Abstract
The effect of temperature (25–80°C), water to seed ratio (25:1–85:1) and pH (3–9) on the yield, apparent viscosity and emulsion stability index of wild sage seed hydrocolloid was investigated. The generated quadratic model showed that the optimum conditions for maximizing the responses were when temperature was 25°C, water to seed ratio was 51:1 and pH was 5.5. All hydrocolloid solutions (1% w/v) showed shear thinning behavior in different extraction conditions, consistency coefficient and flow behavior index varied from 4.455 to 9.435 (Pa.sn), and 0.317 to 0.374, respectively. Besides, the chemical compositions of the seed and extracted gum were determined at optimal conditions.
INTRODUCTION
The food industry has seen a large increase in use of hydrocolloids in recent years. According to the safety, availability and low process costs, plant seeds have a good potential as new sources of hydrocolloids. Most seeds contain starches as the principal reserve food stored for use by the embryonic plant, but many seeds contain other polysaccharide polymers with gum-like functional properties which have served as a useful source of commercial hydrocolloids.[Citation1]
The genus Salvia (Labiatae) contains more than 700 species, which about 200 out of them exist in Iran and is probably found in neighboring countries. Plants belonging to this genus are pharmacologically active and have been used in folk medicine all around the world. Wild sage seed (Salvia macrosiphon) is a small, rounded seed, which readily swells in water to give mucilage,[Citation2] but very few formal studies have looked at this little seed, only the composition of essential oil of this species has been reported by Matloubi-Moghaddam et al.[Citation3] and recently computer image analysis and physic-mechanical properties of the seed investigated by Razavi et al.[Citation4]
Food hydrocolloids are used for thickening, gelling, film forming and stabilizing purposes. Many food products such as sauces, syrups, ice cream, instant foods, beverages and confectionaries, marshmallows, and candies contain hydrocolloids in their formulations. The common property of hydrocolloids is that they impart viscosity or thickening to the aqueous solutions. The rheological behavior of hydrocolloids governs the quality of the end product, as well as the design and evaluation of process equipments.[Citation5] Previous studies showed that the degree of thickening can be influenced by the extraction conditions.[Citation6–11] Hydrocolloids are also added to control the stability of different emulsions such as salad dressings where the effect of extraction conditions on emulsion stability is important to be considered.[Citation12]
Preliminary tests showed that extraction temperature, pH and water to seed ratio have significant influence on the yield, apparent viscosity and emulsion stability index of wild sage seed hydrocolloids. Thus, it is essential to optimize the extraction process in order to obtain the highest yield and quality of this hydrocolloid. The general practice of determining these optima is by varying one parameter while keeping the other at an unspecified constant level. The major disadvantage of this single variable optimization is that it does not include interactive effects among the variables; thus, it does not depict the net effects of various parameters on the reaction rate. In order to overcome this problem, when many factors and interactions affect desired variables, response surface methodology (RSM) is an effective tool for optimizing the process.[Citation7] RSM is an effective statistical method that uses a minimum of resources and quantitative data from an appropriate experimental design to determine and simultaneously solve a multivariate equation.[Citation13] Response surface experiments attempt to identify the response that can be thought of as a surface over the explanatory variables_ experimental space. It usually uses an experimental design such as central-composite experimental design (CCED) to fit an empirical, full second-order polynomial model. A central-composite experimental design, coupled with a full polynomial model, is a very powerful combination that usually provides an adequate representation of most continuous response surfaces over a relatively broad factor domain.[Citation14]
Many researchers have used RSM to optimize hydrocolloid extraction.[Citation6–11] Negligible information is available so far on the extraction and functional properties of wild sage seed hydrocolloid and this paper deals with optimum extraction conditions of wild sage seed hydrocolloid as a novel gum source and some chemical and rheological characterization.
MATERIAL AND METHODS
Sample Preparation
The wild sage seeds used in this study were obtained from a local market in Mashhad, Iran. The seeds were cleaned manually to remove all foreign matter such as dust, dirt, stones and chaff. All chemicals used were reagent grade unless otherwise specified.
Gum Extraction
Wild sage seed gum was extracted from whole seeds using distilled water (water to seed ratio of 25:1–85:1) at pH 3–9. The pH was monitored continuously and adjusted by 0.1 mol/L NaOH and HCl, respectively, while the temperature of the aqueous system ranged from 25–80°C and was controlled within ±2.0°C using an adjustable temperature controlled water bath. Water was preheated to a designated temperature before the seeds were added. Extraction was carried out in three stages; in the first stage, the seeds (40 g) were mixed with 1000 ml water (25:1 W:S) at a specific pH and temperature and enough time (20 min) was given that complete water absorption was occurred. A soaking time of 20 min was selected based on the yield of preliminary trials. Separation of the gum from the swelled seeds was done by passing the seeds through a laboratory extractor (Model 412, Pars Khazar Com., Iran). Crude gum was collected and residual seeds immersed in remaining of water in two stages, according to water to seed ratio proposed for each run, and again was passed through the extractor. The collected crude gum from the different stages was mixed, filtered and dried overnight in a forced convection oven (Model 4567, Kimya Pars Com., Iran) at 70°C.[Citation15] The dried gum was then grounded, filtered and used for analysis.
Experimental Design
The optimization method based on RSM involved three major steps: design of experiment using statistical approach, coefficient estimation based on mathematical model and response prediction and finally model adequacy check. The models were tested with analysis of variance (ANOVA) with 95% degree of confidence. The RSM outputs such as contour and 3D graphic surface plots provide the optimum and most influential variables for hydrocolloid extraction. In this paper, a central-composite experimental design, with three variables, was used to study the response pattern and to determine the optimum combination of variables. The effect of the independent variables x1 (temperature, T), x2 (water to seed ratio, W:S) and x3 (pH), at five variation levels on the responses is shown in . Six replicates at the centre of the design were used to estimate a pure error sum of squares. The responses functions (y) measured were yield, apparent viscosity and emulsion stability index (ESI). Different models were fitted to the responses and their adequacy checked, finally the best model was selected and related coefficients were reported. These values were related to the coded variables (xi, i = 1, 2, and 3) by a second degree polynomial using the equation below:
Table 1 The central composite experimental design and results for yield, apparent viscosity and ESI of crude hydrocolloid extract of wild sage seed
The coefficients of the polynomial were represented by β0 (constant term), β1, β2 and β3 (linear effects), β11, β22 and β33 (quadratic effects), and β12, β13 and β23 (interaction effects). The analysis of variance (ANOVA) tables were generated and the effect and regression coefficients of individual linear, quadratic and interaction terms were determined. The significances of all terms in the polynomial were judged statistically by computing the F-value at a probability (p) level of 0.05. The regression coefficients were then used to make statistical calculation to generate contour maps from the regression models. The data were analyzed using the Design-Expert Software (Version 6.0.2®, 2000, Stat-Ease, Inc., UK).
Flow Behavior
The flow behavior of crude hydrocolloid extract of sage seed was determined using a rotational viscometer (Bohlin Model Visco 88, Bohlin Instruments, UK) equipped with a heating circulator (Julabo, Model F12-MC, Julabo Labortechnik, Germany). Bob and cup measuring spindle (C30) was used during measurements according to the viscosity of dispersion. Prepared samples (1% w/v) were loaded into the cup and allowed to equilibrate for 10 min at desired temperature (25°C) and were then subjected to a programmed logarithmic shear rate ramp increasing from 14 to 300 s−1 during 3 min. The shear stress–shear rate data of wild sage seed gums was tested for power law model as follow (16):
where, τ is the shear stress (Pa); is the shear rate (s−1); k is the consistency coefficient (Pa.sn); and n is the flow behavior index (dimensionless).
Evaluation of Extracted Gum
Extraction yield
The yield of the extracted gum for various extraction conditions was determined by weighting the dried extracted gums and calculating the percentage based on the weight of the seeds.[Citation17]
Apparent viscosity
For Non-Newtonian fluids, viscosity depends on the shear rate, therefore the apparent viscosity (ηa) of crude hydrocolloid extract was calculated by viscometer software at a given shear rate (122s-1) and 25°C for 1% (w/v) solutions.
Emulsion stability index (ESI)
Oil in water emulsions were prepared by mixing 20% canola oil with an aqueous phase containing 0.5% wild sage seed hydrocolloid. The hydrocolloid powder was dissolved in distilled water by stirring at room temperature and kept in a refrigerator overnight to ensure complete hydration. Emulsion was prepared by homogenizing oil and aqueous phase using a high speed blender (Sanyo, Japan). The ESI for the emulsions were determined by the turbidimetric methods.[Citation18] Freshly prepared emulsions (1 ml) were pipetted out at 0 and 10 min after homogenization and diluted with 99 ml SDS (1 g.kg−1). Absorbance of the final dispersion was measured at 500 nm (Spectrophotometer, UV-160A, Shimadzu, Japan). The ESI (min) was determined as follows:
where A0 is the absorbance of the diluted emulsion immediately after homogenization; ΔA is the change in absorbance between 0 and 10 min (A0_A10); and t is the time interval, 10 min in this case.
Analytical Methods
To analyze the chemical composition of the wild sage seeds and that of the extracted gum at optimum condition, moisture content of the seed and the extracted gum at optimum condition was determined by the vacuum oven method (temperature 70°C and pressure 250 mbar) until a constant mass was obtained,[Citation17] total nitrogen and ash contents were determined in duplicate according to AOAC methods (19): 2.061–2.062 (modified Kjeldahl method) and 923.03 (direct method of ash determination in flours), respectively. The Kjeldahl factor used was 6.25. Fat was extracted by a semi-continuous procedure using a Soxhlet device, and diethyl ether and hexane were used as the extraction solvent. Crude fiber (i.e., cellulose, lignin, and part of the total hemi-cellulose) was also determined according to AOAC method No. 920.86. Total carbohydrates were determined as the difference between 100 and the sum of the other components. All measurements were performed at least in triplicate and results expressed as mean ± SD.
RESULTS
Flow Behavior
The dispersions of the hydrocolloid from wild sage seeds showed non-Newtonian pseudoplastic behavior over the entire extraction conditions. Based on the values obtained for R2 , the power law model was well fitted to experimental flow curves (). Both parameters of the power law model, k and n are shown in along with the apparent viscosity values at 46.16 s−1. Values of the flow behavior index (n) were below unity confirming a shear-thinning behavior of the wild sage seed extracts at all extraction conditions tested. The flow behavior ranged from 0.317 to 0.374 with the mean value as 0.347, which showed the high shear thinning tendency of the hydrocolloid solutions. The extraction conditions had no significant effect on the flow behavior index. The values of consistency index, k, ranged from 4.455 to 9.435 Pa sn, while mean value of k was 6.355 Pa sn. The extraction conditions significantly affected the consistency index, increasing pH and descreasing temperature and water to seed ratio had a negative impact on parameter k. As the polysaccharide polymers enhance the thickening properties of the solution, probably this is due to impurities (non polysaccharide compounds) which were probably extracted at higher temperatures and water to seed ratios. Marcotte et al. (16) also reported that an increase in temperature will decrease the consistency coefficient of the hydrocolloids.
Table 2 Rheological data for wild sage seed hydrocolloid solution extracted on different conditions
The apparent viscosity values of sage seed hydrocolloid ranged from 373 to 694 mPa.s at different extraction conditions, while Cui et al. (10) reported the range of 16.74 to 148.50 for flaxseed gum at the same concentration (1% w/w) and shear rate (46.16 s−1).
Statistical Analysis and the Model Fitting
The experimental data for yield, apparent viscosity and emulsion stability index of the extracted hydrocolloid under different treatment conditions are presented in . The analysis of variance of different models showed that adding terms up to quadratic will significantly improved the model (), therefore a quadratic model is the most appropriate model for the three responses.
Table 3 Analysis of variance for different models evaluated for prediction of responses in sage seed hydrocolloid extraction
Model validating parameters and the coefficients of equation terms, which is an empirical relationship between response and the test variables in the regression models after implementing the stepwise ANOVA for response variables, are presented in . The statistical analysis indicated that the proposed regression model for yield, apparent viscosity and ESI was adequate, possessing no significant lack of fit and with satisfactory values of the R2 (coefficient of determination) for all the responses. The R2 values were 0.982, 0.989 and 0.892 for yield, apparent viscosity and ESI, respectively (). The closer the value of R2 to the unity, the better the empirical model fits the actual data.[Citation6] Furthermore, the predicted-R2 is in reasonable agreement with adjusted-R2 for all three responses.
Table 4 ANOVA results showing the variables as a linear, quadratic and interaction terms on each response variables and coefficients for the prediction models
Influence of Variables on Yield
The effect of different extraction conditions on hydrocolloid yield has been reported by the coefficient of the second order polynomials () and to aid visualization, the response surfaces for yield are shown in . As shown in , all variables except linear and quadratic terms of pH and interaction between temperature and pH had significant effect on yield (P < 0.05). The variables with the largest effect on yield were the linear terms of water: seed ratio and temperature, respectively. Higher temperature and water to seed ratio resulted in higher yield due to enhanced mass transfer rate ( and ). Similar results were obtained by Wu et al.,[Citation6] Koocheki et al.,[Citation8] Razavi et al.,[Citation9] Sepulveda et al.,[Citation15] Cui et al.,[Citation10] and Singthong et al.[Citation11] Li et al.[Citation20] reported that the effect of extraction temperature was substantial, but the water to solid ratio had a slight effect on extraction yield. Also, the effect of pH on yield was not significant, Koocheki et al.[Citation8] and Cui et al.[Citation10] also reported the minor effect of pH on extraction yield, but Razavi et al.,[Citation9] Furuta et al.,[Citation21] and Somboonpanyakul et al.[Citation22] reported that pH has a significant effect on the yield. In constant pH (pH = 6.0), increasing water to seed ratio increased the yield. This effect was more pronounced at higher temperatures (). Increased in water to seed ratio in all pH increased the yield except the alkaline pH (). shows the changes of hydrocolloid yield with temperature and pH at a constant water to seed ratio (W:S = 51:1). It was clear that increasing temperature increased the hydrocolloid yield, but pH had no significant effect on yield, however, at high W:S, increasing the pH decreased the yield (). In this study, the lowest yield of gum (7.04%) was obtained at lowest water to seed ratio (24.93:1) at temperature of 52.5°C and pH of 6.00, while the highest value (12.2%) was obtained at the highest temperature (80°C), water to seed ratio of 55:1 and pH of 6.00 ().
Figure 1 Response surface for the effect of temperature, water to seed ratio and pH on yield of wild sage seed hydrocolloid; (a) T and W:S at pH = 6.0; (b) T and pH at W:S = 51:1; (c) W:S and pH at T = 55°C.
Influence of Variables on Apparent Viscosity
The result of analysis of variance and response surface plots showed that the apparent viscosity of hydrocolloid solution was significantly (P < 0.05) affected by the linear, quadratic and the cross terms between all variables (), except the quadratic effect of temperature that was no significant. Based on the sum of squares, water to seed ratio and interaction of water to seed ratio and pH had the largest effect on apparent viscosity (). reveals that at low water to seed ratio, increasing the temperature decreased the apparent viscosity, whereas at higher W:S, temperature had greater effect. At low pH (3–7), increasing temperature decreased the apparent viscosity afterwards the apparent viscosity became constant (). Increasing water to seed ratio at acidic conditions increased the apparent viscosity, while at alkaline conditions it caused reduction in apparent viscosity (). Koocheki et al. (8), Razavi et al. (9) and Ibanez and Ferrero (23) have reported increasing of pH led to decrease in apparent viscosity.
Figure 2 Response surface for the effect of temperature, water to seed ratio and pH on apparent viscosity (at 25°C, 1% (w/v) solution and shear rate 122 s−1) of wild sage seed hydrocolloid. (a) T and W:S at pH = 6.0; (b) T and pH at W:S = 51:1; and (c) W:S and pH at T = 55°C.
Apparent viscosity varied between 198 to 355 mPa.s in different extraction conditions, the lowest apparent viscosity observed when W:S, T and pH were 72.8, 36 and 7.78, respectively, and the highest obtained when W:S, T and pH were 37.1, 36 and 7.78, respectively (). It can be concluded that water to seed ratio substantially affected the apparent viscosity.
Influence of Variables on Emulsion Stability Index
The results showed that only temperature had significant linear effect on ESI and linear effect of water to seed ratio and pH were not significant (). When the temperature increased (), the ESI decreased maybe it was due to decreasing in hydrocolloid solution viscosity as temperature increased. In quadratic terms, only pH and W:S had significant effect and in cross action interaction between pH and temperature, and pH and water to seed ratio had significant effect on ESI (p < 0.05). Increasing temperature at lower pH (3–7) decreased ESI, while at higher pH, it had no significant effect (). Elliptical contour observed in , demonstrated the perfect interaction between pH and water to seed ratio. The maximum predicted value indicated by the surface was confined in the smallest ellipse in the contour diagram (6). In this study, ESI varied between 172 and 450 min under different extraction conditions. The lowest ESI observed when T, W:S and pH were 69°C, 72.8:1 and 4.22, respectively, while the highest ESI obtained when W:S and pH were 54.95 and 6.00 at the lowest T (24.75°C).
Figure 3 Response surface for the effect of temperature, water to seed ratio and pH on ESI (20% O/W emulsion) of wild sage seed hydrocolloid. (a) T and W:S at pH = 6.0; (b) T and pH at W:S = 51:1; and (c) W:S and pH at T = 55°C
Optimization and Verification
Optimization of the extraction procedure was based upon the following: higher extraction yield, apparent viscosity and ESI. The suitability of the models for predicting optimum response values was tested under the conditions: extraction temperature 25°C, water to seed ratio 50.95:1 and pH 5.53. This set of conditions was determined to be optimum by the RSM optimization approach and was also used to validate experimentally and predict the values of the responses using the models (). The experimental and predicted values were found to be not statistically different at 5% level of significance, indicating that the model was adequate for the extraction process.
Table 5 Predicted and experimental values of responses at optimum extraction condition of sage seed hydrocolloid
Most of researchers found higher temperature for optimum extraction,[Citation9,Citation10,Citation15] whereas our study showed that the crude hydrocolloid of sage seed could be extracted in ambient temperature. The yield of crude hydrocolloid of wild sage seed was 9.97%, which was more than values reported at optimum condition for Flaxseed gum[Citation10] and less than Qodumeh seed[Citation8] and Basil seed.[Citation9] The crude hydrocolloid extracted under the optimum conditions was further analyzed for chemical compositions as shown in . In this research, the crude hydrocolloid powder of sage seed contained 79.75% carbohydrates, 2.84% proteins, 0.85% lipid, 6.72% moisture, 1.67% crude fiber, and 8.172% ash ().
Table 6 Chemical compositions of the seeds of wild sage and the hydrocolloid powder
CONCLUSION
The extraction conditions had significant effects on the yield, apparent viscosity and ESI of wild sage seed crude hydrocolloid. Increasing the temperature and water to seed ratio increased the yield of extracted hydrocolloid, while increasing the temperature decreased the ESI and also at high water to seed ratio and alkaline conditions increased the apparent viscosity. The influence of pH on yield and ESI was significant (P < 0.05). Increasing pH at low water to seed ratio increased the apparent viscosity, whereas it decreased at high water to seed ratio. In addition, increasing pH at low temperature decreased the apparent viscosity, while it increased at high temperature. Optimum conditions (temperature 25°C, water to seed ratio at 51:1 and pH at 5.5) for the extraction procedure of crude hydrocolloid from wild seeds were identified. All hydrocolloid solutions (1% w/v) showed shear thinning behavior under different extraction conditions. The hydrocolloid dried powder contain 6.72% moisture, 0.85% lipid, 8.172% ash, 2.84% protein, 1.67% crude fiber, and 79.75% carbohydrate.
Related Research Data
REFERENCES
- Glicksman , M. 1982 . Food Hydrocolloid , Vol. III , 157 – 184 . Boca Raton, FL : CRC Press Inc .
- Razavi , S.M.A. , Farhoosh , R. and Bostan , A. 2007 . “ Functional properties of hydrocolloid extract of some Iranian seeds ” . In Research project No.1475 , Iran : Unpublished report. Ferdowsi University of Mashhad .
- Matloubi-Moghaddam , F. , Amin , Gh. and Safavipoorsohi , E. 2000 . Composition of stembark essential oil from Salvia macrosiphon Boiss . Iranian Journal of Daru , 8 : 28 – 29 .
- Razavi , S.M.A. , Bostan , A. and Rahbari , R. 2010 . Computer image analysis and physic-mechanical properties of wild sage seed (Salvia macrosiphon) . International Journal of Food Properties , 13 : 308 – 316 .
- Ahmed , J. , Ramaswamy , H.S. and Ngadi , M.O. 2005 . Rheological characteristics of arabic gum in combination with guar and xanthan gum using response surface methodology: effect of temperature and concentration . International Journal of Food Properties , 8 : 179 – 192 .
- Wu , Y. , Cui , S.W. , Tang , J. and Gu , X. 2007 . Optimization of extraction process of crude polysaccharides from boat-fruited sterculia seeds by response surface methodology . Food Chemistry , 105 : 1599 – 1605 .
- Triveni , R. , Shamala , T.R. and Rastogi , N.K. 2001 . Optimised production and utilization of exopolysaccharide from Agrobacterium radiobacter . Process Biochemistry , 36 : 787 – 795 .
- Koocheki , A. , Mortazavi , S.A. , Shahidi , F. , Razavi , S.M.A. , Kadkhodaee , R. and Milani , J. 2010 . Optimization of mucilage extraction from Qodume Shirazi seed (Alyssum homolocarpum) using response surface methodology . Journal of Food Process Engineering , In Press. DOI:10.1111/3.1745–4530.2008.00312.X
- Razavi , S.M.A. , Mortazavi , S.A. , Matia-Merino , L. , Hosseini-Parvar , S.H. , Motamedzadegan , A. and Khanipour , E. 2009 . Optimization study of gum extraction from Basil seeds (Ocimum basilicum L.) using response surface methodology . International Journal of Food Science and Technology , 44 : 1755 – 1762 .
- Cui , W. , Mazza , G. , Oomah , B. D. and Biliaderis , C.G. 1994 . Optimization of an aqueous extraction process for flaxseed gum by response surface methodology . LWT , 27 : 363 – 369 .
- Singthong , J. , Ningsanond , S. , Cui , S.W. and Goff , H.D. 2005 . Extraction and physicochemical characterization of Krueo Ma Noy pectin . Food Hydrocolloids , 19 : 793 – 801 .
- Dickinson , E. 2003 . Hydrocolloids at interfaces and the influence on the properties of dispersed system . Food Hydrocolloids , 17 : 25 – 39 .
- Kalaimahan , T. and Tapobrata , P. 1995 . Application of response surface methodology to evaluate the influence of temperature and initial pH on the production of β-1,3-glucanase and carboxymethylcellulase from Trichoderma harzianum . Enzyme and Microbial Technology , 17 ( 12 ) : 1043 – 1049 .
- Deming , S. N. 1990 . Quality by design-part 5 . Chemtech , 20 : 118 – 120 .
- Sepulveda , E. , Saenz , C. , Aliaga , R. and Aceituno , C. 2007 . Extraction and characterization of mucilage in Opuntia spp . Journal of Arid Environment , 68 : 534 – 545.13 .
- Marcotte , M. , Taherian hoshahili , A.R. and Ramaswamy , H.S. 2001 . Rheological properties of selected hydrocolloids as a function of concentration and temperature . Food Research International , 34 : 695 – 703 .
- Singh , K. K. and Goswami , T. K. 1996 . Physical properties of cumin seed . Journal of Agricultural Engineering Research , 64 : 93 – 98 .
- Pearce , K. and Kinsella , J. 1978 . Emulsifying Properties of Proteins: Evaluation of a Turbidimetric Technique . Journal of Agriculture and Food Chemistry , 26 ( 3 ) : 716 – 723 .
- Association of Official Analytical Chemists . 2005 . Official methods of analysis , 2nd , 167 – 179 . Washington, DC : AOAC .
- Li , J. , Ding , S. and Ding , X. 2007 . Optimization of the ultrasonically assisted extraction of polysaccharides from Zizyphus jujuba cv. Jinsixiaozao . Journal of Food Engineering , 80 : 176 – 183 .
- Furuta , H. , Takahashi , T. , Tobe , J. , Kiwata , R. and Maeda , H. 1998 . Extraction of water soluble polysaccharides under acidic conditions . Bioscience Biotechnology Biochemistry , 62 : 2300 – 2305 .
- Somboonpanyakul , P. , Wang , Q. , Cui , W. , Barbut , S. and Jantawat , P. 2006 . Malva nut gum. (Part I): Extraction and physicochemical characterization . Carbohydrate Polymers , 64 : 247 – 253 .
- Ibanez , M.C. and Ferrero , C. 2003 . Extraction and characterization of the hydrocolloid from Prosopis flexuosa DC seeds . Food Research International , 36 : 455 – 460 .