Ronny Horax, Navam Hettiarachchy, and Pengyin Chen
Abstract: Seeds of ripe bitter melon (Momordica charantia) contain approximately 30% protein. However, this protein, which is less functional than soy protein, may have desirable functionalities as a food ingredient after modification. Bitter melon seed protein isolate (BMSPI) was prepared under optimal extraction conditions (defatted meal to 1.3 M NaCl was 1:10 w/v; pH 9.0) and its functional properties were investigated before and after modification by glycosylation. Glycosylation was conducted at varying relative humidities (50%/65%/80%) and temperatures (40 °C/50 °C/60 °C) using a response surface central composite design. Degree of glycosylation (DG) ranged from 39.3 to 52.5%, 61.7 to 70.9%, and 81.2 to 94.8% at 40 °C, 50 °C, and 60 °C, respectively (P values < 0.0001). Denaturation temperatures of all DGs ranged from 111.6 °C to 114.6 °C, while unmodified/native BMSPI had a value of 113.2 °C. Surface hydrophobicity decreased to approximately 60% when the DG was maximal (94.8%). Solubility decreased almost 90% when the DG was maximal in comparison to the native BMSPI (62.0%). Emulsifying activity increased from 0.35 to 0.80 when the DGs were ࣙ80%, while emulsion stability increased from 63 to 72 min when the DGs were greater than 70%. A similar trend was observed with foaming capacity and foaming stability of the glycosylated proteins. This glycosylated BMSPI with improved emulsifying and foaming properties could be used as an ingredient in food products where such properties are required. Keywords: bitter melon, functionality, glycosylation, protein, seed
Introduction Food proteins have been used in many food products for nutritional purposes as well as functional ingredients to improve food quality. Native plant proteins usually do not have desirable functional properties. Hence proteins have been modified by enzymatic and chemical procedures to enhance functional properties including solubility, emulsion, and foam (Nielsen and Olsen 2002). Chemical modifications that have been researched include glycosylation, acylation, alkylation, esterification, amidation, phosphorylation, deamidation, lipophilization, cross-linking with transglutaminase, and others (Schwenhe 1997; Haard 2001). Glycosylation involves interacting proteins with reducing sugars; glycosylation of the food protein can be implemented without adverse effects and is considered to be safe (Kato 2002; Damodaran 2005). Glycosylation is done to make a protein more hydrophilic (Schwenhe 1997). This chemically modified protein is accomplished by covalent attachment of mono- or oligosaccharides to the protein structure through Maillard reactions (Lee and others 1979; Courthaudon and others 1989; Caer and others 1990). By using aldoses such as glucose glycosylation can be reached faster with the protein due to its higher reducing property (Courthaudon and others 1989). A controlled and limited glycosylation of the protein can be carried out to achieve specific functionalities of a protein (Achouri and others 2005). Controlled and limited MS 20141182 Submitted 7/8/2014, Accepted 9/7/2014. Authors Ronny Horax and Navam Hettiarachchy are with Dept. of Food Science, Uni. of Arkansas, 2650 North Young Avenue, Fayetteville, AR 72704, U.S.A. Pengyin Chen is with Dept. of Crop, Soil, and Environmental Sciences, Univ. of Arkansas, 115 Plant Sciences Building, Fayetteville, AR 72701, U.S.A. Direct inquiries to author Dr. Navam Hettiarachchy (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12680 Further reproduction without permission is prohibited
glycosylation can improve emulsifying and foaming properties, water-holding capacity, thermal stability, and solubility properties of many food proteins (Yeboah and others 1999; 2000; Shepherd and others 2000; Chevalier and others 2001; Achouri and others 2005; Wooster and Augustin 2006; Puangmanee and others 2008; Lillard and others 2009; Liu and Zhong 2012). Glycosylation is considered as one of the most promising chemical modifications to improve protein functionalities for food uses (Kato 2002). Bitter melon seed contains more than 30% (dry basis) of protein “as is” (Horax and others 2010a); this may be a good source of protein that meets the minimum preschool children requirements for almost all the amino acid (Horax and others 2010b, 2011). However, results by Horax and others (2011) showed that the functionalities of this protein were deficient in comparison to others, including soy protein that are used in food products. Native plant proteins in general have poor functional properties. However, the functionality of these proteins, such as pea protein, soy glycinin, legumin, soy whey, and rice endosperm protein, have been demonstrated to have improved functional properties by glycosylation (Caer and others 1990; Baniel and others 1992; Achouri and others 2005; Paraman and others 2007; Matemu and others 2009). The bitter melon seed protein may also have desirable functionalities as a food ingredient after its modification by glycosylation. The objectives of this study were to optimize conditions using response surface methodology (RSM) for a protein modification process to produce glycosylated proteins using glucose at varying temperatures and relative humidities (RH), to determine the degrees of glycosylation, surface hydrophobicities, thermal properties, and solubilities of the glycosylated proteins, and to investigate the emulsifying and foaming properties of these proteins.
Vol. 79, Nr. 11, 2014 r Journal of Food Science C2215
C: Food Chemistry
Characteristics and Functionality Enhancement by Glycosylation of Bitter Melon (Momordica charantia) Seed Protein
Glycosylated bitter melon seed protein . . .
Materials and Methods
C: Food Chemistry
analyzed. Degrees of glycosylation, surface hydrophobicities, thermal properties, solubilities at pH 7.0, and emulsifying and foaming Materials properties of the glycosylated proteins were selected as response Bitter melons var. Sri Lanka (Thinneyville White) planted at variables. the Arkansas Agricultural Experiment Station (Fayetteville, Ark., U.S.A.) were harvested from 3 y of crops (2004, 2005, and 2006) DG determination when pericarp (fleshy portion) turned yellow and aril (seed coat or DG of the glycosylated protein isolates were determined usinner tissue) turned red (at 4 to 5 wk after flowering, ripe stage). ing a fluorescamine-based assay described by Yaylayan and others All chemicals for glycosylation, degree of glycosylation (DG), (1992) with slight modification. The isolates (25 mg, protein basis) characterization, and functional property determination were purwere dispersed into 10 mL of DI water and vortexed. The disperchased from VWR Intl., Inc. (Suwanee, Ga., U.S.A.) and Sigmasions were stirred continuously for 30 min and filtered through a Aldrich, Inc. (St. Louis, Mo., U.S.A.). 0.45-μm syringe filter. The filtrates were diluted 20 times, and to 200 μL of the diluted filtrates were added 4 mL of borate buffer Separation of seeds from ripe bitter melon (0.02 M potassium tetraborate, pH 8.5) and 1 mL of fluorescamine The seeds were manually separated from other tissues and oven reagent (15 mg in 100 mL acetone), which was then vortexed. A dried on a stainless steel tray in a dehydrator (Harvest Saver model blank solution was prepared using the buffer without protein. Flu#R-4; Commercial Dehydrator System, Inc., Eugene, Oreg., orescence intensity was read after 5 min of reaction time using U.S.A.) at 40 °C for 24 h. The dried seeds were ground us- a spectrofluorophotometer (Model RF-1501; Shimadzu, Kyoto, R WERKE model M20; Ika Works, Inc., Japan) at excitation and emission wavelengths of 390 and 475 ing a grinder (IKA Wilmington, N.C., U.S.A.), and passed through a 60-mesh sieve nm, respectively. The DG was calculated as follows: DG (%) = (W.S. Tyler Inc., Mentor, Ohio, U.S.A.) to obtain uniform parti- (Ac − Aa)/Ac × 100; where Ac was the fluorescence of uncle size. This bitter melon seed flour (BMSF) was defatted with modified protein and Aa was the fluorescence of the glycosylated hexane (ratio 1:2, w/v) 3 times on a shaker (Lab Line Inc., Fuller- protein. ton, Calif., U.S.A.) at room temperature for 8 h, vacuum-filtered, dried under a hood at ambient temperature to remove the residual Surface hydrophobicity determination hexane, and placed in a 4 °C storage. SH was determined by a hydrophobic fluorescence method
Preparation of protein isolate from defatted bitter melon seed flour Bitter melon seed protein isolate (BMSPI) was prepared from defatted BMSF based on optimum conditions (pH 9.0 in 1.3 M sodium chloride; Horax and others 2011). The defatted BMSF dispersion (defatted BMSF:1.3 M NaCl solution, 1:10 w/v) was stirred for 2 h and centrifuged at 10000 × g for 20 min to separate supernatant from residue. The supernatant was adjusted to pH 4.0 and the precipitated protein was separated from solution by centrifugation at 10000 × g for 15 min. The protein was reextracted twice from the residue. The combined extracted protein was washed twice using deionized (DI) water (pH 4.0), followed by centrifugation. The protein isolate was resolubilized by adjusting to pH 7.0, freeze-dried, and evaluated for surface hydrophobicity (SH), solubility, and thermal, emulsifying, and foaming properties before and after glycosylation. Three replications were obtained from each of 3 y of crops (2004, 2005, and 2006). Glycosylation of BMSPI The experimental design, consisting of 5 replications at the centre point, was constructed by JMP 7.0 software package (SAS Inst. Inc., Cary, N.C., U.S.A.) using a response surface central composite design. Two factors were selected for optimization: RH (in percentage) and temperature. Independent variables were coded as “a” and “A” to represent the lowest and the highest treatment levels, and “0” to represent the center point (Table 1). BMSPI (10 g) and D-glucose (1 g) were dissolved in water to give a 10% (w/v) protein solution. The solution was adjusted to pH 7.0, stirred for 10 min, and freeze-dried. The freeze-dried protein–glucose products (1 g of each) were placed on an aluminum plate and incubated at varying temperatures (40 °C/50 °C/ 60 °C) and RH values (50%/65%/80%) in a humidifier (Hotpack, Philadelphia, Pa., U.S.A.) for 48 h, and then stored at 4 °C until
C2216 Journal of Food Science r Vol. 79, Nr. 11, 2014
described by Hayakawa and Nakai (1985). Protein solutions ranging from 0.001 to 0.015% w/v in 0.01 M phosphate buffer (pH 7) were made, and 4 mL of each was reacted with 50 μL of 1.6 mM 1-anilino-8-naphthalene sulfonate solution (in 0.01 M phosphate buffer, pH 7.0). Fluorescence intensity of the solutions was read using the spectrofluorophotometer at a wavelength of 390 nm excitation and 470 nm emission. A slope from a linear regression between the fluorescence intensities and protein concentrations was considered as the SH of the proteins.
Thermal property determination Thermal properties of the glycosylated proteins were determined using a differential scanning calorimeter (Model Pyris-1; Perkins-Elmer Corp., Norwalk, Conn., U.S.A.). Fifty μL of the protein slurries in DI water (20%, protein basis) in a hermetically sealed large-volume stainless steel pan were scanned with the temperature increasing from 25 °C to 140 °C at a rate of 10 °C/min. A reference of an empty pan was used as comparison, while the instrument was calibrated using indium and zinc prior to measurement. Onset, end, and peak (designated as the denaturation) temperatures, and enthalpy value (delta H) were generated and calculated by thermal analysis software (Version 4.00; Pyris-1-DSC, Perkin-Elmer Corp.). Solubility determination Solubility of the proteins at pH 7.0 was determined using the modified method of Bera and Mukherjee (1989). The protein dispersion (1%) was made in DI water, adjusted to pH 7.0, and stirred for 30 min at room temperature with pH readjustment every 10 min. The dispersion was then centrifuged at 10000 × g for 30 min. Protein contents in the solution before centrifugation and in supernatants after centrifugation were determined based on a modified Kjeldahl method (AACC 1990) using a Kjeltec 2300
Glycosylated bitter melon seed protein . . . Table 1–Response surface design, and actual and predicted values of degrees of glycosylation (percentage) of glycosylated bitter melon seed protein isolates (BMSPI) at varying relative humidity (RH) percentages and temperatures.
Actual value Pattern
RH (percentage)
Temp (°C)
2004a
2005
2006
Average
Predicted value (R2 = 0.98)b
50 50 50 65 65 65 65 65 65 65 80 80 80
40 50 60 40 50 50 50 50 50 60 40 50 60
39.4 65.2 77.9 54.0 66.4 69.0 66.2 73.3 66.6 94.3 50.5 74.3 93.2
40.0 62.4 82.8 48.5 69.4 68.0 68.2 73.2 71.8 90.8 53.7 69.9 95.4
38.5 57.4 82.7 49.2 70.8 67.9 71.2 72.0 68.2 92.1 53.3 68.4 96.0
39.3 61.7 81.2 50.6 68.8 68.3 68.5 72.9 68.9 92.4 52.5 70.9 94.8
40.3 59.8 82.0 50.0 69.7 69.7 69.7 69.7 69.7 92.0 52.0 71.8 94.3
aa a0 aA 0a 00 00 00 00 00 0A Aa A0 AA a
Glycosylated BMSPI extracted from bitter melon seeds obtained from year 2004 crop. P values for RH and temperature against the degree of glycosylation are both
Abstract: Seeds of ripe bitter melon (Momordica charantia) contain approximately 30% protein. However, this protein, which is less functional than soy protein, may have desirable functionalities as a food ingredient after modification. Bitter melon seed protein isolate (BMSPI) was prepared under optimal extraction conditions (defatted meal to 1.3 M NaCl was 1:10 w/v; pH 9.0) and its functional properties were investigated before and after modification by glycosylation. Glycosylation was conducted at varying relative humidities (50%/65%/80%) and temperatures (40 °C/50 °C/60 °C) using a response surface central composite design. Degree of glycosylation (DG) ranged from 39.3 to 52.5%, 61.7 to 70.9%, and 81.2 to 94.8% at 40 °C, 50 °C, and 60 °C, respectively (P values < 0.0001). Denaturation temperatures of all DGs ranged from 111.6 °C to 114.6 °C, while unmodified/native BMSPI had a value of 113.2 °C. Surface hydrophobicity decreased to approximately 60% when the DG was maximal (94.8%). Solubility decreased almost 90% when the DG was maximal in comparison to the native BMSPI (62.0%). Emulsifying activity increased from 0.35 to 0.80 when the DGs were ࣙ80%, while emulsion stability increased from 63 to 72 min when the DGs were greater than 70%. A similar trend was observed with foaming capacity and foaming stability of the glycosylated proteins. This glycosylated BMSPI with improved emulsifying and foaming properties could be used as an ingredient in food products where such properties are required. Keywords: bitter melon, functionality, glycosylation, protein, seed
Introduction Food proteins have been used in many food products for nutritional purposes as well as functional ingredients to improve food quality. Native plant proteins usually do not have desirable functional properties. Hence proteins have been modified by enzymatic and chemical procedures to enhance functional properties including solubility, emulsion, and foam (Nielsen and Olsen 2002). Chemical modifications that have been researched include glycosylation, acylation, alkylation, esterification, amidation, phosphorylation, deamidation, lipophilization, cross-linking with transglutaminase, and others (Schwenhe 1997; Haard 2001). Glycosylation involves interacting proteins with reducing sugars; glycosylation of the food protein can be implemented without adverse effects and is considered to be safe (Kato 2002; Damodaran 2005). Glycosylation is done to make a protein more hydrophilic (Schwenhe 1997). This chemically modified protein is accomplished by covalent attachment of mono- or oligosaccharides to the protein structure through Maillard reactions (Lee and others 1979; Courthaudon and others 1989; Caer and others 1990). By using aldoses such as glucose glycosylation can be reached faster with the protein due to its higher reducing property (Courthaudon and others 1989). A controlled and limited glycosylation of the protein can be carried out to achieve specific functionalities of a protein (Achouri and others 2005). Controlled and limited MS 20141182 Submitted 7/8/2014, Accepted 9/7/2014. Authors Ronny Horax and Navam Hettiarachchy are with Dept. of Food Science, Uni. of Arkansas, 2650 North Young Avenue, Fayetteville, AR 72704, U.S.A. Pengyin Chen is with Dept. of Crop, Soil, and Environmental Sciences, Univ. of Arkansas, 115 Plant Sciences Building, Fayetteville, AR 72701, U.S.A. Direct inquiries to author Dr. Navam Hettiarachchy (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12680 Further reproduction without permission is prohibited
glycosylation can improve emulsifying and foaming properties, water-holding capacity, thermal stability, and solubility properties of many food proteins (Yeboah and others 1999; 2000; Shepherd and others 2000; Chevalier and others 2001; Achouri and others 2005; Wooster and Augustin 2006; Puangmanee and others 2008; Lillard and others 2009; Liu and Zhong 2012). Glycosylation is considered as one of the most promising chemical modifications to improve protein functionalities for food uses (Kato 2002). Bitter melon seed contains more than 30% (dry basis) of protein “as is” (Horax and others 2010a); this may be a good source of protein that meets the minimum preschool children requirements for almost all the amino acid (Horax and others 2010b, 2011). However, results by Horax and others (2011) showed that the functionalities of this protein were deficient in comparison to others, including soy protein that are used in food products. Native plant proteins in general have poor functional properties. However, the functionality of these proteins, such as pea protein, soy glycinin, legumin, soy whey, and rice endosperm protein, have been demonstrated to have improved functional properties by glycosylation (Caer and others 1990; Baniel and others 1992; Achouri and others 2005; Paraman and others 2007; Matemu and others 2009). The bitter melon seed protein may also have desirable functionalities as a food ingredient after its modification by glycosylation. The objectives of this study were to optimize conditions using response surface methodology (RSM) for a protein modification process to produce glycosylated proteins using glucose at varying temperatures and relative humidities (RH), to determine the degrees of glycosylation, surface hydrophobicities, thermal properties, and solubilities of the glycosylated proteins, and to investigate the emulsifying and foaming properties of these proteins.
Vol. 79, Nr. 11, 2014 r Journal of Food Science C2215
C: Food Chemistry
Characteristics and Functionality Enhancement by Glycosylation of Bitter Melon (Momordica charantia) Seed Protein
Glycosylated bitter melon seed protein . . .
Materials and Methods
C: Food Chemistry
analyzed. Degrees of glycosylation, surface hydrophobicities, thermal properties, solubilities at pH 7.0, and emulsifying and foaming Materials properties of the glycosylated proteins were selected as response Bitter melons var. Sri Lanka (Thinneyville White) planted at variables. the Arkansas Agricultural Experiment Station (Fayetteville, Ark., U.S.A.) were harvested from 3 y of crops (2004, 2005, and 2006) DG determination when pericarp (fleshy portion) turned yellow and aril (seed coat or DG of the glycosylated protein isolates were determined usinner tissue) turned red (at 4 to 5 wk after flowering, ripe stage). ing a fluorescamine-based assay described by Yaylayan and others All chemicals for glycosylation, degree of glycosylation (DG), (1992) with slight modification. The isolates (25 mg, protein basis) characterization, and functional property determination were purwere dispersed into 10 mL of DI water and vortexed. The disperchased from VWR Intl., Inc. (Suwanee, Ga., U.S.A.) and Sigmasions were stirred continuously for 30 min and filtered through a Aldrich, Inc. (St. Louis, Mo., U.S.A.). 0.45-μm syringe filter. The filtrates were diluted 20 times, and to 200 μL of the diluted filtrates were added 4 mL of borate buffer Separation of seeds from ripe bitter melon (0.02 M potassium tetraborate, pH 8.5) and 1 mL of fluorescamine The seeds were manually separated from other tissues and oven reagent (15 mg in 100 mL acetone), which was then vortexed. A dried on a stainless steel tray in a dehydrator (Harvest Saver model blank solution was prepared using the buffer without protein. Flu#R-4; Commercial Dehydrator System, Inc., Eugene, Oreg., orescence intensity was read after 5 min of reaction time using U.S.A.) at 40 °C for 24 h. The dried seeds were ground us- a spectrofluorophotometer (Model RF-1501; Shimadzu, Kyoto, R WERKE model M20; Ika Works, Inc., Japan) at excitation and emission wavelengths of 390 and 475 ing a grinder (IKA Wilmington, N.C., U.S.A.), and passed through a 60-mesh sieve nm, respectively. The DG was calculated as follows: DG (%) = (W.S. Tyler Inc., Mentor, Ohio, U.S.A.) to obtain uniform parti- (Ac − Aa)/Ac × 100; where Ac was the fluorescence of uncle size. This bitter melon seed flour (BMSF) was defatted with modified protein and Aa was the fluorescence of the glycosylated hexane (ratio 1:2, w/v) 3 times on a shaker (Lab Line Inc., Fuller- protein. ton, Calif., U.S.A.) at room temperature for 8 h, vacuum-filtered, dried under a hood at ambient temperature to remove the residual Surface hydrophobicity determination hexane, and placed in a 4 °C storage. SH was determined by a hydrophobic fluorescence method
Preparation of protein isolate from defatted bitter melon seed flour Bitter melon seed protein isolate (BMSPI) was prepared from defatted BMSF based on optimum conditions (pH 9.0 in 1.3 M sodium chloride; Horax and others 2011). The defatted BMSF dispersion (defatted BMSF:1.3 M NaCl solution, 1:10 w/v) was stirred for 2 h and centrifuged at 10000 × g for 20 min to separate supernatant from residue. The supernatant was adjusted to pH 4.0 and the precipitated protein was separated from solution by centrifugation at 10000 × g for 15 min. The protein was reextracted twice from the residue. The combined extracted protein was washed twice using deionized (DI) water (pH 4.0), followed by centrifugation. The protein isolate was resolubilized by adjusting to pH 7.0, freeze-dried, and evaluated for surface hydrophobicity (SH), solubility, and thermal, emulsifying, and foaming properties before and after glycosylation. Three replications were obtained from each of 3 y of crops (2004, 2005, and 2006). Glycosylation of BMSPI The experimental design, consisting of 5 replications at the centre point, was constructed by JMP 7.0 software package (SAS Inst. Inc., Cary, N.C., U.S.A.) using a response surface central composite design. Two factors were selected for optimization: RH (in percentage) and temperature. Independent variables were coded as “a” and “A” to represent the lowest and the highest treatment levels, and “0” to represent the center point (Table 1). BMSPI (10 g) and D-glucose (1 g) were dissolved in water to give a 10% (w/v) protein solution. The solution was adjusted to pH 7.0, stirred for 10 min, and freeze-dried. The freeze-dried protein–glucose products (1 g of each) were placed on an aluminum plate and incubated at varying temperatures (40 °C/50 °C/ 60 °C) and RH values (50%/65%/80%) in a humidifier (Hotpack, Philadelphia, Pa., U.S.A.) for 48 h, and then stored at 4 °C until
C2216 Journal of Food Science r Vol. 79, Nr. 11, 2014
described by Hayakawa and Nakai (1985). Protein solutions ranging from 0.001 to 0.015% w/v in 0.01 M phosphate buffer (pH 7) were made, and 4 mL of each was reacted with 50 μL of 1.6 mM 1-anilino-8-naphthalene sulfonate solution (in 0.01 M phosphate buffer, pH 7.0). Fluorescence intensity of the solutions was read using the spectrofluorophotometer at a wavelength of 390 nm excitation and 470 nm emission. A slope from a linear regression between the fluorescence intensities and protein concentrations was considered as the SH of the proteins.
Thermal property determination Thermal properties of the glycosylated proteins were determined using a differential scanning calorimeter (Model Pyris-1; Perkins-Elmer Corp., Norwalk, Conn., U.S.A.). Fifty μL of the protein slurries in DI water (20%, protein basis) in a hermetically sealed large-volume stainless steel pan were scanned with the temperature increasing from 25 °C to 140 °C at a rate of 10 °C/min. A reference of an empty pan was used as comparison, while the instrument was calibrated using indium and zinc prior to measurement. Onset, end, and peak (designated as the denaturation) temperatures, and enthalpy value (delta H) were generated and calculated by thermal analysis software (Version 4.00; Pyris-1-DSC, Perkin-Elmer Corp.). Solubility determination Solubility of the proteins at pH 7.0 was determined using the modified method of Bera and Mukherjee (1989). The protein dispersion (1%) was made in DI water, adjusted to pH 7.0, and stirred for 30 min at room temperature with pH readjustment every 10 min. The dispersion was then centrifuged at 10000 × g for 30 min. Protein contents in the solution before centrifugation and in supernatants after centrifugation were determined based on a modified Kjeldahl method (AACC 1990) using a Kjeltec 2300
Glycosylated bitter melon seed protein . . . Table 1–Response surface design, and actual and predicted values of degrees of glycosylation (percentage) of glycosylated bitter melon seed protein isolates (BMSPI) at varying relative humidity (RH) percentages and temperatures.
Actual value Pattern
RH (percentage)
Temp (°C)
2004a
2005
2006
Average
Predicted value (R2 = 0.98)b
50 50 50 65 65 65 65 65 65 65 80 80 80
40 50 60 40 50 50 50 50 50 60 40 50 60
39.4 65.2 77.9 54.0 66.4 69.0 66.2 73.3 66.6 94.3 50.5 74.3 93.2
40.0 62.4 82.8 48.5 69.4 68.0 68.2 73.2 71.8 90.8 53.7 69.9 95.4
38.5 57.4 82.7 49.2 70.8 67.9 71.2 72.0 68.2 92.1 53.3 68.4 96.0
39.3 61.7 81.2 50.6 68.8 68.3 68.5 72.9 68.9 92.4 52.5 70.9 94.8
40.3 59.8 82.0 50.0 69.7 69.7 69.7 69.7 69.7 92.0 52.0 71.8 94.3
aa a0 aA 0a 00 00 00 00 00 0A Aa A0 AA a
Glycosylated BMSPI extracted from bitter melon seeds obtained from year 2004 crop. P values for RH and temperature against the degree of glycosylation are both
