Mona I. Massoud (2001).Chemical and Technological Studies on Natural Sweeteners from Stevia
Title of Thesis
Arabic Title :
دراسات كيماوية وتكنولوجية علي المحليات الطبيعية من نبات الاستيفيا" "
Mona I. Massoud (2001).Chemical and Technological Studies on Natural sweeteners from Stevia Plant. Ph.D. Thesis in Food Technology, Food Science and Technology Department, Faculty of Agriculture, AlexandriaUniversity.
***(The Unilever Mashreq Prize provided by the Scientific Society for Food Industries, for the bestPh.D. thesis in food technology for the year 2002).
Summery
Nonnutritive sweeteners, are compounds that provide sweet taste for food and beverages avoiding the calories. Due to ban of some artificial sweeteners on the basis that it is a human carcinogen. It has been essentially to the toxicological safeness and excessive sugar consumption, natural sweeteners have met with growing interest. In Egypt, there is a great interest on the strategical plan for the cultivation of a new crop used as source of natural sweeteners. Some trials for stevia plant cultivation just has been started in Egypt. Therefore, this investigation aimed to evaluate this new sweetener crop through characterization of its chemical composition of this plant during it’s growth period, extraction the sweeteners compound, identification, chemical structure study, physical properties, evaluation the sweetness intensity of these compounds and it’s uses in some foods and beverages. The results obtained from this study can be summarized in the following points:
(1) / The data of chemical composition of the stevia plant during growth stage showed that there is changes in proximate composition especially for carbohydrate content which increased to reach the highest level (58.17%) of dry leaves at 4 months of growth period then decreased again to (53.67 and 50.36%) at 5 and 6 months of growth period. It can be concluded that the most suitable time for harvesting at 4 months as plant began blooming.(2) / Highest amount of carbohydrate (57.91%) in stevia dry leaves were obtained by drying the plant at sunshine time (29-31°C) followed by drying in electric oven at 50°C (57.42%) while the lowest amount of carbohydrate (54.40%) obtained at sun (19-31°C) during all the day at open air.
(3) / Proximate composition of dried stevia leaves from different sources was carried out and the highest carbohydrate content (62.34%) was obtained in imported leaves followed by dry leaves obtained from the plants grown in green house. The variation in carbohydrate and markedly differences in ash content between green house plants and imported plants could be attributed to varieties and agricultural conditions.
(4) / The technique of Infrared Spectrometry was used for the identification and determination of sweeteners in stevia leaves. The data showed that the highest concentration of stevia sweeteners appeared in leaves obtained from the plant of 4 months growth age.
(5) / Dry leaves of stevia were extracted with three different solvents namely MeoH, MeoH : H2O and H2O. Stevioside and rebaudioside A (R.A) as the major compounds of stevia sweeteners were determined by HPLC in each extract obtained. The results indicated that the highest amount of stevioside and R.A (22 and 12.2% respectively) were obtained in MeoH extract followed by MeoH : H2O 17.99 and 9.83%, respectively and H2O extract which gave the lowest amount of stevioside and R.A (16.98 and 8.40, respectively).
(6) / The results obtained by HPLC analysis for each stevia sweeteners extracted from dry leaves at 2, 3, 4, 5, 6 and 7th months of growth with the three different solvent used revealed that the highest amount of stevia sweeteners was obtained always at 4 months of growth period for the plant cultivated in the green house.
(7) / Stevioside and R.A as determined in stevia leaves of different sources revealed that highest amount of stevioside and R.A content were obtained in stevia leaves of green house, while the lowest amount were obtained in leaves from open field. Also, stevia sweeteners content in leaves was more than five folds of stevia sweeteners content found in the stems of the plant.
(8) / The data obtained for stevia sweeteners (%) during growth period showed that after completely blooming (5 months) of growth, the stevia sweeteners content dropped to 25.12% which represented a decreasing percent of 34.92% of sweetener percent (38.96%) obtained at 4 months of growth as extracted by MeoH.
(9) / Sweeteners compounds namely ducloside A, stevioside, R.A., R.C. and R.E. were 0.06, 20.03, 11.78, 6.63 and 0.46%, respectively at 4 months of plant growth period. Which found to be the highest amount during the growth period of the plant.
(10) / The purification methods of stevia sweetener extract used revealed that alumina oxide or active carbon were the best method for the elimination of pigments or any colour polar constituents from the stevia extract to obtain the pure stevia sweeteners.
(11) / Stevia sweeteners were identified by TLC. TLC illustrated that the best separation of sweeteners compounds were obtained by solvent system EtoAC, HoAC : H2O (8 : 3 : 2) and the Rf values for stevioside, R.A, R.C, R.E and steviolbioside were 0.42, 0.30, 0.10, 0.16 and 0.50, respectively.
(12) / Identification and chemical structure study for purified stevia sweeteners were carried out by different techniques namely Infrared, Mass and H-NMR spectrometry. The mass spectrum of stevia sweeteners did not show the expected molecular ion (calculated [M]+ = 804.89). But some fragments were observed. The relative position of the oxygen functions was further supported by the mass spectrum which showed mass spectral ion peaks at m/z 318 (C20H30O3) and 342 (C12H22O11) and 180 (C6 H12O6). Also, 1H-NMR spectra were recorded for stevia sweeteners and the chemical shift values were given. H-NMR spectrum showed a singlet at 0.68 mg/L due to CH3 groups at carbon 4 and 10. A multiplet is observed at 0.9 mg/L due to one CH2 group and a singlet at 1.1 mg/L due to one CH2 group three multiplets are observed in the range from 1.15-2.05 mg/L due to 7 CH2 groups and 24 groups at carbon 5 and 9.
(13) / The crystal form and structure of the crystallize stevia sweeteners was examined and visualized by polarized light and electron scanning microscopy. The photomicrographs illustrated the difference in appearance of sweeteners crystal in shape, size and aggregation. Photomicrographs of polarized light also illustrated that stevia sweeteners are occurred in different forms, needle, clusters, bundles and polygonal crystals and that could be attributed to the different chemical composition of the different sweetener compound of stevia sweeteners.
(14) / The melting point of stevia sweeteners crystals was determined to be 197°C which was close to the melting point of stevioside (198°C) Specific optical rotation was –38.5°C (5.7% of aqueous solution) and stevia sweeteners was found to be highly soluble in MeoH, EtoH and soluble in water (about one gram in 10.2 ml water).
(15) / Stability of pure stevia sweeteners were obtained in aqueous solution by heating at different pH values. The results indicated that pure stevia sweeteners were stable in neutral or acidic aqueous solutions at ambient or 60°C and also it was observed that stevia sweeteners in neutral aqueous solution were more stable than in acidic aqueous solution.
(16) / The results of sensory evaluation revealed that the sweetness equivalency of pure stevia sweeteners extracted from dry leaves was about 200 times more sweet than sucrose of 0.4% solution. Also, the data showed that with increasing the stevia sweeteners concentration the sweetness increased up to 100 mg/100 ml.
(17) / The data of sensory evaluation showed that there is no significant difference in the mean of sensory score for taste, flavour and acceptability of tea, anise and peppermint (150 ml) sweetened with sucrose (5g/150 ml), or with three to five dry leaves of stevia about (0.31-0.40 g of dry leaves).
(18) / Tea, peppermint, anise, Lemonade, Nescafe and Cappuccino were sweetened with (100 mg/150 ml) stevia sweeteners or with (5 g/150 ml) sucrose. Taste (sweetness), flavour and acceptability were evaluated by the panelists. The results showed that there is no significant difference for taste (sweetness), flavour and acceptability among the beverages sweetened with stevia sweeteners or with sucrose.
(19) / The biscuits were prepared with the same recipe except sugar, one made with sugar (sucrose) as the control samples and for other sugar was replaced with stevia sweeteners with different ratio of replacement 50, 75 and 100%. The results of objective and subjective tests of biscuits revealed that sensory scores for biscuits made from 50% stevia sweeteners as a replacement of sugar were the same like biscuits made of sugar but there was differences occurred for the sensorial properties of biscuits replaced with 75 and 100% stevia sweeteners.
(20) / Cake also was made and the sugar was replaced with 50, 75 and 100% stevia sweeteners. Non significant changes in surface appearance (shape and colour, eating properties were found among cake with 50% stevia sweeteners and cake with 100% sucrose but there was a significant effect in interior appearance was observed.
(21) / The apple and carrot jams which prepared with 50% stevia sweeteners as a sucrose replacement had the same sensorial properties and were accepted as the jams prepared with sucrose.
(22) / Stevia sweeteners was also used as sweetener for Yoghurt (50 mg/100 ml). The results of the sensory evaluation revealed that there is no significant difference for the mean scores of taste, flavour, texture and acceptability for Yoghurt sweetened with stevia sweeteners and Yoghurt sweetened with sucrose.
تم إرسال نسخة من الرسالة إلي :
Prof. Dr. J. BH. Linssen
Agrotechnology and Food Sciences
Lab. of Food Chemistry,
WageningUniversity, P.O. Box 8129
6700 EV wageningen,
The Netherlands.
والذي جاء في تقريره أن الرسالة في مجملها مقبولة بدون أي تعديلات وأعرب عن سعادته بالعمل حيث أن الرسالة تعطي معلومات عن المركبات المحلية من أصل طبيعي وأيضا تطبيقات هذه المحليات في الأغذية ، والرسالة تفي بالمستوى العلمي الكافي للبحث في علوم الأغذية وأنه راضي تماماً عن الرسالة ويعتبرها رسالة جيدة في مجال علوم الأغذية