty acid peroxy radicals involved oxidation of cholesterol. These radicals attack the 7- and 5- position of cholesterol. The correlations between acid number and ascorbic acid, 25 - OHC level, and the sum of oxysterols and the correlation 25 - OHC level with the sum of oxysterols in both sausages were also noteworthy. At the same time when acid number increase, the ascorbic acid level decreased and the level of 25 - OHC and the sum of oxysterols increased. In the "piwna" type of sausage the correlation acid number with 7 b - OHC, b epoxy - C and nitrite was found. In both sausages the correlation between the sum of oxysterols and 7 b - OHC was found additionally. In the "mortadela" sausage, the correlation of 7 b - OHC with 25 - OHC and the "piwna" 7 b - OHC with the nitrite leve were found. The level of 7 b - OHC increased while nitrite level decreased. The statistical analysis showed a statistically high influence of nitrite on b epoxy - C, 25 - OHC and ascorbic acid. Similarly as in the case of oxysterols the decrease in the nitrite level was correlated with the oxysterols increase (Fig. 7) and the ascorbic acid decrease. Only in the "mortadela" type of sausage the correlation between the pH level, nitrite and ascorbic acid was found. While pH values increased, the level of ascorbic acid and nitrite decreased.

Fig. 5. The correlation of sum of oxysterols with the peroxide value in the "mortadela" and the "piwna" types of sausages

Fig. 6. Correlation of sum of oxysterols with acid number in the "mortadela" and the "piwna" types of sausages

Fig. 7. Correlation of nitrite level with sum of oxysterols in the "piwna" type of sausage

CONCLUSIONS

Storage time statistically influenced the peroxide value, 25 - OHC, the sum of oxysterols and the ascorbic acid level in both of sausages.
In the "mortadela" type of sausage all transformations undergo faster and more rapidly than in the "piwna" type of sausage
High correlations between the sum of oxysterols and the rest of parameters were found

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PN - ISO 3960

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PN-74/A-82114

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Zofia Zaborowska
Institute of Agricultural and Food Biotechnology,
Departament of Food Concentrates, Poznan, Poland
Starołecka 40, 61-361 Poznan, Poland
ph: (+4861) 879 32 41
fax: (+4861) 879 34 83

Waldemar Uchman
Institute of Meat Technology,
University of Life Sciences in Poznań, Poland
Wojska Polskiego 31, 60-624, Poznań, Poland
ph: (+48 61) 846 72 61
fax: (+48 61) 846 72 54
email: waluchm@au.poznan.pl

Agnieszka Bilska
Institute of Meat Technology,
University of Life Sciences in Poznań, Poland
Wojska Polskiego 31, 60-624, Poznań, Poland
phone: (+48 61) 846 72 61
email: abilska@au.poznan.pl

Henryk Jeleń
Institute of Plant Products Technology,
August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 31, 60-624 Poznan, Poland

Magdalena Rudzińska
Institute of Plant Products Technology,
August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 31, 60-624 Poznan, Poland

Erwin Wąsowicz
Institute of Plant Products Technology,
August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 31, 60-624 Poznan, Poland

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