Eur Food Res Technol (2000) 211 : 393–395
Q Springer-Verlag 2000
ORIGINAL PAPER
Pramila Rao 7 D. Sujatha 7 K. Rathina Raj S. Vishwanatha 7 K. Narasimhamurthy 7 P. Saibaba D. Narasimha Rao 7 S. Divakar
Safety aspects of residual b-cyclodextrin in egg treated for cholesterol removal Received: 28 September 1999
Abstract Subchronic toxicity dietary studies were conducted in albino rats on the safety aspects of residual b-cyclodextrin in egg samples treated for cholesterol removal at concentrations of 250, 125 and 20 ppm for a period of 90 days. Daily food intake, body weight gain, relative organ weights, histopathology, haematology and serum enzyme activities were studied. The study however, did not reveal any toxicity at any of the levels tested. It can therefore be concluded from the present study that b-cyclodextrin-treated egg samples do not cause any toxicity. Key words b-Cyclodextrin 7 Cholesterol 7 Egg yolk
Introduction b-Cyclodextrin is a cyclic oligosaccharide consisting of seven glucose units joined in the form of a ring in a(I]4) fashion. b-Cyclodextrin possesses remarkable abilities to form stable inclusion complexes with a wide variety of organic molecules [1]. Cholesterol (cholest 5ene 3-ol) can be included in the b-cyclodextrin cavity to form a stable 1 : 2 complex [2]. This phenomenon has formed the basis for the development of a technology for the removal of cholesterol from chicken egg yolk and whole egg [3–5]. Although the functional proper-
P. Rao 7 D. Narasimha Rao Department of Meat, Fish and Poultry Technology, Central Food Technological Research Institute, Mysore-570 013, India D. Sujatha 7 K. Rathina Raj 7 S. Vishwanatha K. Narasimhamurthy 7 P. Saibaba Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore-570 013, India S. Divakar (Y) Department of Fermentation Technology and Bioengineering, Central Food Technological Research Institute, Mysore-570 013, India
ties of the cholesterol reduced samples are not affected [6], the process leaves residues of b-cyclodextrin in egg. Some countries, like Japan, Hungary, France, Spain and Holland have approved the usage of b-cyclodextrin in food systems, but approval by the Food and Drug Administration of the United States of America is still awaited. The LD50 value for b-cyclodextrin in rats (oral) is 0.6 g/Kg of body weight (bw) [7]. It is reported that when administered orally, only insignificant amount of b-cyclodextrin in the intact form is absorbed, and most of it is metabolized by the microflora in the colon to maltose and glucose [8]. However, residual b-cyclodextrin in egg samples has been separated by enzymatic degradation using bacterial and fungal aamylases [9–11]. Bellringer et al. [12] observed that bcyclodextrin present at above 12 500 ppm levels can be toxic and Toyoda et al. [13] reported hepatic lesions in F344 rats treated with b-cyclodextrin at dietary levels above 10% during a 13 week study. Contrary to the above, Olivier et al. [14] reported no toxicity during a subchronic toxicity study with b-cyclodextrin in rats at 10% dietary levels. On optimization of conditions for the removal of cholesterol from chicken egg using b-cyclodextrin [15], our study revealed that 1100–1800 ppm of residual bcyclodextrin is still present in egg samples [16]. However, after digestion of residual b-cyclodextrin in treated egg by the enzyme a-glucoamylase obtained from a mutant strain of Aspergillus niger (CFTRI 1105) [11], it was found that 25–250 ppm of b-cyclodextrin still persisted in the egg samples. In the light of the above, the present study was carried out to determine the safety aspects of egg containing residual b-cyclodextrin.
Materials and methods Animals and diets. Twenty-one day-old male and female albino rats (Wistar-CFT strain, 40 g) obtained from the animal house facility of our Institute were grouped by randomized design into
394 various groups of eight animals each. They were housed individually in stainless steel cages with screen bottoms in a room maintained at 22B2 7C with relative humidity of 60% and exposed to a light and dark cycle of 12 h duration each. Rats were fed a diet containing 26.8% wheat flour, 26.8% ragi flour, 28.0% roasted bengal gram, 5.0% casein, 0.6% calcium carbonate, 2.0% common salt, 4.0% skimmed milk powder and 6.8% peanut oil. In addition to this, the diet was supplemented with vitamins A, D3, E and C in the form of a multivitamin mixture at 0.03 ml for every 100 g diet (Famitone water-soluble vitamin mixture). All the groups of rats were fed this same control diet. Whereas group I rats were fed the control diet without any supplementation, group II rats were fed the control diet supplemented with 250 ppm egg yolk. The remaining three experimental groups i.e. groups III, IV and V were fed the same control diet along with 250 ppm egg yolk treated with b-cyclodextrin at 250, 125 and 20 ppm levels respectively. The egg yolk was treated with b-cyclodextrin at the respective doses as per the procedure of Manohar et al. [15]. The amount of egg yolk supplemented was calculated on the basis of one egg per day for a 60Kg human subject. b-Cyclodextrin was obtained from M/S Cyclolabs, Budapest, Hungary. The animals were provided water and food ad libitum throughout the experimental period of 90 days. The animals were monitored regularly for food intake and weekly body weights were recorded. Haematology and histopathology. At the end of 90 days, the animals were starved overnight and sacrificed under mild ether anaesthesia. Blood was collected by cardiac puncture over EDTA for the measurement of various haematological parameters. Haemoglobin (Hb) was measured according to the cyanmethaemoglobin method [17]; peripheral counts of red blood cells (RBCs) and total white blood cells (WBCs) according to Bharucha et al. [18]; differential counts of WBCs according to Bharucha et al. [18]; and packed cell volume (PCV) using microcapillaries supplied with microhaematocrit centrifuge (Thomas Scientific, USA). Portions of various tissues fixed in 10% neutral formalin were processed and 6mm thick paraffin sections were stained with haematoxylin and eosin for histopathological examination [19]. Serum enzymes. Serum obtained after centrifugation was analysed for the serum enzymes, glutamate oxaloacetate transaminase (GOT), or aspartate aminotransferase, glutamate pyruvate transaminase (GPT) or alanine aminotransferase, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH), from readymade kits obtained from M/S Span Diagnostics, Surat, India. Statistical analysis of the data was carried out using analysis of variance (ANOVA) and Student’s t-test [20].
Table 1 Haematological profile of male and female rats. Values are mean B SEM of eight rats; there were no significant differences between control and experimental groups (ANOVA). Hb Group
Hb (g/dl)
RBC (10 6/ml)
WBC (10 3/ml)
Results and discussion Dietary feeding of b-cyclodextrin for a period of 3 months did not produce any obvious signs of toxicity or mortality during the experimental period at any of the levels in either sex of the animals. Neither did incorporation of b-cyclodextrin cause any reduction in food intake. However, group III rats (250 ppm b-cyclodextrin) showed marginal reduction in food intake, especially during the 1st week of the experiment. Data on absolute weight and relative organ weights expressed as percentage of body weights (data not shown) of both sexes among various groups did not show any significant change and were comparable to controls. The results of various haematological indices are presented in Table 1. No marked changes were observed in any of the parameters studied. Gross examination of the various tissues at necropsy did not reveal any abnormalities that could be attributed to b-cyclodextrin treatment. Further, on microscopic examination, no treatment related histological changes were observed in any of the tissues in any of the group of rats. Data on the activities of the serum enzymes, GOT, GPT, ALP, and LDH in both sexes of rats are presented in Table 2. While the activities of GOT, GPT and ALP were comparable to those of controls in both sexes, the activity of ALP was significantly higher in group I females (P~0.05) and in group II females (P~0.01) and in group IV females (P~0.001) than in the respective controls. Although plenty of literature is available on the toxicity of b-cyclodextrin per se (up to 10% dietary levels), the present study was aimed at unequivocally establishing the safety aspects of b-cyclodextrin in treated egg samples at the levels employed in the present study. In this context, the present study assumes relevance. Although there was a marginal reduction in food intake in group III rats (250 ppm b-cyclodextrin treated) during the 1st week of the study, it did not result in Haemoglobin, RBC red blood cells, WBC white blood cells, PCV packed cell volume, DC differential counts, L lymphocytes, N neutrophils, M monocytes PCV (%)
DC (%) L
N
M
Males I II III IV V
13.9B0.74 12.9B0.27 13.8B0.49 14.3B1.04 13.9B0.40
10.63B0.54 11.68B1.17 11.62B0.85 11.73B1.58 11.10B1.33
12.84B1.63 13.02B7.40 14.64B4.10 14.36B8.16 12.43B5.01
39.4B2.1 33.8B2.4 40.8B2.3 39.2B1.1 41.6B1.8
72.4B4.15 74.2B2.60 73.0B2.90 74.2B3.51 77.6B2.07
25.0B4.0 24.6B2.0 25.2B1.9 23.6B3.6 19.8B3.1
1.7B2.6 1.2B1.3 1.8B0.8 2.2B1.1 2.6B1.5
Females I II III IV V
14.1B1.20 13.3B0.37 13.2B0.57 14.5B0.5 13.0B0.69
11.22B1.38 11.69B0.96 10.43B0.72 11.60B1.21 11.83B1.32
9.29B2.61 5.88B1.85 7.34B4.02 9.61B4.95 7.41B5.75
38.0B3.7 34.6B2.5 39.2B2.9 40.2B1.1 38.2B2.1
74.3B2.24 75.0B2.12 74.4B3.56 73.4B4.06 72.6B3.11
24.4B2.4 23.4B2.1 23.8B3.1 24.0B3.5 24.6B3.3
1.3B0.5 1.4B0.5 1.8B0.5 2.6B1.5 2.8B1.1
395 Table 2 Serum enzyme activities in male and female rats. Values are mean B SEM of eight rats. LDH Lactate dehydrogenase, GOT glutamate oxaloacetate transaminase, GPT glutamate pyruvate transaminase, ALP alkaline phosphatase Group
LDH (change in OD/min/ml serum)
GOT (units/ml serum)
GPT (units/ml serum)
ALP (mmol p-nitrophenol/h/ml serum)
Males I II III IV V
1.45B0.28 1.27B0.23 1.22B0.16 1.35B0.23 1.19B0.14
18.57B0.79 20.83B0.75 18.50B0.93 19.50B0.55 20.29B1.11
27.75B0.89 28.00B0.53 26.75B0.71 29.57B0.53 28.50B0.53
37.43B7.98 49.80B5.72*** 41.85B8.88 40.00B9.94 45.60B4.33
Females I II III IV V
1.93B0.35 1.14B0.17 1.32B0.19 1.30B0.13 1.10B0.15
5.14B0.38 6.62B0.74 5.50B0.53 6.00B0.93 5.75B0.89
6.87B2.10 8.37B0.52 7.25B0.71 9.75B0.71 8.62B1.51
104.37B13.07 97.87B13.19** 103.50B12.79 89.87B13.31* 100.00B11.69
* P~0.001; ** P~0.01; *** P~0.05
reduced growth rate and was comparable to controls, suggesting a normal growth rate. No significant alterations in both absolute and relative organ weights were observed in either sex of the rats under various groups and they were comparable to respective controls. Minor variations observed in the various haematological indices have been attributed to the occurrence of normal biological variations. Absence of any significant histological alterations in any of the tissues in the treatment group at the doses tested relates to the low dosage employed [13]. This is also in agreement with the reports by Olivier et al. [14] who reported no toxicity even after feeding b-cyclodextrin at a 10% level in the diet. Although significant alterations in the serum alkaline phosphatase activity were observed in both male and female rats in certain groups, it did not, however, elicit any significant liver damage as evidenced by histological studies. Further, these changes were not observed in b-cyclodextrin treated males or females at the 250 ppm level. An egg has approximately 15 g of yolk and the diets computed consisted of 250 ppm of egg yolk treated with 250 ppm of b-cyclodextrin at the highest dosage level. The b-cyclodextrin consumed by an average 300 g body weight male or female rat at 15 g of diet consumption per day amounted to 3.75 ppm of b-cyclodextrin. Consumption of treated egg containing 1100 ppm of b-cyclodextrin [16] by a human subject (Average weight 60 Kg), corresponds to 0.75–1.5 mg/ Kg.bw (0.75–1.5 ppm) which is much lower than the dosage employed in this study. We have also observed that treatment of egg yolk by the enzyme a-glucoamylase further reduces the residual b-cyclodextrin in egg samples [11]. Additionally, the levels of b-cyclodextrin employed in the present study are well within the acceptable daily intake of 0–5 mg per Kg.bw as established by both FAO/WHO (1993) and the Commission of the European Communities reports of the Scientific Committee for Foods (SCF, 41 series, 1997). Hence, it can be concluded from the present study that b-cyclodextrin-treated egg samples do not cause any toxicity in
rats as evidenced by our subchronic toxicity studies at the doses tested. Acknowledgements One of us thanks the Department of Biotechnology for the financial support of this project.
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