The possibility of using jojoba safe extract as natural food preservation
Makpoul .K .R, Ibraheem.A.A
Food science and technology – Agricultural industrialization unite
Department of Plant Production – Desert Research Center ‘ Cairo ‘Egypt
Abstract
The effect of crude aqueous extract of jojoba meal and leaves with simmondsin (MS and LS) and without simmondsin (MS-1 and LS-1) at various concentrations on Physio chemical, microbiological and sensory quality of mango drink during storage was assessed. Inhibitory activity of the jojoba extracts as preservatives and their effect on chemical and sensory attributes was tested periodically by simulating the industrial mango drink storage in the room temperature for a period of 180 days. Protein, fats, decreased while ash content and total soluble solid (TSS) increased during the storage period. A slight progressive decline in pH was observed with a proportional increase (p<0.05) in the acidity of the stored pulp samples. Significant inhibition of the total bacterial count (TBC) was observed on applying the specified concentration. Storage time significantly (p<0.05) increased the CFU/g of the drink samples as the maximum growth was observed after 180 days of storage. Sensory characteristics of the drink prepared from treated mango pulp samples were affected negatively on addition of preservatives however, the samples were accepted by the judges even after six months of storage.
Keywords : jojoba, defatted meal, jojoba leaves, simmondosin, food preservation, mango drink
corresponding author / khaledmakpoul@hotmail.com
1- INTRODUCTION
Food spoilage and food-borne pathogens development on food products has a direct effect on the decrease of the nutritional quality by consuming protein, fat and carbohydrate present in these products which subsequently causes food discolor ration, biochemical changes and toxicity, in addition of their adverse economic consequences. Many bacterial and fungal contaminants are able to produce some highly toxic secondary metabolites, like mycotoxins, that are capable of causing disease and death in humans (You, 2006). Actually, many preservation methods were employed in food industry including low-temperature storage, vacuum package, irradiation’ but the use of chemical preservatives remains the most employed method in agroindustry. However, the safety problems related to the use of chemical preservatives are receiving growing attention. Therefore, many research teams have focused on the development of safety preservation procedures employing naturally derived substances such as salt, sugars, vinegar and natural extracts from dietary plants. The richness of these dietary plants on phenolic compounds such as tannins and flavonoids, known for their several biological effects including antimicrobial properties, can have a direct impact on reducing the health hazards and economic losses due to food-borne pathogens(Feten et al, 2014). Natural plants have a high potential for producing The most important of bioactive constituents, which are mainly secondary metabolites, such as alkaloids, saponin, flavonoids,
tannins and phenolic compounds. These phytochemicals could be toxic to microbial cells [Dash et al, 2008).
Simmondsia chinensis (jojoba) is a semiarid evergreen shrub. It grows wild in the desert south-western United States and north-western Mexico. However, the plant is cultivated in some of the Middle East and Latin American countries (Borlaug et al., 1985; Bellirou et al., 2005). Jojoba seeds contain about 50’60% of a unique wax ester oil which is composed mainly of straight chain monoesters in the range of C40’C44 (Ellinger et al., 1973). Jojoba oil has good markets in the cosmetics and lubricant industries (Cokelaere et al., 1992a), and recently, it has been reported that the jojoba seeds possess anti-inflammatory activity (Habashy et al., 2005). After oil extraction of jojoba seeds, a protein rich residue remains, known as defatted jojoba meal. The meal contains 20’32% of protein, consisting mainly of albumins (79%) and globulins (21%) (Shrestha et al., 2002). This meal also contains approximately 15% of a group of glucosides, known as simmondsins (Ellinger et al., 1973; Van Boven et al., 2000). Eight glucoside compounds (simmondsin and seven simmondsin derivatives) have been isolated and identified form jojoba seeds (Bellirou et al., 2005). Among these the methylated compounds simmondsin and simmondsin 2_-ferulate exhibited food-intake inhibition in rodents and chickens. In our search of the literature we have found no studies on the bioactivity of extracts and glucosides isolated from jojoba against agricultural pests (Moustafa et al., 2007). Besides Jojoba seeds and meal have been shown to contain considerable amounts of tannins (2.5%) (Wiseman, 1987 a, b). In addition, Jojoba contains anthocyanins namely malvidin (Sharp, 1974), alkaloids (Hultin, 1966), while the leaves contain two major flavonoids which are isorhamnetin 3- rutinoside (narcissin) and isorhamnetin 3, 7-dirhamnoside (Orwa et al., 2009). Originally, it was believed that the jojoba plant was toxic because of the presence of simmondsin. It was stated that the -CN part would give rise to HCN in the body when digesting the simmondsin compound (Booth et al., 1974; Verbiscar et al., 1980; Williams, 1980), thus leading to emaciation. Meanwhile, it has been proven that digestion of simmondsin does not lead to liberation of cyanides into the body (Cokelaere et al., 1992b), but rats fed with 3% de-oiled jojoba flour still showed a lower weight gain than the pair-fed animals (Cokelaere et al., 1993).
In 1980, Verbiscar et al. reported that five mice died when fed simmondsin at 750 mg/kg for 14 days and three surviving mice showed signs of hepatotoxicity and possible intestinal hemorrhage. Intraperitoneal administration of the same dose, however, did not decrease body weight of rats nor were there any other drug-induced effects. Subsequent pair-feeding studies, using more moderate levels of simmondsin, suggested that its effects are primarily because of decreased voluntary food consumption. Rats fed 250 mg/kg for 5 days showed no toxicological influences on biochemical parameters of the liver, pancreas and kidneys and no pathological changes were found in kidney, liver, pancreas, stomach, intestine, testis and seminal vesicle (Moyad, 2014). The LD50 of the aqueous extract of Simmondsia chinensis was 4.14 g kg-1 body weight, method which represents 20.54 g of crude powdered plant material for 1 kg body weights (Litchfield and Wilcoxon, 1949).
The aim of the present investigation is to achieve the effective utilization of water extracts of jojoba leaves and jojoba defatted meal for food preservation.
Determine total polyphenol, simmondsin, and tannin contents at (leaves, and defatted meal) of jojoba. Chemical preservatives are being replaced with water extracts from jojoba leaves and defatted meal. Therefore, the biological activity of the extracts will be studied. Several concentrations of water extract, evaluated as food preservation in mango juice during storage. followed by investigated to the antimicrobial effect of the different concentrations of water extract. determine the effect of simmondsin in food preservation.
2. Materials and methods
2-1 Plant material preparation:
Jojoba defatted meal and leaves were obtained from Middle Sinai research station (El Maghara in Sinai)- desert research center-Egypt. The defatted meal samples were ground to pass through a 60-mesh sieve using an analytical mill to fine powder. Jojoba leaves were dried at 50”C then ground to pass through a 60-mesh sieve using an analytical mill to fine powder. Mango fruit was obtained from the local market of Egypt. The fruit was thoroughly washed to remove dirt, dust, pesticide residues and microflora on the surface of the fruit.
2.2. Preparation of Extracts
The fine powdered samples (20g) of defatted meal and leaves were extracted with 100 ml of boiling water until cooled, then saved at room temperature for 24 h and filtered using Whatman No. 1 filter paper. This crude extracts with simmondsin were labeled as (MS) for meal with simmondsin and (LS) for leaves with simmondsin. For extracts without simmondsin the fine powdered samples (20g) of defatted meal and leaves were extracted with 100 ml of acetone and water (80:20, (v/v)) for 24 h at ( 25”C). After filtered The residues of defatted meal and leaves were extracted with 100 ml of boiling water until cooled, then saved at room temperature for 24 h and filtered using Whatman No. 1 filter paper. This crude extracts without simmondsin were labeled as (MS-1) for meal without simmondsin and (LS-1) for leaves without simmondsin.
2-3 Determination of phenols, tannins and simmondsins in extracts
Total phenols were determined with the Folin-Ciocalteau reagent (Makkar, et al., 1993, and Makkar 2003). Extractable tannins were determined as the differences in total phenols (measured by Folin-Ciocalteau reagent) before and after treatment with insoluble polyvinyl polypyrrolidone (PVPP), as this polymer binds strongly to tannins (Makkar et al., 1995). Total phenols TP and total tannins (TT) TT were expressed as tannic acid equivalents. Condensed tannins were measured by the HCl-butanol method and results were expressed as leucocyanidin equivalent (Makkar, 2003). Simmondsins were determined in the defatted jojoba meal and jojoba leaves extracts using HPLC apparatus with a L-6200 pump (Merck- Hitachi, Germany) equipped with a L-3000 photo diode array detector (Merck-Hitachi, Germany). Total simmondsins (TS) determined as summation of (Simmondsin, simmondsin ferulate, demethylsimmondsin (DMS), and didemethylsimmondsin (DDMS)).
2-4 Preparation of mango drink samples:
Mangoes were passed to separate pulp from the stones and skin and the pulp obtained was ready to serve drinks (pulp 8%, acid 0.2% and sugar 16 Brix), and mixed with deferent concentration (1%, 2% and 3%) of jojoba extract (MS, MS-1, LS and LS-1). Mango drinks with chemical preservatives sodium benzoate (NaC6H5CO2) (0.1 %) was control sample (SB). The mango drinks samples were transferred to lock glass bottle (1 litter) and stored at room temperature (25”C) for a period of 180 days.
2-5 Sensory evaluation of mango drinks
Each sample of mango drink were presented to a panel of judges for sensory evaluation for color, taste, flavor, stickiness, and overall acceptability. The panel members were selected on the basis of their ability to discriminate and scale a broad range of different attributes of mango and mango products. An orientation program was organized for the panel members to brief them the objective of the study. The judges were provided with prescribed questionnaires to record their observation. The information contained on the performa was 9 = Like extremely; 8 = Like very much; 7 = Like moderately; 6 = Like slightly; 5 = Neither like nor dislike; 4 = Dislike slightly; 3 = Dislike moderately; 2 = Dislike very much; 1 = Dislike extremely. The panelists expectorated the drinks and rinsed mouth using distilled water between samples the method described by Larmond (1977). Samples were carried out after 30 days for analyses. The experiment was repeated twice and the values are presented as means (SD”).
2-6 Microbiological assay:
The determination of the total microbial contamination of the drink samples were performed after 30 days until six months by the method outlined in compendium of methods for the microbiological examination of foods (Anon., 1992). Nutrient agar, (The media used in the present investigation were obtained from Microbiological Resources Center (Cairo) Faculty of Agriculture, Ain Shams University) was used for periodical determination of total bacterial count (TBC) in the stored mango drink samples. Nutrient medium was suspended/litre of distilled water, mixed thoroughly, pH adjusted at 7.2 (25”C) (Jenway 3510-UK), heated with frequent agitation and boiled for 1 minute to completely dissolve the ingredients and autoclaved at 121”C for 15 minutes. One gram sample was taken from each treated drink sample using aseptic techniques, placed in labeled sterile dilution bottles and made into a volume of 100 ml by distilled water to achieve 10-1 suspension under sterile conditions. The contents were mixed thoroughly and aliquots were serially diluted and enumerated onto Nutrient agar. Plates were subsequently incubated (Memmert 100-Germany) for 48h at 37”C and TBC was calculated using colony counter. Samples were carried out after every 30 days for analyses. The experiment was repeated twice and reported data represent mean values (CFU/ml) of these measurements (Saeed et al, 2010).
2-7 Physio chemical analyses of the drink
The main physical-chemical characteristics of the juices have been established, thus: the relative density of the juices was picnometrically measured, the refraction index with ABB” refractometer, the sugar content with a Carlzeiss Jena portable refractometer, the pH through the potentiometric method using pH-meter equipped with a SenTix81 combined glass electrode. The glass electrode was calibrated using standard buffer solutions. The turbidity of the juices was measured with a TURB 355 IR/T turbidimeter (Corina et al, 2006).
2-8 Hemolysis assay
The hemolytic activity of aqueous jojoba meal extract and aqueous jojoba leaves extract were evaluated using human erythrocytes. Different extracts at the concentrations ranging from 0.05 to 10 mg ml-1, were incubated with washed erythrocytes (108 cells) in PBS (Dulbecco’s phosphate-buffered saline) pH 7.4 (100 ”l) for 1 h at 37 ”C. After centrifugation (1000 g for 5 min), the absorbance at 450 nm of the supernatant was measured. A parallel erythrocytes incubation in the presence of Triton X 0.1% and PBS served as controls inducing 100% and 0% hemolysis, respectively. Extracts hemolytic activities were expressed as LC50 corresponding to the concentration inducing 50% hemolysis (Feten et al, 2014).
2-9 Statistical analysis
Data were statistically analyzed, using analysis of variance (Steel et al., 1997). Duncan’s Multiple Range Test was applied to assess the difference between means. Significance was defined at p’0.05. Values are means of two experiments (SD”).
3- RESULTS AND DISCUSSION
3-1 Determination of phenols, tannins and simmondsins in extracts
Most studies on jojoba have focused on the extraction or transformation of simmondsin as the principal toxic constituent. Other components may contribute to the toxicity and unpalatability of jojoba leaves and meal. Phenolic compounds may impart astringency and bitterness (Ozawa et al 1987). The simmondsin content in aqueous extract of jojoba defatted meal (MS) was (41.6 mg/ml) and contained (24.9 mg/ml) phenols, whereas the aqueous extract of jojoba leaves (LS) contained (3.9 mg/ml) simmondsin and (10.3 mg/ml) phenols as shownin Fig (1).
Acetone was found effective in removing 85% of the simmondsins and 35% of phenols from jojoba meal and leaves (LUIS and AUGUSTO, 1990). The simmondsin was extracted at levels (5.4 mg/ml) in aqueous extract of jojoba defatted meal after treated with acetone (MS-1), and phenols were extracted at levels (19.7 mg/ml). Whereas the aqueous extract of jojoba leaves after treated with acetone (LS-1) contained (0.3 mg/ml) simmondsin and (8.2 mg/ml) phenols as shown in Fig (1). Tannins are the polyphenolic compounds the level of tannins in aqueous extract of jojoba defatted meal (MS) was (0.98 mg/ml) and (7.8 mg/ml) in aqueous extract of jojoba leaves (LS). Whereas the aqueous extract of jojoba defatted meal after treated with acetone (MS-1) contained (0.74 mg/ml) and aqueous extract of jojoba leaves after treated with acetone (LS-1) contained (5.6 mg/ml) tannins.
3-2 Sensory evaluation of mango drinks
Mango drinks prepared from the treated pulp samples was carried out for colour, flavour, taste, stickiness and overall acceptability. It is evident that addition of jojoba extracts preservatives greatly influences these attributes with a little loss in drink quality (Fig 2, 3 and 4). The results pertaining to the effect of the addition of jojoba aqueous extract of meal and leaves with simmondsins (MS and LS) and after removed simmondsins (MS-1 and LS-1) as food preservatives to mango drink are presented in fig (2and 3). Concentration and synergistic addition of MS 1% and LS 1% seem to have slight effect on their ability to act differently for deteriorating the taste, color, flavor, stickiness and overall acceptability of stored mango drink, the drink samples were still liked very much by the judges for color and flavor as shown in fig (4).
However, the exception was noticed in scores showing greater variability in relation to treatment and the concentration of these extracts. The maximum deterioration was noticed with (MS 3% and LS 3%) in the drink sample for all parameter and overall acceptability as a function of storage for the time, therefore must be rejected. Color score 2 for MS 3% and LS 3%, overall acceptability score for MS 3% and LS 3% respectively. The panelists clearly identified the changes in parameter profile of the (LS 2% and LS-1 2%) samples rating the stored sample inferior as compared to the freshly prepared drinks. Storage time perpetually decrease flavor score until 30 days storage, nevertheless, the drink samples were still liked by the judges for overall acceptability. A uniform pattern of decline in these sensory attributes of the mango drink samples treated with MS 2%, MS-1 1% , MS-1 2% , MS-1 3% and LS 3% were evident in relation to storage time which makes the samples were rejected.
3-3 Microbiological assay:
Table. 1 revealed inhibitory effects of jojoba aqueous extract (MS, MS-1, LS and LS-1 ) on the microbial growth of the mango drink at different concentrations (1,2 and 3 %) used in the food industry. The highest inhibitory effects on bacterial growth in mango drink samples were exerted by MS and LS at a concentration of 3% followed by concentration of 2% of MS and LS of each. Increasing of MS-1 and LS-1 concentration from 1 to 3% reduced the growth velocity and concentration 1% of MS and LS were shown to be equally effective as compared to 1 mg/kg of the SB (Fig. 5). The results of the present study also demonstrated an inhibitory effect of MS, MS-1, LS and LS-1 in mango drink stored for a period of six months.
The highest level of contamination in mango drink samples was observed in control (no preservative added) after 180 days of storage while the minimum growth was shown in the presence of LS 3%. Periodical analysis of the mango drink samples for the TBC showed a progressive increase in the growth though the rate of growth varied with different treatment for 180 days suggesting the jojoba extract to be relatively inhibitor in mango drink. The results of the present study substantiated that none of the extracts used as preservatives were able to completely inhibit the bacterial growth in all concentrations for a period of 180 days storage.
3-4 Physicochemical analyses of the drink
Incorporation of jojoba extract preservatives exhibited a significant (p<0.05) effect on physicochemical profile of mango drink (Table 2). Addition of MS, MS-1, LS and LS-1 at all concentration did not show a non-significant effect on the fat content of the samples. Increasing of extracts concentration from 1 to 3% reduced the protein content in mango drink sample. The results revealed that jojoba aqueous extracts increased the acidity of mango drink with a corresponding decrease in pH value of the samples. Storage time had shown a pronounced effect on physicochemical attributes of chemically preserved mango drink. Progressive decrease in turbidity, density and protein content of drink sample was observed over the entire storage period of 180 days. The high sugar content of drink might be attributed to the transformation of starch into soluble sugars under the action of enzymes during ripening, however, the increase in TSS was apparent in the last period of 180 days storage.
3-5 Hemolysis assay
Reports dealing about aqueous jojoba extract toxicity, we have observed that the (LS, LS -1 and MS-1) extracts don’t exhibited hemolytic activity against human erythrocytes at concentrations ranging from 1 to 20 ml/kg body weight except for MS extract (LC50 = 3.5 ml kg body weight)
4- DISCUSSION
Beside phenolics and tannins have antimicrobial activity that is affected by factors such as the aglycone, number, position and chemical structure of sugar side chains (Maier 2008). Tannins are the phenolic compounds act as antibacterial agent against many pathogenic bacteria such as Staphylococcus aureus, Staphylococcus epidermis, Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumonia, Salmonella typhi and Escherichia coli (Kamal et al, 2010) .
The antimicrobial properties of aqueous jojoba extracts can be due to the richness of its organs on polyphenolic compounds. Many studies have demonstrated that good linear relationships exist between antibacterial activity and the high level of phenolic components, and emphasized the importance of several classes of polyphenol such as phenolic acids and tannins. Phenolic compounds in plant defense mechanism against pathogenic microorganisms, insects, and herbivores. However, we have observed that aqueous jojoba extracts from defatted meal and leaves exhibiting the highest antibacterial activity in food preservation, as compared with the chemical preservatives sodium benzoate. These findings can be explained by the nature of the components implicated in the food preservation as antimicrobial activity.
The present results indicate that the isolated glucosides; Total simmondsin (Simmondsin, simmondsin ferulate, demethylsimmondsin (DMS), and didemethylsimmondsin (DDMS)) have remarkable antimicrobial activities against. Moreover, they have moderate antifungal activity against . This can be explained that the simmondsin may have a role in the food preservation. Furthermore, the crude aqueous extracts of jojoba defatted meal and leaves with simmondsin were more potent than other extracts of the meal and leaves after removed simmondsin. It is concluded from these results that the isolated glucosides might be considered as key compounds for developing safe alternative food protection agents according to the IC50 values.
5- Conclusions
This study demonstrated the inhibitory effects of crude aqueous extract of jojoba meal and leaves with simmondsin (MS and LS) and without simmondsin (MS-1 and LS-1) on microbial growth in the mango drink stored under room temperature. Suggested that MS and LS at concentrations of 1% for each had been equally effective at 100 ppm sodium benzoate (SB) used individually. Further, these aqueous extracts as food preservatives had significantly affected the physic chemically profile of the drink samples with a pronounced increase in acidity and corresponding decrease in pH during storage for six months. Addition of jojoba extracts adversely influenced the sensory attributes of the stored drink however, the product remained acceptable after six months storage. The effect of jojoba aqueous extracts as preservatives on physic chemical profile, microflora and organoleptic properties of stored mango drink shown in this work constitutes a major contribution that can help the development of a safer and viable storage of mango drink at industrial scale according to the IC50 values.
References
1. Anonymous. (1992). Compendium of Methods for the Microbiological Examination of Foods.American Public Health Association, Washington, DC.
2. Bellirou, A., Bouali, A., Bouammali, B., Boukhatem, N., Elmtili, B.N., Hamal, A., El-Mourabit, M., (2005). Extraction of simmondsin and oil in one step from jojoba seeds. Ind. Crops Prod. 21, 229′ 233.
3. Booth A.N., Elliger C.A., Waiss A.C. Jr. (1974) Isolation of a toxic factor from jojoba flour. Life Sci. 15:1115-1120
4. Borlaug, N., Baldwin, A.R., Estefan, R., Harris, M., Plucknett, D.L., (1985). Jojoba a New Crop for Arid Lands. New Raw Material for Industry. National Academy Press, Washington, DC, pp. 6’13.
5. Cokelaere M.M., Buyse J., Daenens P., Decuypere E., K”hn E. and Van Boven M. (1993) Influence of jojoba fl our supplementation on growth and organ function in rats. J. Agric. Food Chem. 41:1444-1448.
6. Cokelaere M.M., Dangreau H.D., Daenens P., Bruneel N., Arnouts S., Decuypere E. M.-P. and K”hn E. (1992b) Investigation of possible toxicological influences of simmondsin after subacute administration in the rat. J. Agric. Food Chem. 40:2443-2445
7. Cokelaere, M.M., Dangreau, H.D., Arnauts, S., Kuhn, E.R., Decuypere, E., (1992a). Effect of pure simmondsin on food intake in rats. J. Agric. Food Chem. 40, 1839’1842.
8. Corina costescu, d. p”rvu, a. rivi”. (2006) . The Determination Of Some Physical-Chemical Characteristics For Orange, Grapefruit And Tomato Juices. journal of agroalimentary processes and technologies, volume xii, no. 2
9. Dash, M.; Kumar, P.J.; Panda, P. (2008). Phytochemical and antimicrobial screening of extracts of Aquilaria agallocha Roxb. Afr. J. Biotechnol.
10. Ellinger, C.A., Waiss, A.C., Lundin, R.E., (1973). Simmondsin, an unusual 2-cyanomethylenecyclohexyl glucoside from Simmondsia californica. J. Chem. Soc. Perkin Trans. 1, 2209’2212.
11. Feten Abbassi, Besma Ayari, Baya Mhamdi and Lamjed Toumi, (2014). Phenolic contents and antimicrobial activity of squirting cucumber – (Ecballium elaterium) extracts against food-borne pathogens. Pak. J. Pharm. Sci., Vol.27, pp.475-479.
12. Habashy, R.R., Abdelnaim, A.B., Khalifa, A.E., Al-Azizi, M.M., (2005). Anti-inflammatory effects of jojoba liquid wax in experimentalmodels. Pharmacol. Res. 51, 95’105.
13. Hultin Eskil (1966), Alkaloid-screening of Plants from Boyce Thompson Southwestern Arboretum, Acta Chemica Scandinavica, Vol. 19, pp. 1297- 1300.
14. Kamal, A.; Arif, J.M.; Ahmad, I.Z. (2010), Potential of Nigella sativa L. seed during different phases of germination on inhibition of bacterial growth. J. Biotechnol. Pharm. Res. 1, 009-013.
15. Larmond, E. (1977). Laboratory Methods for Sensory Evaluation of Food. Canada Deptt Agri Pub. 1637.
16. Litchfield. J.T. and F. Wilcoxon, (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther., 96: 99-133.
17. Luis A. Medina And Augusto Trejo-Gonzalez. (1990). Detoxified and Debittered Jojoba Meal: Biological Evaluation and Physical-Chemical Characterization. Cereal Chem. 67(5):476-479.
18. Maier, M.S. (2008), Biological activities of sulfated glycosides from echinoderms. Stud. Nat. Prod. Chem. 35, 311-354.
19. Makkar, H.P.S. (2003) Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds, Small Rumin. Res., 49: 241’256.
20. Makkar, H.P.S., Bl”mmel, M. and Becker, K. (1995) In vitro effects of and interactions between tannins and saponins and fate of tannins in the rumen, J. Sci. Food Agric., 69: 481.
21. Makkar, H.P.S., Bl”mmel, M., Borowy, N.K. and Becker, K. (1993) Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods, J. Sci. Food Agric., 61: 161’165.
22. Moustafa A. Abbassy , Samir A.M. Abdelgaleil , Abdel-Salam H. Belal, Mona A.A. Abdel Rasoul., (2007). Insecticidal, antifeedant and antifungal activities of two glucosides isolated from the seeds of Simmondsia chinensis . Industrial Crops and Products 26, 345’350.
23. Moyad Jamal Shahwan. (2014). antihyperlipidimic effect of simmondsia chinensis seeds extracts in rabbits. IJABPT V 5, Issue 1, 1-3.
24. Ozawa, T., Lilley, T. H., And Haslam, E. (1987). Polyphenol Interactions: Astringency and the Loss of Astringency in Ripening Fruits. Phytochemistry 26:2937.
25. Saeed Akhtar, Muhammad Riaz, Anwaar Ahmad And Atif Nisar. (2010). Physico-Chemical, Microbiological And Sensory Stability Of Chemically Preserved Mango Pulp. Pak. J. Bot., 42(2): 853-862, 2010.
26. Sharp P.B. (1974), an anthocyanin in Simmondsia chimensis: genetic and taxonomic implications, Master's Theses, University of Arizona,
27. Shrestha, M.K., Peri, I., Smirnoff, P., Birk, Y., Goldhirsh, A.G., (2002). Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities. J. Agric. Food Chem. 50, 5670’5675.
28. Steel, R., J. Torrie and D. Dickey. (1997). Principles and Procedures of Statistics. A Biometrical Approach, 3rd Ed. McGraw Hill Book Co. New York, USA.
29. Tucson, USA. Orwa C, Mutua A, Kindt R, Jamnadass R and Simons A. (2009), Simmondsia chinensis, Agroforestry Database: a tree reference and selection guide version 4.0, [Online], Available at: http://www.worldagroforestry.org/af/treedb/, Accessed on 15-11-2012.
30. Van Boven, M., Holser, R., Cokelaere, M., Flo, G., Decuypere, E., (2000). Gas chromatographic analysis of simmondsin and simmondsin ferulates in jojoba meal. J. Agric. Food Chem. 48, 4083’4086.
31. Verbiscar A.J., Banigan T.F., Weber C.W., Reid B.L., Trei J.E., Nelson E.A., Raffauf R.F., Kosersky D. (1980) Detoxification of jojoba flour. J. Agric. Food Chem. 28:571-578.
32. Williams R.R. (1980) The toxicity of simmondsin, a glucoside found in jojoba (Simmondsia chinensis). M. Sci. Thesis, University of Arizona.
33. Wiseman M. O. and Price R. L. (1987 a), Characterization of protein concentrates of jojoba meal, Cereal Chemistry, Vol. 64, pp. 91-93.
34. Wiseman M. O. and Price R. L. (1987 b), Functional properties of protein concentrates from pressed jojoba meal, Cereal Chemistry, Vol. 64, pp. 94- 97.
35. You X (2006). Food safety and food additive of antiseptic. Food. Sci. Technol., 1: 1-4.