EFFECT OF STORAGE TIME ON CHOLESTEROL OXIDATION PRODUCTS IN METKA TYPE SAUSAGE

Zofia Zaborowska, Waldemar Uchman, Agnieszka Bilska, Henryk Jeleń, Magdalena Rudzińska, Erwin Wąsowicz

ABSTRACT

The effect of storage on the cholesterol oxidation products (COPs) content, lipid oxidation, hydrolytic changes, pH, ascorbic acid and nitrite contents in metka sausage was studied. All parameters were examined after 1, 7, 14 days of storage at 4°C in darkness. Statistical analysis showed influence of storage time on all parameters. Significant increase (p = 0.05) of peroxide value, acid number and pH value was noted. Storage time had a significant effect (p = 0.05) on the decrease of ascorbic acid and nitrite level. The content of particular oxysterols and sum of oxysterols statistically decreased (p = 0.05).

Key words: cholesterol oxidation products, peroxide value, acid number, metka sausage, storage time..

INTRODUCTION

Cholesterol is the main sterol in animal fats and oils and can generate many oxidation products (COPs) under prevailing conditions. Cholesterol oxidation products commonly found in food are: 7b-hydroksycholesterol (7b-OHC), 7a-hydroksycholesterol (7a-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) and cholestane-3b-5a,6b -triol (triol-C) [21]. Much attention has been paid to the levels of the polar COPs due to their possible adverse health effects and their levels in food. In both in vivo and in vitro studies the COPs have been shown to have a variety of potentially atherogenic, cytotoxic, mutagenic and possibly carcinogenic effects [2].

It has been reported that small amounts of some COPs are presented in row material and processed meat. It is also convincing that oxysterols associated with lipid oxidation in meat arise from heating [8, 12], during storage [8], at various stages of processing, and type of meat product [6]. Moreover, cholesterol readily undergoes oxidation in the presence of oxygen, light, metal ions, radiation and other compounds which could generate reactive components [10, 13, 14, 22]. 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 [20] 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 [5].

Despite the widespread existence of oxysterols in foods and their adverse effect on health, little work has been done on meat products. Particularly in metka type 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 metka type sausage.

MATERIALS AND METHODS

Materials
Metka sausage was a model sausage which was produced on small laboratory scale. In this sausage all parameters were examined after 1, 7, 14 days of storage. The sausage was stored at 4°C in darkness.

Reagents
Hexane, acetonitrile, isopropanol, methyl tert-butyl ether (MTBE), dichlorohydroxy-N-(1-naftylo)-etyldiamine and a 30% methanolic solution of sodium methylate and pyridine were purchased from Sigma-Aldrich (USA). 2,6-dihydrofenoloindofenol was purchased from Loba (Germany). A derivatization reagent, BSTFA (Bis(trimethylsilyl)trifluoroacetamide), was obtained from Supelco, Inc. (Bellefonte, PA). 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 Chemical Co.(St. Louis, MO). 7a-hydroksycholesterol (7a-OHC) was obtained from Steraloids Inc. (Wilton, NH).

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 [3] method: 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 shaker 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: 7-ketocholesterol (7-keto-C), 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
Lipid peroxidation was measured in according with PN-ISO 3960 and expressed in terms of miliomols of 0₂ /kg of sample. Acid number was measured in according with PN- 84/A – 85803 and expressed in milligrams of KOH/ 1 g of fat.

Nitrite determination
Nitrite levels were determined in triplicate in according with PN-74/A-82114 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 ERH 111 electrode [9].

Statistical analysis
The experimental design was intended to determine the influence of storage time on lipid fraction, selected chemical factors and cholesterol oxidation products content in Polish raw sausage – metka. The correlations between storage time and all parameters was calculated. Data was analyzed using a simple regression analysis. Significance was defined at p = 0.05. The experiment was carried out in three replications.

RESULTS AND DISCUSSION

Mean values of experimental peroxide value, acid number, pH, ascorbic acid and nitrite dates are presented in Table 1.

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 to measure hydroperoxides, which are of transistory nature and are the primary products of lipid oxidation, intermediate in the formation of hydroxyl and carbonyl compounds [7]. The hydrolysis changes of samples were measured namely by determination of acid number.

Statistical treatment of analytical data showed significant influence of storage time on peroxide value and acid number.

The peroxide value and acid number increased markedly upon storage at 4°C for all the time of storage.

The pH analysis is the most commonly method used to determine the quality of meat and meat products. Storage time influenced on increase of pH values. The initial pH value was 6.17 and grow up during teen days to 6.26.

Ascorbic acid level the same as nitrite content decrease for all the storage time. These decreases were statistically significant. The reason of decrease of ascorbic acid level is his oxidation to dehydroascorbic acid and the other mean products.

As previously shown different researches oxidative changes of lipids are closely associated with cholesterol oxidation [1, 4] and therefore in the experiment we determined the degree of cholesterol oxidation products formation.

In the metka type sausage we determined the content of 7b- hydroxycholesterol (7b-OHC), 7–keto-C and sum of oxysterols (Table 1). 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.

Statistical treatment of analytical data showed significant influence of storage time metka type sausage on all oxysterols. On storage in the refrigeration (4°C) amounts of both cholesterol oxidation products decreased over the teen days period as shown at Figure 1 and Figure 2 respectively from [1.76 µg/1 g of sample] to [1.31 µg/1 g of sample] for 7b-OHC and from [1.79 µg/1 g of sample] to [1.47 µg/1 g of sample] for 7 keto-C. Decrease of 7b-OHC content may be correlated with his transformation to secondary cholesterol oxidation product 7-ketocholesterol. The reason of decrease of 7 keto-C may be decomposition this oxysterol during storage. Total sum of oxysterols showed the same behavior as the individual ones (Fig. 3). Decrease in COPs could be caused by their destruction or by their reaction with other molecules [19].

Data collected in Table 1 were used to calculated correlation coefficient between all mentioned parameters. Obtained results have been presented in Table 2.

The thicken correlation coefficients are statistically significant.

Presented results indicate high correlation between all parameters except correlation between peroxide value and acid number, correlation between pH value and acid number and nitrite level and acid number correlation. Increase of peroxide value paralelled the increase pH value and decrease of both oxysterols, their sum (Fig. 4) and decrease nitrite and ascorbic acid levels. Acid number was correlated with 7b-OHC, 7 keto – C , sum of oxysterols (Fig. 5) and ascorbic acid level. Increase of acid number paralelled the decrease all mentioned parameters. 7b-OHC and 7 keto-C showed high correlations between all measured parameters. Decrease the contents both oxysterols was correlated with decreased the rest of parameters. The same as individual oxysterols decrease of sum of oxysterol was correlated with nitrite and ascorbic acid decrease (Fig. 6 and 7) and pH increase (Fig. 8). Addition of ascorbic acid increased pigment and lipid stability in ground pork and ground beef [11]. Ascorbic acid functioned as an antioxidant with some substrates by scavenging oxygen and inhibiting radical formation at double bounds. Nitrite plays an important role both in color development and as a preservative exerting an anticlostridial effect in cured meat. It was also recognized that the addition of nitrite during the curing process decreased lipid peroxidation [7]. Nitrites, through nitric oxide, have autooxidation effect mainly by acting on the iron of the heme proteins and stabilizing unsaturated lipids within the membrane. The increase of pH value was correlated with nitrite and ascorbic acid decreased. Nitrite content was namely correlated with ascorbic acid.

1. Storage time statistically influenced on all measured parameters in metka type sausage.

2. All parameters were correlated each other except correlation between acid number and: peroxide value, pH and nitrite content.

3. In metka type sausage concentrations of oxysterols decreased for all the time.

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