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Studies on the physicochemical characteristics of heated honey, honey mixed with ghee and their food consumption pattern by rats.
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PMID:  22131701     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
Honey and ghee are the two food substances used widely in our diet. In Ayurveda, it is quoted that heated honey and honey mixed with equal amount of ghee produce deleterious effects. Hence, it was of our interest to study the physicochemical characteristics and chemical constituents of heated honey and honey mixed with ghee, and their effect on daily food intake and organ weights of rats. The specific gravity of samples showed a significant decrease in honey and ghee samples heated to 140°C. The pH of honey heated to 140°C was elevated with a reduction in the specific gravity. There was a significant rise in hydroxymethyl furfuraldehyde (HMF) in 60º and 140°C heated honey samples. The browning and total antioxidant of honey mixed ghee samples was significantly higher when compared to ghee samples. Further, the authors have also evaluated the effects of consumption of heated honey, ghee, honey mixed with equal amount of ghee and heated honey mixed with heated ghee in rats. The feeding of heated honey and honey mixed with ghee for 6 weeks showed no significant change in the food intake, weight gain and relative organ weights. The study revealed that the heated honey mixed with ghee produces HMF which may cause deleterious effects.
Authors:
A Annapoorani; K R Anilakumar; Farhath Khanum; N Anjaneya Murthy; A S Bawa
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Ayu     Volume:  31     ISSN:  0976-9382     ISO Abbreviation:  Ayu     Publication Date:  2010 Apr 
Date Detail:
Created Date:  2011-12-01     Completed Date:  2012-10-02     Revised Date:  2013-05-29    
Medline Journal Info:
Nlm Unique ID:  7605807     Medline TA:  Ayu     Country:  India    
Other Details:
Languages:  eng     Pagination:  141-6     Citation Subset:  -    
Affiliation:
Government Ayurveda Medical College, Defence Food Research Laboratory, Mysore, India.
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Journal Information
Journal ID (nlm-ta): Ayu
Journal ID (publisher-id): AYU
ISSN: 0974-8520
ISSN: 0976-9382
Publisher: Medknow Publications Pvt Ltd, India
Article Information
Copyright: © AYU (An International Quarterly Journal of Research in Ayurveda)
open-access:
Print publication date: Season: Apr-Jun Year: 2010
Volume: 31 Issue: 2
First Page: 141 Last Page: 146
ID: 3215355
PubMed Id: 22131701
Publisher Id: Ayu-31-141
DOI: 10.4103/0974-8520.72363

Studies on the physicochemical characteristics of heated honey, honey mixed with ghee and their food consumption pattern by rats
A. Annapooraniaff1
K. R. Anilakumar1
Farhath Khanum1
N. Anjaneya Murthyaff1
A. S. Bawa1
Government Ayurveda Medical College, Defence Food Research Laboratory, Mysore, India
1Biochemistry and Nutrition Discipline, Defence Food Research Laboratory, Mysore, India
Correspondence: Address for correspondence: Dr. K. R. Anilakumar, Biochemistry and Nutrition Discipline, Defence Food Research Laboratory, Mysore, India. E-mail: anilakumarkr@gmail.com

Introduction

In Ayurveda, food is considered as God Brahma and also one of the three basic pillars (food, sleep and celibacy, i.e., ahara, nidra and Brahmacharya) for a healthy life.[1] Food-related diseases include nutritional deficiency, food contamination, food intolerance and also food incompatibility. Food incompatibility may develop because of contradictory qualities, contradictory combination, opposed processing procedures and the nature of food itself. Incompatible food habits lead to a number of diseases including sterility, herpes, eye diseases, skin eruptions, ascites, fistula, leprosy, sprue, edema, fever, rhinitis, fetal distress and may even cause death.[2]

Honey is a sweet, sticky, yellowish fluid made by bees from nectar.[3] Ghee is the Indian name for clarified butter fat, and is usually prepared from cow's milk, buffalo's milk or from mixed milk.[3] Honey and ghee are the two food substances used widely in our diet. The commercial honey available in the market is heat-processed, which absolutely contradicts the accepted Ayurvedic concept. Heating of honey is much discussed under quality deterioration, wherein certain enzymes and nutrients are lost. Honey cakes, honey candies, etc., that are baked are also available for consumption. In some instances, honey and ghee are mixed and served as food. In Ayurveda, Acharya Charaka has quoted that heated honey and honey mixed with equal ghee produce deleterious effects in the body and may cause death also.[4] Hence, the authors have evaluated the physicochemical characteristics of heated honey mixed with heated ghee and its food consumption pattern by rats.


Materials and Methods
Materials

All the biochemicals used in these investigations were of highest purity and procured from Sigma company, St. Louis street, MO, USA; Merck, Darmstadt, Germany, Sisco Research Laboratory (Mumbai, India), Across Organics (Mumbai, India), Spectrochem (Mumbai, India) and S.D. Fine Chemicals (Mumbai, India). All the organic solvents were of analytical reagent grade.

Unprocessed natural honey (raw) was procured from Madikeri forest, Karnataka. The honey samples were heated to 60 and 140°C in order to generate hydroxymethyl furfuraldehyde (HMF). Cow's ghee was procured from Salem, Tamil Nadu. Processed honey (Dabur) was procured from the market. Processed ghee (Nandini) was procured from Mysore Dairy.

Honey and ghee mixture: Ghee samples were mixed with equal quantity of honey and similarly for the 60 and 140°C heated samples.

Methods

Inbred male rats (Rattue norvegicus) of Wistar strain reared in the Defence Food Research Laboratory, Mysore, in the weight range of 100–150 g were used for the entire study. Male Wistar rats were divided into six groups of six rats in each. Group I was fed with normal pellet diet, and served as a control. Group II rats were fed with honey along with pellet diet. Group III rats were fed with heated (140°C) honey with pellet diet. Group IV rats received with ghee with pellet diet. Group V rats were fed with equal amount of honey mixed ghee with pellet diet. Group VI rats were fed with heated honey (140°C) mixed with heated ghee with diet. Clearance of experimental design by the Institutional Ethical committee for rats was taken.

Dose fixation

Ayurvedic classics do not contain the dose of honey and ghee that can be taken together as a part of regular diet. Hence, the dose was fixed based on clinical practice. As per Ayurvedic practice, for a 70 kg man, the normal dose of honey recommended is 48 g/day (1 pala matra) and recommended dose of ghee is 30 ml/day (hraseeyasi matra). Accordingly, the dose of honey was fixed at 102 mg of honey/day and that of ghee was calculated to be 64 μl/day for the Wistar rats of about 150 g body weight.

The animals were kept at ambient temperature and exposed to light–dark cycle of 12 hours duration each. The leftover diet was collected, dried and weighed to determine the food intake. Weekly food intake and weight gain were monitored. Blood was collected from the orbital plexus of the rats on 0 day and at the end of 6 weeks before sacrifice. The reagents used in the biochemical assays were of highest purity and procured from AGAPPE Diagnostics (Kerala, India), Crest Bio-Systems (Goa, India) and Span Diagnostics (Surat, India). After the completion of 6 weeks, the rats were sacrificed under mild anesthesia (sodium pentobarbitone, 50 mg/kg body weight i.p.) and liver, Kidney, heart, brain and colon tissues were quickly excised.

Total sugars and reducing sugars in honey and ghee samples were estimated as per the prescribed procedure.[5] Total ash, acid insoluble ash and specific gravity were estimated as per the method of AOAC International.[6] HMF and browning index in honey were studied spectrophotometrically. The amount of total polyphenolic compounds was measured.[7] The determination of flavonoids was performed according to a colorimetric assay.[8] The antioxidant activity was determined by the standard method prescribed.[9] Peroxide value (PV) was determined according to the AOAC method.[10] Free fatty acid (FFA) value[11] and Thiobarbituric acid value (TBA) value[12] in ghee were also estimated.


Results and Discussion

Heating of honey is contraindicated according to Ayurveda as it causes deleterious effects. Hence, to evaluate this concept, honey has been heated beyond the permissible temperature, i.e., 140°C for 2 minutes. On heating ghee, there is formation of more peroxides. Hence, the study has been conducted to evaluate the effects of heating honey, honey mixed ghee and heated honey mixed heated ghee on Wistar rats.

Table 1 shows the total and reducing sugar and moisture content of honey and ghee samples. All the honey samples showed total and reducing sugars within the prescribed limits. The moisture content of honey was 17% and did not exceed 25% as prescribed by P.F.A. act A.07.03.[13] It was observed that on heating the moisture content decreased significantly. The moisture content of ghee was found to be within the prescribed limits of AGMARK standards not >0.3%.[14]

Table 2 depicts the ash value, acid insoluble ash, pH, specific gravity and organoleptic characteristics of honey and ghee samples. These results showed no significant difference in ash value of unprocessed, processed and heated honey samples. However, the ash value of ghee was significantly low. The acid insoluble ash was found to be within the normal range for all the samples. The pH of honey and ghee samples showed no significant difference. The pH of 140°C heated honey (both processed and unprocessed) was more as compared to the unprocessed, processed and 60°C heated unprocessed and processed honey samples, but not statistically significant. The specific gravity of honey and ghee samples showed significant decrease in specific gravity of 140°C heated honey samples (unprocessed and processed honey) and ghee samples (cow's ghee and Nandini ghee). This may probably be due to the increase in the density of honey and reduced moisture content.

The organoleptic characteristics of honey and ghee samples showed the following results: unprocessed honey – “very good”, processed honey – “good”, 60°C heated, unprocessed and processed honey samples – “good above fair” and 140°C heated honey (both processed and unprocessed) – “fair”, on a 9-point Hedonic scale. However, the “taste” was not included as a part of the organoleptic evaluation.

According to P.F.A act A.07.03, honey should not contain more than (a) 25% of moisture, (b) 0.5% ash and (c) 5% of sucrose. The minimum reducing sugar content should be 65%. The normal pH should be 3.2–4.5 and specific gravity of honey should be 1.3–1.5. Normal FFA value of ghee is 0.5–3.0. The samples used for the assay were found to be well within these values in the raw form. However, on heating, the pH of 140°C heated honey (both processed and unprocessed) was at an elevated level, which may indicate the decrease in acid content of honey. At the same time, specific gravity of 140°C heated honey (both processed and unprocessed) sample was reduced considerably, showing the decreased quality of the honey on heating. It was reported that the boundary thermal treatment, assumed as the most severe condition able to produce a permissible quality loss, was found to be 140°C for 15 seconds in the transient stage and 30 seconds in the isothermal stage.[15]

Table 3 depicts the chemical characteristics of honey samples, viz., HMF, browning index, total phenolic compounds, flavonoids and total antioxidant activity. The table shows a significant rise in HMF in 60°C heated unprocessed and processed honey and 140°C heated unprocessed and processed honey. The European Union, in order to simplify and update the legislation in some food sectors and to follow the new standards of codex on honey, published the EU Directive 2001/110/CE (L 10/47), where in Annex II honey description and chemical composition are listed. The EU Directive follows sections 2 and 3 of ALINORM, 01/25, but with differences. Point 3.2 of ALINORM stresses the effect of overheating on chemical composition changes and quality loss.[16] The most important difference is given in the HMF level. White (1994) proposed the HMF level to be the only reliable heating/storage index in honey. The normal values of HMF must not exceed 80 mg/kg of honey coming from tropical regions with ambient temperature.[17] There was a significant increase in browning and also a rise in antioxidant activity in heated honey samples when compared to unheated honey samples. The browning observed was non-enzymatic due to the Maillard reaction which occurs when the sugars condense with free amino acids. It is believed that the Maillard Reaction Products (MRPs) act as non-nutrient antioxidants.[18] The antioxidant properties of MRPs have been reported to be strongly affected by the physicochemical properties of the system and by the processing conditions. The polyphenols, ascorbic acid and other carbonyl compounds, even if formed during oxidative reactions, can take part in the Maillard reaction itself.[19] As reported earlier, the antioxidant activity and brown pigment formation increases with temperature and time and there is a correlation between the antioxidant activity and increased brown pigment formation.[20] There is also a significant increase in total phenolic compounds and flavonoids in 140°C heated unprocessed and processed honey when compared to other samples of honey. This also suggests the increase in antioxidant activity of honey samples with respect to heat.

Table 4 shows the PV, FFA, TBA value, browning index, total phenols, flavonoids and total antioxidant activity of cow's ghee, Nandini ghee, and honey and ghee mixture. The results show no statistical difference in PV, FFA, TBA, browning index, total phenols, flavonoids and total antioxidant activity of the ghee samples and ghee mixed honey samples. However, the browning and total antioxidant of honey mixed ghee samples was significantly high when compared to ghee samples. This may possibly be due to the mixture of honey in the ghee sample, wherein phenols present in honey impart brown color on oxidation and due to the correlation between antioxidant activity and brown pigment formation, respectively. The data also showed no change taking place in saturated to unsaturated fatty acid ratio by mixing honey with ghee.

Figures 1 and 2 give the weekly food intake and weight gain pattern, respectively, of rats fed with ghee and honey. The results show no change in food intake pattern of rats fed with ghee and honey samples. The relative organ weights, viz., liver, Kidney, heart and spleen were also not altered by the intake of honey and ghee samples either in raw or in heated forms [Figures 36].


Conclusion

The study has shown that heating of honey reduces the specific gravity with a subsequent raise in ash value, pH, HMF, browning, phenolics and antioxidant activity. The mixing of honey with ghee brings about enhancement in browning, antioxidants and specific gravity without altering the food consumption and organ weights of the rats. The study revealed that heated honey (>140°C) mixed with ghee produces HMF which may produce deleterious effects and act as a poison in due course (Ushnam cha samagrutham madhu marayati).


References
1. Sharma RK,Dash BAgnivesha, Sutra sthana11th chapter, 35th Shloka, Charaka Samhita with Ayurveda Deepika commentary by ChakrapanidattaYear: 20017th edVaranasiChaukhambha Sanskrit Sansthana21920
2. Acharya YTAgnivesha, Sutra Sthana26th Chapter, 102- 104th Shlokas, Charaka Samhita with Ayurveda Deepika commentary by ChakrapanidattaYear: 20015th edVaranasiChaukhambha Sanskrit Sansthana3615
3. Kindersley D. Illustrated Oxford dictionaryYear: 2007Reprinted edLondonOxford university Press341390
4. AcharyaAgnivesha YTAgnivesha, Sutra sthana1st Chapter, commentary for 6th Shloka, Charaka Samhita with Ayurveda Deepika commentary by ChakrapanidattaYear: 20015th edVaranasiChaukhambha Sanskrit Sansthana4
5. Ranganna S. Hand book of Analysis and Quality control for fruit and vegetable productsYear: 19992nd edBangaloreTata Mc Graw- Hill publishing company8, 145 (237).
6. Patrica cumiff. Official methods of Analysis of AOAC Internationalrd revisionYear: 19972, 3rd revision16th edUSAAOAC International Science15, 35
7. Singleton VL,Orthofer R,Lameula-Ravento R. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin- Ciocalteu reagentMethods EnzymolYear: 199929915278
8. Singh RP,Chidambara M,Jayaprakasha GK. Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using invitro modelsJ Agric Food ChemYear: 20025081611754547
9. Eberhardt MV,Lee CY,Liu RH. Antioxidant activity of fresh appleNatureYear: 2000405903410879522
10. AOCS, Official methods and recommended practices of the American oil chemist's societyAmerican Oil Chemist's Society, Champaign, IIIYear: 199324th ed 2nd print. Method cd.8.53 (including 1990 and 1992 additions and revisions).
11. Kuruppu DP,Schmidt K,Langerak DI,Van Duren MD,Farkas JB. The effect of irradiation and fumigation on the antioxidative properties of some spicesIFFIT Report No: 37Year: 198322
12. Tatledgis BG,Pearson KM,Dugan LR Jr. A distillation method for the qualitative determination of malonaldehyde in rancid foodJ Am Oil Chemi Soc,Year: 1960374447
13. Prevention of Food Adulteration Act 1954, Rules 1955 and commodity index with exhaustive short notesYear: 1999215th edDelhiInternational law book company182, 251
14. Beena TK. Studies on variation in the quality of ghee sold by Mysore DiaryYear: 1995MysoreDFRL
15. Lucero H,Bulacio L,Tossi EA. Effect of honey high temperature short-time heating on parameters related to quality, crystallization phenomena and fungal inhibitionYear: 2004ArgentinaUni Tech Nac
16. ALINORM 01/25Report of 70th session of the codex committee on Sugars, LondonYear: 2000Month: 2
17. White JW. The role of HMF and diastase assays in honey quality evaluationBee worldYear: 19947510417
18. Nicoli MC,Anese M,Parpinel MT,Franceschi S,Lerici CR. Loss and or formation of antioxidants during food processing and storageCancer LettYear: 19971147149103257
19. Manzocco L,Calligaris S,Mastrocola D,Nicoli MC,Lerici CR. Review of non- enzymatic browning and antioxidant capacity in processed foodsTrends Food Sci TechnolYear: 2001113406
20. Turkmen N,Sari F,Poyrazoglu ES,Velioglu YS. Effects of prolonged heating on antioxidant activity and colour of honeyFood ChemYear: 2006956537

Figures

[Figure ID: F1]
Figure 1 

Food intake of rats fed with honey and ghee; values are mean ± SD of six rats



[Figure ID: F2]
Figure 2 

Weight gain of rats fed with honey and ghee; values are mean ± SD of six rats



[Figure ID: F3]
Figure 3 

Effects of feeding of honey and ghee on relative weight of rat liver. Values are mean ± SD of six rats; values bearing same superscript (a) are not significantly different from each other



[Figure ID: F4]
Figure 4 

Effects of feeding of honey and ghee on relative weight of rat kidney. Values are mean ± SD of six rats; SD ranged from 0.075 to 0.090 and values bearing same superscript (a) are not significantly different from each other



[Figure ID: F5]
Figure 5 

Effects of feeding of honey and ghee on relative weight of rat heart. Values are mean ± SD of six rats; values bearing same superscripts (a) are not significantly different from each other



[Figure ID: F6]
Figure 6 

Effects of feeding of honey and ghee on relative weight of rat spleen. Values are mean ± SD of six rats; values bearing same superscript (a) are not significantly different from each other



Tables
[TableWrap ID: T1] Table 1 

Changes in sugar and moisture contents of honey and ghee on heating



[TableWrap ID: T2] Table 2 

Changes in physicochemical characteristics of honey and ghee on heating



[TableWrap ID: T3] Table 3 

Changes in phytoconstituents of heated honey



[TableWrap ID: T4] Table 4 

Changes in stability parameters and phytoconstituents of ghee




Article Categories:
  • Original Article

Keywords: Ghee, heated honey and heated ghee, honey mixed with ghee, honey, hydroxymethyl furfuraldehyde.

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