ASSESSMENT OF NITRITE AND SORBIC ACID SALTS LEVEL IN SOME MEAT PRODUCTS AND THEIR PUBLIC HEALTH SIGNIFICANCE

Dept. of Food Hygiene, Port-Said Lab.,

Animal Health Research Institute, Dokki, Giza, Egypt.

Assessment of Nitrite and Sorbic Acid Salts Level in Some Meat Products and Their Public Health Significance

(With 8 Tables and One Figure)

By

H. El-S.M. Farag and Noha R.M. Abd-El-Fatah*

*Dept. of Food Hygiene, Animal Health Research Institute,

Dokki, Giza, Egypt.

(Received at 8/1/2011)

تقدير مستوي النيتريت وأملاح السوربيک في بعض منتجات اللحوم 

وتأثيرهما علي الصحة العامة

حسن السيد محمد فرج ، نهي رشدي محمد عبد الفتاح

في دراسة لتقدير مستوي النترات والنيتريت وأملاح السوربيک في بعض منتجات اللحوم تم جمع مائة وعشرون عينة بواقع 30 عينة لکل من السجق ، البسطرمة، البلوبيف ، اللانشون المعلب من أسواق مدينة بورسعيد. وبقياس مستوي النيتريت في عينات منتجات اللحوم موضع الدراسة وجد أن متوسط مستوي النيترايت کان 120.40±7.03، 142.15 ± 9.13 ، 186.27 ± 4.42 ، 159.96 ±  6.73 جزء في المليون لعينات السجق ، البسطرمة، البلوبيف ، اللانشون المعلب علي التوالي.، بينما کان متوسط مستوي النترات وأملاح السوربيک  73.49 ± 6.46 ، 62.54 ± 19.44، 39.82  ± 9.96 و 28.52 ± 10.86، جزء في المليون و0.92 ± 0.11، 1.25± 0.12، 0.0 ، 0.0 جرام /کجم لعينات السجق ، البسطرمة، البلوبيف ، اللانشون المعلب علي التوالي. ووجد أن 100% من العينات موضع الدراسة تحتوي علي نيتريت، بينما 100%، 3.33%، 43.33%، ، 26.67 % من عينات السجق ، البسطرمة، البلوبيف ، اللانشون المعلب علي التوالي تحتوي علي النترات. وعلي الجانب الأخر وجد أن 100% من عينات السجق والبسطرمة تحتوي علي أملاح السوربيک بينما لم يستدل علي هذه الأملاح في عينات البلوبيف واللانشون المعلب. وبمقارنه النتائج بالحد المسموح للنيتريت حسب دستور منظمة الزراعة والأغذية ومنظمة الصحة العالمية في هذا الشأن وجد أن 3 (10%)، 7 (23.33%) ، 4 (13.33%) من عينات البسطرمة، البلوبيف ، اللانشون المعلب علي التوالي تعدت الحد المسموح به، بينما مستوي النترات في جميع العينات موضع الدراسة لم يتعد الحد المسموح. أيضا وجد أن 2 (6.67%) من عينات السجق ، 5 (16.67%) من عينات البسطرمة تعدت الحد المسموح به لأملاح السوربيک. وتم مناقشة تأثير مستوي النترات والنيتريت وأملاح السوربيک علي الصحة العامة.

SUMMARY

One hundred twenty samples of some meat products (30 each of sausage, pastrami, corned beef and canned luncheon beef) were randomly purchased from Port-Said markets. The samples were examined for assessment the levels of nitrite, nitrate and sorbic acid salts. The obtained results revealed that the mean values of nitrite level in the examined meat products were 120.40 ± 7.03, 142.15 ± 9.13, 186.27 ± 4.42 and 159.96 ± 6.73 ppm for sausage, pastrami, corned beef and canned luncheon beef respectively. While that of nitrate and sorbic acid salts were 73.49 ± 6.46, 62.54 ±19.44, 39.82 ± 9.96 and 28.52 ±10.86 ppm and 0.92 ±0.11, 1.25 ±0.12, 0.00 and 0.00 g/kg for sausage, pastrami, corned beef and canned luncheon beef respectively. For all samples nitrite was detected in 100% of the examined samples, while nitrate was detected in 100, 3.33, 43.33 and 26.67% of the sausage, pastrami, corned beef and canned luncheon beef samples respectively. On the other hand sorbic acid salts was detected in 100% of sausage and pastrami samples and could not be detected in the corned beef and canned luncheon beef samples. For all specimens 3 (10%), 7 (23.33%) and 4 (13.33%) of pastrami, corned beef and canned luncheon beef samples exceeded the permissible limits of nitrite established by JECFA "FAO/WHO" (1974)  respectively, while nitrate not exceed the permissible limits. In case of sorbic acid salts 2 (6.67%) of sausage and 5 (16.67%) of pastrami exceeded the permissible limits of sorbic acid salts.The effects of nitrite, nitrate and sorbic acid salts levels on public health significance were discussed.

Key words: Meat products, preservatives, nitrate, nitrite, sorbic acid.

Introduction

Meat and meat products are important component of diet for a large majority of people and considered the vital part of any balanced and nutritious diet. They have a highly nutritive value and palatability thus considered a highly perishable foods and an excellent environmental source for microbial growth(Garcia et al., 1995; Kalalou et al., 2004).

The potential of food contamination and spoilage makes it necessary to add preservatives to foods (Lindsay, 1985). Food preservation has become an increasingly important practice in modern food technology with the increase in the processed and convenience foods (Saad et al., 2005). These preservatives are added to stop or delay the nutritional losses and meat spoilage due to microbiological, enzymatic or chemical changes of foods and to prolong shelf life and quality of food(Mota et al., 2003).

The most commonly used preservatives in meat processing are nitrite, nitrate and sorbic acid (Mihyar et al., 1999). Sodium and potassium salts of nitrite and nitrate are widely used as preservatives in meat processing. The nitrite is either added as salts or produced from nitrate by microbial reduction. Nitrite and/or nitrate plays a crucial role in the curing process, which results in the typical sensory properties and an appealing pinkish reddish color to the meat products that is well know by consumers consequently prevent meat products from turning brown or grey colored. (Dahle, 1979; Noel et al., 1990; Hyytia et al., 1997). The coloring of the processed meat results from the conversion of nitrate to nitrite by chemical reaction that reacts with the myoglobin to form a bright red nitrosomyoglobin. This bright red color is converted into permanent pink pigment nitrosohemochrome under effect of cooking (Hyytia et al., 1997).Generally, nitrite has antimicrobial properties that prevent pathogenic and non-pathogenic bacteria. Nitrite control or prevent the growth of spores particularly of Clostridium botulinum, these spores are a real concern in the food industry because they can survive normal heat processing under right conditions, they can produce vegetative cells which resulted in food poisoningby their lethal toxin (Olsman, 1977; Roberts and Ingram, 1977; Noel et al., 1990; Hyytia     et al., 1997).Besides the coloring and antimicrobial effect of nitrite and nitrate, they have antioxidant properties and can preserve the taste and flavoring and prevent warmed over (undesirable flavors), off odors and rancidity of several meat and meat products during their storage (AMI, 1978; Dahle, 1979; Honikel, 2008).

The other most common used food preservatives were sorbic acid and its water-soluble salts specially potassium sorbate. The microbiological safety of food products was best ensured by addition of potassium sorbates(Shahidi et al., 1988; Ferrand et al.,2000 a). Sorbates are naturally occurring unsaturated fatty acid and considered the best antibacterials agents because their broad spectrum of action. They effectively inhibit certain bacteria and food born yeast and mold species (Sofos and Busta, 1993).The inhibitory effect of sorbates on microorganisms may be lethal or static. The mechanism by which sorbates inhibits microbial growth may be due to its effect on enzymes such as dehydrogenase involved in fatty acid oxidation and sulfhdryl enzyme such as succinic dehydrogenase and yeast alcohol dehydrogenase (Davidson and Juneja, 1990; Sofos and Busta, 1993).  

Many epidemiological studies have showed that diets plays                a substantial role in the etiology of many forms of cancer and it has been estimated that as much as 40-60% of cancer cases may be attributed to factors associated with diets (Miller, 1985; Twombly, 1995).Thus, a specific attention has been focused on the possible role of nitrite and nitrate, in cancer (Jakszyn and Gonzalez, 2006). The potential health dangerous of nitrite referred to its ability to reacts with secondary amines and amides present in the meat products under acidic condition to form nitrosamine. This compound is strong mutagens and powerful carcinogenic in animals and potential in humans causing cancer in certain body organs and tissues e.g. stomach, urinary bladder and others organs (Lijinsky and Kovatch, 1982; Mirvish, 1995). Nitrosamine may be ingested directly with cured meat or may be formed in vivo in the stomach (Howe et al., 1986; Nijinsky, 1999). Ingested nitrates can be reduced to nitrite in the saliva and thus these compounds are considered as potential precursor of endogenously formed nitrosamine (Shapiro      et al., 1991; Grosse et al., 2006).Nitrites also have a vasodilator effect cause intravascular hemolysis in human specially that with glucose-6-phosphate dehydrogenase deficiency (Chan, 1996), headaches (Henderson and Raskins, 1972), hypotension, abdominal pain, acidosis (Gosselin et al., 1984) and linked with triggering migraines (FDA, 1998). In infants, epidemiological evidence showed a relationship between consumption of cured meats and incidence of childhood leukemia, brain tumors, methaemoglobinaemia (blue baby syndrome) and cyanosisdue to the reaction ofnitrite with hemoglobin in the red blood cells. Some of these signs may appear also in young children and elder person(Sen and Baddoo, 1997).

On the other hand, sorbates are low or non-toxic even in large quantities (Shahidi et al., 1988; Ferrand et al., 2000 a)and considered the lowest allergenic potential of all food preservatives.They do not accumulate in the body and being rapidly metabolized by pathway similar to those of other fatty acids through ß-oxidation to form CO₂ and H₂O (Lindsay, 1996; Walker, 1990). Sorbic acid act as a nucleophile and form complex with amino compounds under normal food processing conditions with the resulting adducts being devoid of genotoxic activity (Ferrand et al., 2000 b).In human, few cases of idiosyncratic intolerance to sorbic acid have been reported such as non-immunological contact urticaria and pseudo-allergy (Safford et al., 1990; Walker, 1990)

Therefore, the objective of the present study aimed to investigate the level of nitrite, nitrate and sorbic acid salts in sausage, pastrami, corned beef and canned luncheon beef that mostly consumed by people to evaluate the overall safety of these preservatives in such meat products.  

Materials and methods

1: Samples collection:

A total of 120 random samples of some meat products (30 each of sausage, pastrami, corned beef and canned luncheon beef) were purchased from Port-Said markets. Each individual sample was placed separately into sealed plastic bags, thoroughly identified and delivered to the laboratory. All specimens were processed for assessment the levels of the nitrite, nitrate and sorbic acid and its salts.

2: Chemical examination:

2-1: Preparation of the samples:

All specimens of sausage, pastrami, and the entire contents of the corned beef and canned luncheon beef meat, were separately passed rapidly three times through food chopper with plate opening equal to 1/8^th inch (3 mm), mixed thoroughly after each grinding to obtain a uniform mass and finally began all determinations promptly. The homogenate sample was transferred to a wide mouth glass or other suitable container with an airtight stopper. The analysis was carried out as soon as possible, if any delay, the sample was chilled to inhibit the decomposition (A.O.A.C., 2000).

2-2: Determination of nitrite and nitrate levels:

2-2-1: Reagents:

All reagents used were of analytical grade purchased from Merck (Merck, Darmstadt, Germany) and Sigma-Aldrich (Sigma, St. Louis, MO). Nitrite standard were obtained from Sigma-Aldrich.

2-2-2: Sample extraction:

In 50 ml beaker, 5 gm of the prepared sample were weighed to which about 40 ml of heated water (at 80^°C) were added and mixed thoroughly with glass rod then transferred to 500 ml volumetric flask. Beaker and glass rod were thoroughly washed with successive portions of hot water (at 80^°C) and all washings water were added to flask. Enough hot water was added to bring volume to about 300 ml. The flask was put in steam bath and let stand for 2 hours with shaking occasionally. The samples were cooled to room temperature, diluted to volume with water and remixed then filtered (by free nitrite and nitrate filter paper); if turbidity remained after filtration, centrifuging will usually clear the solution (I.S.O., 1975; A.O.A.C., 2000).

2-2-3: Standard curve of nitrite:

In 50 ml volumetric flask, 2.5 ml of sulphanilamide solution (0.5 gm of sulphanilamide were dissolved in 150 ml of 15% acetic acid (v/v) then filtered "by free nitrite and nitrate filter paper" and stored in a glass stoppered brown bottle) were added and thoroughly mixed with aliquot containing 5-50 μg NaNO₂ (was prepared by added 10, 20, 30, 40 ml of nitrite working solution). After 5 minutes, 2.5 ml NED reagent (0.2 gm N- "1 Napthyl" ethylenediamine dihydrochloride were dissolved in 150 ml of 15% (v/v) acetic acid), were added and diluted to volume with mixing and  let color developed 15 minutes. A portion of the solution was transferred to photometer cell and determined absorbance at 540 nm against blank. Standard curve is straight line up to 1 ppm Na NO₂ in final solution according to A.O.A.C. (2000).

2-2-4: Reduction of nitrate to nitrite:

The nitrate residue in the sample aliquot was reduced to nitrite by acid reduction technique according to Narayana and Sunil (2009).5 ml concentrated HCl and 2 ml of Zn / NaCl granular mixture were added to 10 ml of the sample aliquot. The mixture was allowed to stand for 30 minutes with occasionally stirring to form nitrite and then filtered the solution to 100 ml volumetric flask using Whitman filter paper No. 41. The filtrate was diluted up to the mark and the reduced nitrate levels was assessed and calculated according to A.O.A.C. (2000).

2-2-5: Assessment of the sample for nitrate and nitrite levels:

In 50 ml volumetric flask, 2.5 ml of sulphanilamide solution were added and mixed with 45 ml of the sample filtrate. After 5 minutes, 2.5 ml NED reagent were added and diluted to volume with mixing and let color develop 15 minutes. A portion of sample solution was transferred to photometer cell and determined absorbance at 540 nm against blank of 45 ml water and 2.5ml sulphanilamide reagent and 2.5ml of NED reagent. Nitrite concentrations in the samples were determined by comparison with standard curve. All the estimations were carried out in duplicate for each of reduced and non reduced aliquot according to A.O.A.C. (2000).

2-2-6: Calculation (A.O.A.C., 2000):

C = Concentration of sodium nitrite in μg/ml read from the calibration curve that corresponds with the absorption of the solution prepared from the sample.

M = Mass in gm of sample taken.

V= Volume in ml of aliquot portion of filtrate taken for test.

1.23= factor to convert nitrite to nitrate

2-3: Determination of Sorbic acid salts:

2-3-1: Reagent:

All reagents used were of analytical grade purchased from Merck (Merck, Darmstadt, Germany) and Sigma-Aldrich (Sigma, St. Louis, MO). Sorbic acids standard were obtained from Sigma-Aldrich.

2-3-2: Sample extraction (oxidation methods):

In one liter steam distillation flask, 50 gm of the prepared sample were weighed and 100 gm of magnesium sulphate hexahydrate and 100 ml 1N sulphuric acid were added. Rapidly the prepared sample (do not heat the distilling flask) was steam distilled and the distillates were received in the receiving flask containing 10 ml of 1N NaOH. About 450 ml of distillate were collected in about 30 minutes, cooled and transferred to a 500 ml volumetric flask. 15 ml 1N sulphuric acid were added to the sample aliquot, then diluted to the volume with water and mixed well.

2-3-3: Standard curve of sorbic:

In 500 ml volumetric flask, 25 ml of the standard sorbic acid stock solution (1.0 gm of sorbic acid was dissolved in a small volume of 1N NaOH and diluted to 1 liter with water "1 mg/ml") were pipetted and diluted to volume with water (50 μg/ml). Next 0.0, 10.0, 20.0, 50.0 and 80.0 ml of this solution were pipetted into five 100 ml volumetric flasks and diluted to volume with water (range 0, 5, 10, 25, and 40 μg/ml respectively). Then 2 ml of each of the working standards and blank were pipetted into 5 test tubes and 2 ml of dichromate solution (freshly prepared solution of equal volumes of 0.3 N sulphuric acid mixed with a solution of 0.5 gm potassium dichromate in 1 liter of water) were added, followed by heating in a boiling water bath for 5 minutes. After cooling, 2 ml of 0.5% TBA solution (0.5 gm of thiobarbituric acid was dissolve in 25 ml water + 20 ml 0.5 N NaOH then added 11 ml 1 N HCL and diluted to 100 ml with water) were added and heated in a boiling water bath for 10 minutes. The mixture was cooled rapidly, transferred to 50 ml volumetric flask and diluted to the volume with water. The absorbance of the solution was measured at 532 nm against blank using 1 cm cuvette. All the estimations were carried out in duplicate and the mean of the duplicate was taken. Plot absorbance μg sorbic acid for a standard curve (μg sorbic acid = 0, 10, 20, 50, 80 in 2 ml aliquots) according to FAO (1986).

2-3-4: Assessment of the sample levels:

Two ml of the aliquots sample were pipetted into a test tube and 2 ml of dichromate solution were added, followed by heating in a boiling water bath for 5 minutes. After cooling 2 ml of 0.5% TBA solution (0.5 gm of thiobarbituric acid was dissolve in 25 ml water + 20 ml 0.5 N NaOH then, added 11 ml 1 N HCl and diluted to 100 ml with water) were added and heated in a boiling water bath for 10 minutes, then cooled rapidly and transferred to 50 ml volumetric flask and diluted to the volume with water. The absorbance of the solution was measured at 532 nm using 1 cm cuvette. All the estimations were carried out in duplicate and taken the mean of the duplicate according to FAO (1986).

2-3-5: Calculation (FAO, 1986):

Where A = μg sorbic acid corresponding to sample absorbance, taken from the standard curve

             S = sample weight in gm

3: Statistical methods

Minimum, maximum, mean, standard error and standard deviation of mean as well as frequency distribution were used to describe data. One-Way ANOA test was used to compare the mean of the nitrite, nitrate and sorbic acid salts levels in sausage, pastrami, corned beef and canned luncheon beef. P value was considered significant if less than 0.05 and 0.01 at 95% and 99% respectively. These tests were analyzed using the Statistical Package for Social Scientists (SPSS) for windows 16.0 (SPSS Inc., Chicago, IL, and USA).

Results

Table 1: Statistical analytical results of the nitrite levels (expressed as ppm of NaCO₂) recovered from the examined meat products (n=30 of each).

ND = Non-Detectable.   D = Detectable.  Min. = Minimum.    Max. = Maximum.       SE = Standard Error    SD = Standard Deviation.

Table 2: Statistical analytical results of the nitrate levels (expressed as ppm of NaCO₃) recovered from the examined meat products (n=30 of each).

ND= Non-Detectable.   D= Detectable.  Min. = Minimum.    Max. = Maximum.      

SE = Standard Error    SD = Standard Deviation.

Table 3: Statistical analytical results of the sorbic acid salts levels (expressed as ppm of sorbic acid) recovered from the examined meat products (n=30 of each).

ND= Non-Detectable.   D= Detectable.  Min. = Minimum.    Max. = Maximum.      

SE = Standard Error    SD = Standard Deviation.

Table 4: Frequency distribution of the examined meat products based on their nitrite levels (n=30 of each)

ND = Non detectable level.

Table 5: Frequency distribution of the examined meat products based on their nitrate levels (n=30 of each).

ND = Non detectable level.

Table 6: Frequency distribution of the examined meat products based on their sorbic acid salts levels (n=30 of each)

ND = Non detectable level.

Table 7: Correlation between the different mean values of nitrite, nitrate and sorbic acid salts levels in the examined meat products samples (at p = 0.05 "95%" and p = 0.01 "99 %").

(**) = Highly significant relationship at 0.01 (99%).

(*) = Significant relationship at 0.05 (95%).

Table 8: Comparison between the mean values of nitrite, nitrate and sorbic acid salts with the permissible limits of each in the examined meat products.